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Sample records for angiotensin receptor binding

  1. Structural determinants for binding to angiotensin converting enzyme 2 (ACE2 and angiotensin receptors

    Directory of Open Access Journals (Sweden)

    Daniel eClayton

    2015-01-01

    Full Text Available Angiotensin converting enzyme 2 (ACE2 is a zinc carboxypeptidase involved in the renin angiotensin system (RAS and inactivates the potent vasopressive peptide angiotensin II (Ang II by removing the C-terminal phenylalanine residue to yield Ang1-7. This conversion inactivates the vasoconstrictive action of Ang II and yields a peptide that acts as a vasodilatory molecule at the Mas receptor and potentially other receptors. Given the growing complexity of RAS and level of cross-talk between ligands and their corresponding enzymes and receptors, the design of molecules with selectivity for the major RAS binding partners to control cardiovascular tone is an on-going challenge. In previous studies we used single β-amino acid substitutions to modulate the structure of Ang II and its selectivity for ACE2, AT1R and angiotensin type 2 (AT2R receptor. We showed that modification at the C-terminus of Ang II generally resulted in more pronounced changes to secondary structure and ligand binding, and here we further explore this region for the potential to modulate ligand specificity. In this study, 1 a library of forty-seven peptides derived from the C-terminal tetra-peptide sequence (-IHPF of Ang II was synthesised and assessed for ACE2 binding, 2 the terminal group requirements for high affinity ACE2 binding were explored by and N- and C-terminal modification, 3 high affinity ACE2 binding chimeric AngII analogues were then synthesized and assessed, 4 the structure of the full-length Ang II analogues were assessed by circular dichroism, and 5 the Ang II analogues were assessed for AT1R/AT2R selectivity by cell-based assays. Studies on the C-terminus of Ang II demonstrated varied specificity at different residue positions for ACE2 binding and four Ang II chimeric peptides were identified as selective ligands for the AT2 receptor. Overall, these results provide insight into the residue and structural requirements for ACE2 binding and angiotensin receptor

  2. Localization and characterization of angiotensin II receptor binding and angiotensin converting enzyme in the human medulla oblongata.

    Science.gov (United States)

    Allen, A M; Chai, S Y; Clevers, J; McKinley, M J; Paxinos, G; Mendelsohn, F A

    1988-03-08

    Angiotensin II receptor and angiotensin converting enzyme distributions in the human medulla oblongata were localised by quantitative in vitro autoradiography. Angiotensin II receptors were labelled with the antagonist analogue 125I-[Sar1, Ile8] AII while angiotensin converting enzyme was labelled with 125I-351A, a derivative of the specific converting enzyme inhibitor, lisinopril. Angiotensin II receptor binding and angiotensin converting enzyme are present in high concentrations in the nucleus of the solitary tract, the dorsal motor nucleus of vagus, the rostral and caudal ventrolateral reticular nucleus, and in a band connecting the dorsal and ventral regions. In the rostral and caudal ventrolateral reticular nucleus, angiotensin II receptors are distributed in a punctate pattern that registers with neuronal cell bodies. The distribution and density of these cell bodies closely resemble those of catecholamine-containing neurones mapped by others. In view of the known interactions of angiotensin II with both central and peripheral catecholamine-containing neurons of laboratory animals, the current anatomical findings suggest similar interactions between these neuroactive compounds in the human central nervous system. The presence of angiotensin II receptors and angiotensin converting enzyme in the nucleus of the solitary tract, dorsal motor nucleus of vagus, and rostral and caudal ventrolateral reticular nucleus demonstrates sites for central angiotensin II to exert its known actions on vasopressin release and autonomic functions including blood pressure control. These data also suggest a possible interaction between angiotensin II and central catecholeminergic systems.

  3. Receptor binding radiotracers for the angiotensin II receptor: radioiodinated [Sar1, Ile8]angiotensin II

    International Nuclear Information System (INIS)

    Gibson, R.E.; Beauchamp, H.T.; Fioravanti, C.; Brenner, N.; Burns, H.D.

    1994-01-01

    The potential for imaging the angiotensin II receptor was evaluated using the radioiodinated peptide antagonist [ 125 I][Sar 1 , Ile 8 ]angiotensin II. The radioligand provides a receptor-mediated signal in several tissues in rat (kidneys, adrenal and liver). The receptor-mediated signal of 3% ID/g kidney cortex should be sufficient to permit imaging, at least via SPECT. The radiotracer is sensitive to reductions in receptor concentration and can be used to define in vivo dose-occupancy curves of angiotensin II receptor ligands. Receptor-mediated images of [ 123 I][Sar 1 , Ile 8 ]angiotensin II were obtained in the rat kidney and Rhesus monkey liver. (author)

  4. Does protein binding modulate the effect of angiotensin II receptor antagonists?

    Directory of Open Access Journals (Sweden)

    Marc P Maillard

    2001-03-01

    Full Text Available IntroductionAngiotensin II AT 1-receptor antagonists are highly bound to plasma proteins (≥ 99%. With some antagonists, such as DuP-532, the protein binding was such that no efficacy of the drug could be demonstrated clinically. Whether protein binding interferes with the efficacy of other antagonists is not known. We have therefore investigated in vitro how plasma proteins may affect the antagonistic effect of different AT1-receptor antagonists.MethodsA radio-receptor binding assay was used to analyse the interaction between proteins and the ability of various angiotensin II (Ang II antagonists to block AT1-receptors. In addition, the Biacore technology, a new technique which enables the real-time monitoring of binding events between two molecules, was used to evaluate the dissociation rate constants of five AT1-receptor antagonists from human serum albumin.ResultsThe in vitro AT 1-antagonistic effects of different Ang II receptor antagonists were differentially affected by the presence of human plasma, with rightward shifts of the IC50 ranging from one to several orders of magnitude. The importance of the shift correlates with the dissociation rate constants of these drugs from albumin. Our experiments also show that the way that AT1-receptor antagonists bind to proteins differs from one compound to another. These results suggest that the interaction with plasma proteins appears to modulate the efficacy of some Ang II antagonists.ConclusionAlthough the high binding level of Ang II receptor antagonist to plasma proteins appears to be a feature common to this class of compounds, the kinetics and characteristics of this binding is of great importance. With some antagonists, protein binding interferes markedly with their efficacy to block AT1-receptors.

  5. IN SILICO EVALUATION OF ANGIOTENSIN II RECEPTOR ANTAGONIST’S PLASMA PROTEIN BINDING USING COMPUTED MOLECULAR DESCRIPTORS

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    Jadranka Odović

    2014-03-01

    Full Text Available The discovery of new pharmacologically active substances and drugs modeling led to necessity of predicting drugs properties and its ADME data. Angiotensin II receptor antagonists are a group of pharmaceuticals which modulate the renin-angiotensin-aldosterone system and today represent the most commonly prescribed anti-hypertensive drugs. The aim of this study was to compare different molecular properties of seven angiotensin II receptor antagonists / blockers (ARBs, (eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and their plasma protein binding (PPB data. Several ARBs molecular descriptors were calculated using software package Molinspiration Depiction Software as well as Virtual Computational Chemistry Laboratory (electronic descriptor – PSA, constitutional parameter – Mw, geometric descriptor – Vol, lipophilicity descriptors - logP values, aqueous solubility data – logS. The correlations between all collected descriptors and plasma protein binding data obtained from relevant literature were established. In the simple linear regression poor correlations were obtained in relationships between PPB data and all calculated molecular descriptors. In the next stage of the study multiple linear regression (MLR was used for correlation of PPB data with two different descriptors as independent variables. The best correlation (R2=0.70 with P<0.05 was established between PPB data and molecular weight with addition of volume values as independent variables. The possible application of computed molecular descriptors in drugs protein binding evaluation can be of great importance in drug research.

  6. The Fifth Transmembrane Domain of Angiotensin II Type 1 Receptor Participates in the Formation of the Ligand-binding Pocket and Undergoes a Counterclockwise Rotation upon Receptor Activation*

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    Domazet, Ivana; Martin, Stéphane S.; Holleran, Brian J.; Morin, Marie-Ève; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT1, N200C-AT1, I201C-AT1, G203C-AT1, and F204C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant I201C-N111G-AT1 became more sensitive to MTSEA, whereas mutant G203C-N111G-AT1 lost some sensitivity. Our results suggest that constitutive activation of AT1 receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side. PMID:19773549

  7. The fifth transmembrane domain of angiotensin II Type 1 receptor participates in the formation of the ligand-binding pocket and undergoes a counterclockwise rotation upon receptor activation.

    Science.gov (United States)

    Domazet, Ivana; Martin, Stéphane S; Holleran, Brian J; Morin, Marie-Eve; Lacasse, Patrick; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2009-11-13

    The octapeptide hormone angiotensin II exerts a wide variety of cardiovascular effects through the activation of the angiotensin II Type 1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein- coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. The role of the fifth transmembrane domain (TMD5) was investigated using the substituted cysteine accessibility method. All of the residues within Thr-190 to Leu-217 region were mutated one at a time to cysteine, and after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of L197C-AT(1), N200C-AT(1), I201C-AT(1), G203C-AT(1), and F204C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD5 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant I201C-N111G-AT(1) became more sensitive to MTSEA, whereas mutant G203C-N111G-AT(1) lost some sensitivity. Our results suggest that constitutive activation of AT(1) receptor causes an apparent counterclockwise rotation of TMD5 as viewed from the extracellular side.

  8. The angiotensin II type 1 receptor antagonist Losartan binds and activates bradykinin B2 receptor signaling

    DEFF Research Database (Denmark)

    Bonde, Marie Mi; Olsen, Kristine Boisen; Erikstrup, Niels

    2011-01-01

    The angiotensin II type 1 receptor (AT1R) blocker (ARB) Losartan has cardioprotective effects during ischemia-reperfusion injury and inhibits reperfusion arrhythmias -effects that go beyond the benefits of lowering blood pressure. The renin-angiotensin and kallikrein-kinin systems are intricately...

  9. Pharmacological significance of the interplay between angiotensin receptors: MAS receptors as putative final mediators of the effects elicited by angiotensin AT1 receptors antagonists.

    Science.gov (United States)

    Pernomian, Larissa; Pernomian, Laena; Gomes, Mayara S; da Silva, Carlos H T P

    2015-12-15

    The interplay between angiotensin AT1 receptors and MAS receptors relies on several inward regulatory mechanisms from renin-angiotensin system (RAS) including the functional crosstalk between angiotensin II and angiotensin-(1-7), the competitive AT1 antagonism exhibited by angiotensin-(1-7), the antagonist feature assigned to AT1/MAS heterodimerization on AT1 signaling and the AT1-mediated downregulation of angiotensin-converting enzyme 2 (ACE2). Recently, such interplay has acquired an important significance to RAS Pharmacology since a few studies have supporting strong evidences that MAS receptors mediate the effects elicited by AT1 antagonists. The present Perspective provides an overview of the regulatory mechanisms involving AT1 and MAS receptors, their significance to RAS Pharmacology and the future directions on the interplay between angiotensin receptors. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. The Second Transmembrane Domain of the Human Type 1 Angiotensin II Receptor Participates in the Formation of the Ligand Binding Pocket and Undergoes Integral Pivoting Movement during the Process of Receptor Activation*

    Science.gov (United States)

    Domazet, Ivana; Holleran, Brian J.; Martin, Stéphane S.; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-01-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT1) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT1 receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT1 receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT1, L81C-AT1, A85C-AT1, T88C-AT1, and A89C-AT1 mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT1 receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT1 receptor background. Indeed, mutant D74C-N111G-AT1 became insensitive to MTSEA, whereas mutant L81C-N111G-AT1 lost some sensitivity and mutant V86C-N111G-AT1 became sensitive to MTSEA. Our results suggest that constitutive activation of the AT1 receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket. PMID:19276075

  11. The second transmembrane domain of the human type 1 angiotensin II receptor participates in the formation of the ligand binding pocket and undergoes integral pivoting movement during the process of receptor activation.

    Science.gov (United States)

    Domazet, Ivana; Holleran, Brian J; Martin, Stéphane S; Lavigne, Pierre; Leduc, Richard; Escher, Emanuel; Guillemette, Gaétan

    2009-05-01

    The octapeptide hormone angiotensin II (AngII) exerts a wide variety of cardiovascular effects through the activation of the angiotensin II type-1 (AT(1)) receptor, which belongs to the G protein-coupled receptor superfamily. Like other G protein-coupled receptors, the AT(1) receptor possesses seven transmembrane domains that provide structural support for the formation of the ligand-binding pocket. In order to identify those residues in the second transmembrane domain (TMD2) that contribute to the formation of the binding pocket of the AT(1) receptor, we used the substituted cysteine accessibility method. All of the residues within the Leu-70 to Trp-94 region were mutated one at a time to a cysteine, and, after expression in COS-7 cells, the mutant receptors were treated with the sulfhydryl-specific alkylating agent methanethiosulfonate-ethylammonium (MTSEA). MTSEA reacts selectively with water-accessible, free sulfhydryl groups of endogenous or introduced point mutation cysteines. If a cysteine is found in the binding pocket, the covalent modification will affect the binding kinetics of the ligand. MTSEA substantially decreased the binding affinity of D74C-AT(1), L81C-AT(1), A85C-AT(1), T88C-AT(1), and A89C-AT(1) mutant receptors, which suggests that these residues orient themselves within the water-accessible binding pocket of the AT(1) receptor. Interestingly, this pattern of acquired MTSEA sensitivity was altered for TMD2 reporter cysteines engineered in a constitutively active N111G-AT(1) receptor background. Indeed, mutant D74C-N111G-AT(1) became insensitive to MTSEA, whereas mutant L81C-N111G-AT(1) lost some sensitivity and mutant V86C-N111G-AT(1) became sensitive to MTSEA. Our results suggest that constitutive activation of the AT(1) receptor causes TMD2 to pivot, bringing the top of TMD2 closer to the binding pocket and pushing the bottom of TMD2 away from the binding pocket.

  12. Angiotensin type 2 receptor (AT2R) and receptor Mas

    DEFF Research Database (Denmark)

    Villela, Daniel; Leonhardt, Julia; Patel, Neal

    2015-01-01

    The angiotensin type 2 receptor (AT2R) and the receptor Mas are components of the protective arms of the renin-angiotensin system (RAS), i.e. they both mediate tissue protective and regenerative actions. The spectrum of actions of these two receptors and their signalling mechanisms display striki...

  13. Receptor Autoradiography Protocol for the Localized Visualization of Angiotensin II Receptors.

    Science.gov (United States)

    Linares, Andrea; Couling, Leena E; Carrera, Eduardo J; Speth, Robert C

    2016-06-07

    This protocol describes receptor binding patterns for Angiotensin II (Ang II) in the rat brain using a radioligand specific for Ang II receptors to perform receptor autoradiographic mapping. Tissue specimens are harvested and stored at -80 °C. A cryostat is used to coronally section the tissue (brain) and thaw-mount the sections onto charged slides. The slide-mounted tissue sections are incubated in (125)I-SI-Ang II to radiolabel Ang II receptors. Adjacent slides are separated into two sets: 'non-specific binding' (NSP) in the presence of a receptor saturating concentration of non-radiolabeled Ang II, or an AT1 Ang II receptor subtype (AT1R) selective Ang II receptor antagonist, and 'total binding' with no AT1R antagonist. A saturating concentration of AT2 Ang II receptor subtype (AT2R) antagonist (PD123319, 10 µM) is also present in the incubation buffer to limit (125)I-SI-Ang II binding to the AT1R subtype. During a 30 min pre-incubation at ~22 °C, NSP slides are exposed to 10 µM PD123319 and losartan, while 'total binding' slides are exposed to 10 µM PD123319. Slides are then incubated with (125)I-SI-Ang II in the presence of PD123319 for 'total binding', and PD123319 and losartan for NSP in assay buffer, followed by several 'washes' in buffer, and water to remove salt and non-specifically bound radioligand. The slides are dried using blow-dryers, then exposed to autoradiography film using a specialized film and cassette. The film is developed and the images are scanned into a computer for visual and quantitative densitometry using a proprietary imaging system and a spreadsheet. An additional set of slides are thionin-stained for histological comparisons. The advantage of using receptor autoradiography is the ability to visualize Ang II receptors in situ, within a section of a tissue specimen, and anatomically identify the region of the tissue by comparing it to an adjacent histological reference section.

  14. Reduced glomerular angiotensin II receptor density in diabetes mellitus in the rat: time course and mechanism

    International Nuclear Information System (INIS)

    Wilkes, B.M.

    1987-01-01

    Glomerular angiotensin II receptors are reduced in number in early diabetes mellitus, which may contribute to hyperfiltration and glomerular injury. The time course and role of the renin-angiotensin-aldosterone system in the pathogenesis of the receptor abnormality were studied in male Sprague-Dawley rats made diabetic with streptozotocin (65 mg, iv). Glomerular angiotensin II receptors were measured by Scatchard analysis; insulin, renin activity, angiotensin II, and aldosterone were measured by RIA. Diabetes mellitus was documented at 24 h by a rise in plasma glucose (vehicle-injected control, 133 +/- 4; diabetic, 482 +/- 22 mg/dl and a fall in plasma insulin (control, 53.1 +/- 5.7; diabetic, 35.6 +/- 4.0 microIU/ml. At 24 h glomerular angiotensin II receptor density was decreased by 26.5% in diabetic rats (control, 75.5 +/- 9.6 X 10(6); diabetic, 55.5 +/- 8.3 X 10(6) receptors/glomerulus. Receptor occupancy could not explain the defect, because there was reduced binding in diabetic glomeruli after pretreatment with 3 M MgCl 2 , a maneuver that caused dissociation of previously bound hormone. There was a progressive return of the receptor density toward normal over the 60 days following induction of diabetes, with diabetic glomeruli measuring 22.7%, 14.8%, and 3.7% fewer receptors than age-matched controls at 11 days, 1 month, and 2 months, respectively

  15. Angiotensin type 2 receptors

    DEFF Research Database (Denmark)

    Sumners, Colin; de Kloet, Annette D; Krause, Eric G

    2015-01-01

    In most situations, the angiotensin AT2-receptor (AT2R) mediates physiological actions opposing those mediated by the AT1-receptor (AT1R), including a vasorelaxant effect. Nevertheless, experimental evidence vastly supports that systemic application of AT2R-agonists is blood pressure neutral...

  16. Angiotensin receptors and angiotensin I-converting enzyme in rat intestine

    International Nuclear Information System (INIS)

    Duggan, K.A.; Mendelsohn, F.A.; Levens, N.R.

    1989-01-01

    The purpose of this study was to map the distribution of angiotensin II (ANG II) receptors and ANG I-converting enzyme (ACE) in rat intestine. ANG II binding sites were visualized by in vitro autoradiography using iodinated [Sar1, Ile8]ANG II. The distribution of ACE was mapped using an iodinated derivative of lisinopril. Male Sprague-Dawley rats were killed and the interior of the whole intestine washed with ice-cold saline. Segments of duodenum, jejunum, ileum, and colon were quickly frozen in a mixture of isopentane and dry ice. Twenty-micron frozen sections were thaw-mounted onto gelatin-coated slides, incubated with either ligand, and exposed to X-ray film. After exposure and subsequent development, the films were quantitated by computerized densitometry. ANG II receptors were most dense in the colon, followed by the ileum, duodenum, and jejunum. Within each segment of intestine, specific ANG II binding sites were localized exclusively to the muscularis. In contrast, ACE was present in both the mucosa and the muscularis. The colocalization of ANG II receptors and ACE may suggest a role for locally generated ANG II in the control of intestinal function. The luminal orientation of ACE in the mucosa of the small intestine may suggest that at this site ACE serves primarily to hydrolyze dietary peptides

  17. Two distinct calmodulin binding sites in the third intracellular loop and carboxyl tail of angiotensin II (AT(1A receptor.

    Directory of Open Access Journals (Sweden)

    Renwen Zhang

    Full Text Available In this study, we present data that support the presence of two distinct calmodulin binding sites within the angiotensin II receptor (AT(1A, at juxtamembrane regions of the N-terminus of the third intracellular loop (i3, amino acids 214-231 and carboxyl tail of the receptor (ct, 302-317. We used bioluminescence resonance energy transfer assays to document interactions of calmodulin with the AT(1A holo-receptor and GST-fusion protein pull-downs to demonstrate that i3 and ct interact with calmodulin in a Ca²⁺-dependent fashion. The former is a 1-12 motif and the latter belongs to 1-5-10 calmodulin binding motif. The apparent Kd of calmodulin for i3 is 177.0±9.1 nM, and for ct is 79.4±7.9 nM as assessed by dansyl-calmodulin fluorescence. Replacement of the tryptophan (W219 for alanine in i3, and phenylalanine (F309 or F313 for alanine in ct reduced their binding affinities for calmodulin, as predicted by computer docking simulations. Exogenously applied calmodulin attenuated interactions between G protein βγ subunits and i3 and ct, somewhat more so for ct than i3. Mutations W219A, F309A, and F313A did not alter Gβγ binding, but reduced the ability of calmodulin to compete with Gβγ, suggesting that calmodulin and Gβγ have overlapping, but not identical, binding requirements for i3 and ct. Calmodulin interference with the Gβγ binding to i3 and ct regions of the AT(1A receptor strongly suggests that calmodulin plays critical roles in regulating Gβγ-dependent signaling of the receptor.

  18. Two Distinct Calmodulin Binding Sites in the Third Intracellular Loop and Carboxyl Tail of Angiotensin II (AT1A) Receptor

    Science.gov (United States)

    Zhang, Renwen; Liu, Zhijie; Qu, Youxing; Xu, Ying; Yang, Qing

    2013-01-01

    In this study, we present data that support the presence of two distinct calmodulin binding sites within the angiotensin II receptor (AT1A), at juxtamembrane regions of the N-terminus of the third intracellular loop (i3, amino acids 214–231) and carboxyl tail of the receptor (ct, 302–317). We used bioluminescence resonance energy transfer assays to document interactions of calmodulin with the AT1A holo-receptor and GST-fusion protein pull-downs to demonstrate that i3 and ct interact with calmodulin in a Ca2+-dependent fashion. The former is a 1–12 motif and the latter belongs to 1-5-10 calmodulin binding motif. The apparent Kd of calmodulin for i3 is 177.0±9.1 nM, and for ct is 79.4±7.9 nM as assessed by dansyl-calmodulin fluorescence. Replacement of the tryptophan (W219) for alanine in i3, and phenylalanine (F309 or F313) for alanine in ct reduced their binding affinities for calmodulin, as predicted by computer docking simulations. Exogenously applied calmodulin attenuated interactions between G protein βγ subunits and i3 and ct, somewhat more so for ct than i3. Mutations W219A, F309A, and F313A did not alter Gβγ binding, but reduced the ability of calmodulin to compete with Gβγ, suggesting that calmodulin and Gβγ have overlapping, but not identical, binding requirements for i3 and ct. Calmodulin interference with the Gβγ binding to i3 and ct regions of the AT1A receptor strongly suggests that calmodulin plays critical roles in regulating Gβγ-dependent signaling of the receptor. PMID:23755207

  19. Distinct angiotensin II receptor in primary cultures of glial cells from rat brain

    International Nuclear Information System (INIS)

    Raizada, M.K.; Phillips, M.I.; Crews, F.T.; Sumners, C.

    1987-01-01

    Angiotensin II (Ang-II) has profound effects on the brain. Receptors for Ang-II have been demonstrated on neurons, but no relationship between glial cells and Agn-II has been established. Glial cells (from the hypothalamus and brain stem of 1-day-old rat brains) in primary culture have been used to demonstrate the presence of specific Ang-II receptors. Binding of 125 I-Ang-II to glial cultures was rapid, reversible, saturable, and specific for Ang-II. The rank order of potency of 125 I-Ang-II binding was determined. Scatchard analysis revealed a homogeneous population of high-affinity binding sites with a B/sub max/ of 110 fmol/mg of protein. Light-microscopic autoradiography of 125 I-Ang-II binding supported the kinetic data, documenting specific Ang-II receptors on the glial cells. Ang-II stimulated a dose-dependent hydrolysis of phosphatidylinositols in glial cells, an effect mediated by Ang-II receptors. However, Ang-II failed to influence [ 3 H] norepinephrine uptake, and catecholamines failed to regulate Ang-II receptors, effects that occur in neurons. These observations demonstrate the presence of specific Ang-II receptors on the glial cells in primary cultures derived from normotensive rat brain. The receptors are kinetically similar to, but functionally distinct from, the neuronal Ang-II receptors

  20. Crystal structure of NL63 respiratory coronavirus receptor-binding domain complexed with its human receptor

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Kailang; Li, Weikai; Peng, Guiqing; Li, Fang; (Harvard-Med); (UMM-MED)

    2010-03-04

    NL63 coronavirus (NL63-CoV), a prevalent human respiratory virus, is the only group I coronavirus known to use angiotensin-converting enzyme 2 (ACE2) as its receptor. Incidentally, ACE2 is also used by group II SARS coronavirus (SARS-CoV). We investigated how different groups of coronaviruses recognize the same receptor, whereas homologous group I coronaviruses recognize different receptors. We determined the crystal structure of NL63-CoV spike protein receptor-binding domain (RBD) complexed with human ACE2. NL63-CoV RBD has a novel {beta}-sandwich core structure consisting of 2 layers of {beta}-sheets, presenting 3 discontinuous receptor-binding motifs (RBMs) to bind ACE2. NL63-CoV and SARS-CoV have no structural homology in RBD cores or RBMs; yet the 2 viruses recognize common ACE2 regions, largely because of a 'virus-binding hotspot' on ACE2. Among group I coronaviruses, RBD cores are conserved but RBMs are variable, explaining how these viruses recognize different receptors. These results provide a structural basis for understanding viral evolution and virus-receptor interactions.

  1. Trends in co-prescribing of angiotensin converting enzyme inhibitors and angiotensin receptor blockers in Ireland.

    LENUS (Irish Health Repository)

    Wan Md Adnan, Wan A H

    2011-03-01

    (i) To examine the trends in co-prescribing of angiotensin converting enzyme inhibitor (ACEI) and angiotensin-II receptor blocker (ARB) therapy and (ii) to examine the influence of major clinical trials (CALM, COOPERATE, VALIANT and ONTARGET) on co-prescribing.

  2. Renal graft failure after addition of an angiotensin II receptor antagonist to an angiotensin-converting enzyme inhibitor

    DEFF Research Database (Denmark)

    Kamper, Anne-Lise; Nielsen, Arne Høj; Baekgaard, Niels

    2002-01-01

    Combined treatment with an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin II (Ang II) receptor blocker (ARB) has been suggested in order to achieve a more complete blockade of the renin-angiotensin-aldosterone system in cardiovascular and renal disease. The present report descri...

  3. Angiotensin receptor blockers: Focus on cardiac and renal injury.

    Science.gov (United States)

    Arumugam, Somasundaram; Sreedhar, Remya; Thandavarayan, Rajarajan A; Karuppagounder, Vengadeshprabhu; Krishnamurthy, Prasanna; Suzuki, Kenji; Nakamura, Masahiko; Watanabe, Kenichi

    2016-04-01

    Angiotensin II, an important component of renin angiotensin system, is a potent vasopressor and its actions are mostly mediated via angiotensin II type 1 receptor (AT1R) and role of AT2R in counterbalancing the actions of AT1R stimulation are under extensive research. In addition to its physiological actions, angiotensin II plays important roles in the pathogenesis of atherosclerosis, hypertension, left ventricular hypertrophy, and heart failure. The effects of angiotensin II can be blocked by either suppressing its production by blocking angiotensin converting enzyme or by antagonizing its actions on AT1R using angiotensin II receptor blockers (ARBs). Instead of the extensive use of ARBs in the treatment of various cardiovascular diseases, proper selection of a particular ARB is crucial as the clinical condition of individual patient is different and also their economic status would play an essential role in medication compliance. Thus a critical review of the proven and promising actions of ARBs against various pathological conditions will be of great importance for the clinicians as well as for the researchers. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS

    DEFF Research Database (Denmark)

    Leonhardt, Julia; Villela, Daniel C.; Teichmann, Anke

    2017-01-01

    The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may in...

  5. Estrogen effects on angiotensin receptors are modulated by pituitary in female rats

    International Nuclear Information System (INIS)

    Douglas, J.G.

    1987-01-01

    The present studies were designed to test the hypothesis that changes in angiotensin II (ANG II) receptors might modulate the layered target tissue responsiveness accompanying estradiol administration. Estradiol was infused continuously in oophorectomized female rats. Aldosterone was also infused in control and experimental animals to avoid estrogen-induced changes in renin and ANG II. ANG II binding constants were determined in radioreceptor assays. Estradiol increased binding site concentration in adrenal glomerulosa by 76% and decreased binding sites of uterine myometrium and glomeruli by 45 and 24%, respectively. There was an accompanying increase in the affinity of ANG II binding to adrenal glomerulosa and uterine myometrium. Because estrogen is a potent stimulus of prolactin release from the pituitary of rodents, studies were also designed to test the hypothesis that prolactin may mediate some or all of the estrogen-induced effects observed. Hypophysectomy abolished estradiol stimulation of prolactin release and most ANG II receptor changes. Prolactin administration to pituitary intact rats was associated with a 50% increase in receptor density of adrenal glomerulosa simulating estradiol administration. However, the changes in glomeruli and uterine myometrium were opposite in that both tissues also increased receptor density, suggesting that prolactin was not the sole mediator of the estrogen-induced receptor changes. In conclusion, regulation of ANG II receptors in a number of diverse target tissues by estradiol is complex with contributions from estrogens and pituitary factors, which include but do not exclusively involve prolactin

  6. Combined Angiotensin Receptor Antagonism and Neprilysin Inhibition

    Science.gov (United States)

    Hubers, Scott A.; Brown, Nancy J.

    2016-01-01

    Heart failure affects approximately 5.7 million people in the United States alone. Angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, beta-blockers, and aldosterone antagonists have improved mortality in patients with heart failure and reduced ejection fraction, but mortality remains high. In July 2015, the FDA approved the first of a new class of drugs for the treatment of heart failure; valsartan/sacubitril (formerly known as LCZ696 and currently marketed by Novartis as Entresto) combines the angiotensin receptor blocker valsartan and the neprilysin inhibitor prodrug sacubitril in a 1:1 ratio in a sodium supramolecular complex. Sacubitril is converted by esterases to LBQ657, which inhibits neprilysin, the enzyme responsible for the degradation of the natriuretic peptides and many other vasoactive peptides. Thus, this combined angiotensin receptor antagonist and neprilysin inhibitor addresses two of the pathophysiologic mechanisms of heart failure - activation of the renin-angiotensin-aldosterone system and decreased sensitivity to natriuretic peptides. In the Prospective comparison of ARNI with ACEI to Determine Impact on Global Mortality and morbidity in Heart Failure (PARADIGM-HF) trial, valsartan/sacubitril significantly reduced mortality and hospitalization for heart failure, as well as blood pressure, compared to enalapril in patients with heart failure, reduced ejection fraction, and an elevated circulating level of brain natriuretic peptide or N-terminal pro-brain natriuretic peptide. Ongoing clinical trials are evaluating the role of valsartan/sacubitril in the treatment of heart failure with preserved ejection fraction and hypertension. We review here the mechanisms of action of valsartan/sacubitril, the pharmacologic properties of the drug, and its efficacy and safety in the treatment of heart failure and hypertension. PMID:26976916

  7. Glucocorticoid-Induced Fetal Programming Alters the Functional Complement of Angiotensin Receptors Subtypes within the Kidney

    OpenAIRE

    Gwathmey, TanYa M.; Shaltout, Hossam A.; Rose, James C.; Diz, Debra I.; Chappell, Mark C.

    2011-01-01

    We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80–81 days gestation with full term delivery. Renal nuclear and plasma membrane fractions were isolated from 1.0–1.5 year old sheep for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were sign...

  8. Valsartan attenuates intimal hyperplasia in balloon-injured rat aortic arteries through modulating the angiotensin-converting enzyme 2-angiotensin-(1-7)-Mas receptor axis.

    Science.gov (United States)

    Li, Yonghong; Cai, Shanglang; Wang, Qixin; Zhou, Jingwei; Hou, Bo; Yu, Haichu; Ge, Zhiming; Guan, Renyan; Liu, Xu

    2016-05-15

    The role of the Mas receptor in the activity of valsartan against intimal hyperplasia is unclear. Herein, we investigated the role of the angiotensin-converting enzyme 2 (ACE2)-angiotensin-(1-7)-Mas receptor axis on the activity of valsartan against intimal hyperplasiain balloon-injured rat aortic arteries. Wistar rats were randomized equally into the sham control group, injured group, and injured plus valsartan (20 mg/kg/d)-treated group. Valsartan significantly attenuated the vascular smooth muscle cell proliferation and intimal and medial thickening on days 14 and 28 after injury. The angiotensin-(1-7) levels as well as ACE2 and Mas receptor mRNA/protein expression were significantly decreased in the injured rats, compared to the uninjured rats; meanwhile, the angiotensin II level as well as the ACE and AT1 receptor mRNA/protein expression were increased (all P valsartan significantly increased the angiotensin-(1-7) levels as well as ACE2 and Mas receptor mRNA/protein expression but decreased the angiotensin II level, ACE and AT1 receptor mRNA/protein expression, as well as the p-ERK protein expression, compared to the injured group (all P valsartan attenuates neointimal hyperplasiain balloon-injured rat aortic arteries through activation of the ACE2-angiotensin-(1-7)-Mas axis as well as inhibition of the ACE-angiotensin II-AT1 and p-ERK pathways. Copyright © 2016 Elsevier Inc. All rights reserved.

  9. Antidiabetic mechanisms of angiotensin-converting enzyme inhibitors and angiotensin II receptor antagonists: beyond the renin-angiotensin system

    Czech Academy of Sciences Publication Activity Database

    Kurtz, T. W.; Pravenec, Michal

    2004-01-01

    Roč. 22, č. 12 (2004), s. 2253-2261 ISSN 0263-6352 R&D Projects: GA ČR GA301/03/0751 Grant - others:HHMI(US) HHMI55000331 Institutional research plan: CEZ:AV0Z5011922 Keywords : angiotensin II receptors * metabolic syndrome * peroxisome proliferator activated receptors Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.871, year: 2004

  10. Molecule-specific Effects of Angiotensin II–Receptor Blockers Independent of the Renin–Angiotensin System

    Czech Academy of Sciences Publication Activity Database

    Kurtz, T. W.; Pravenec, Michal

    2008-01-01

    Roč. 21, č. 8 (2008), s. 852-859 ISSN 0895-7061 R&D Projects: GA MŠk(CZ) 1M0520; GA MZd(CZ) NR8545 Grant - others:EURATOOLS(XE) LSHG-CT-2005-019015; HHMI(US) 55005624 Institutional research plan: CEZ:AV0Z50110509 Keywords : angiotensin-receptor blockers * cardiovascular protection * renin-angiotensin system Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 3.122, year: 2008

  11. Nonpeptidic angiotensin II AT₁ receptor antagonists derived from 6-substituted aminocarbonyl and acylamino benzimidazoles.

    Science.gov (United States)

    Zhang, Jun; Wang, Jin-Liang; Yu, Wei-Fa; Zhou, Zhi-Ming; Tao, Wen-Chang; Wang, Yi-Cheng; Xue, Wei-Zhe; Xu, Di; Hao, Li-Ping; Han, Xiao-Feng; Fei, Fan; Liu, Ting; Liang, Ai-Hua

    2013-11-01

    Both 6-substituted aminocarbonyl and acylamino benzimidazole derivatives were designed and synthesized as nonpeptidic angiotensin II AT₁ receptor antagonists. Compounds 6f, 6g, 11e, 11f, 11g, and 12 showed nanomolar AT₁ receptor binding affinity and high AT₁ receptor selectivity over AT₂ receptor in a preliminary pharmacological evaluation. Among them, the two most active compounds 6f (AT₁ IC₅₀ = 3 nM, AT₂ IC₅₀ > 10,000 nM, PA₂ = 8.51) and 11g (AT₁ IC₅₀ = 0.1 nM, AT₂ IC₅₀ = 149 nM, PA₂ = 8.43) exhibited good antagonistic activity in isolated rabbit aortic strip functional assay. In addition, they were orally active AT₁ receptor antagonists in spontaneous hypertensive rats. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  12. Differential clinical profile of candesartan compared to other angiotensin receptor blockers

    Directory of Open Access Journals (Sweden)

    Zimlichman R

    2011-12-01

    Full Text Available Relu Cernes1,2, Margarita Mashavi1,3, Reuven Zimlichman1,31The Brunner Institute for Cardiovascular Research, Wolfson Medical Center and Tel Aviv University, Tel Aviv, Israel; 2Department of Nephrology, Wolfson Medical Center, Holon, Israel; 3Department of Medicine, Wolfson Medical Center, Holon, IsraelAbstract: The advantages of blood pressure (BP control on the risks of heart failure and stroke are well established. The renin-angiotensin system plays an important role in volume homeostasis and BP regulation and is a target for several groups of antihypertensive drugs. Angiotensin II receptor blockers represent a major class of antihypertensive compounds. Candesartan cilexetil is an angiotensin II type 1 (AT[1] receptor antagonist (angiotensin receptor blocker [ARB] that inhibits the actions of angiotensin II on the renin-angiotensin-aldosterone system. Oral candesartan 8–32 mg once daily is recommended for the treatment of adult patients with hypertension. Clinical trials have demonstrated that candesartan cilexetil is an effective agent in reducing the risk of cardiovascular mortality, stroke, heart failure, arterial stiffness, renal failure, retinopathy, and migraine in different populations of adult patients including patients with coexisting type 2 diabetes, metabolic syndrome, or kidney impairment. Clinical evidence confirmed that candesartan cilexetil provides better antihypertensive efficacy than losartan and is at least as effective as telmisartan and valsartan. Candesartan cilexetil, one of the current market leaders in BP treatment, is a highly selective compound with high potency, a long duration of action, and a tolerability profile similar to placebo. The most important and recent data from clinical trials regarding candesartan cilexetil will be reviewed in this article.Keywords: angiotensin receptor blockers, candesartan, candesartan cilexetil, clinical trials, efficacy studies, safety, blood pressure

  13. Local angiotensin II promotes adipogenic differentiation of human adipose tissue mesenchymal stem cells through type 2 angiotensin receptor

    Directory of Open Access Journals (Sweden)

    Veronika Y. Sysoeva

    2017-12-01

    Full Text Available Obesity is often associated with high systemic and local activity of renin-angiotensin system (RAS. Mesenchymal stem cells of adipose tissue are the main source of adipocytes. The aim of this study was to clarify how local RAS could control adipose differentiation of human adipose tissue derived mesenchymal stem cells (ADSCs. We examined the distribution of angiotensin receptor expressing cells in human adipose tissue and found that type 1 and type 2 receptors are co-expressed in its stromal compartment, which is known to contain mesenchymal stem cells. To study the expression of receptors specifically in ADSCs we have isolated them from adipose tissue. Up to 99% of cultured ADSCs expressed angiotensin II (AngII receptor type 1 (AT1. Using the analysis of Ca2+ mobilization in single cells we found that only 5.2 ± 2.7% of ADSCs specifically respond to serial Ang II applications via AT1 receptor and expressed this receptor constantly. This AT1const ADSCs subpopulation exhibited increased adipose competency, which was triggered by endogenous AngII. Inhibitory and expression analyses showed that AT1const ADSCs highly co-express AngII type 2 receptor (AT2, which was responsible for increased adipose competency of this ADSC subpopulation.

  14. Angiotensin-II type 1 receptor gene polymorphism and diabetic microangiopathy

    DEFF Research Database (Denmark)

    Tarnow, L; Cambien, Francois; Rossing, P

    1996-01-01

    with proliferative retinopathy and without diabetic retinopathy was found either: 77 (50%) / 66 (42%) / 13 (8%) vs. 42 (63%) / 22 (33%) / 3 (4%) had AA/AC/CC genotypes, respectively. CONCLUSIONS: The A1166-->C polymorphism in the angiotensin-II type 1 receptor gene does not contribute to the genetic susceptibility...... is present particularly in vascular smooth muscle cells, myocardium and the kidney. A transversion of adenine to cytosine at nucleotide position 1166 in the gene coding for the angiotensin-II type 1 receptor has been associated with hypertension in the non-diabetic population. METHODS: We studied...... the relationship between the A1166-->C polymorphism in the angiotensin-II type 1 receptor gene in patients with insulin dependent diabetes mellitus (IDDM) and diabetic nephropathy (121 men, 77 women, age 41 +/- 10 years, diabetes duration 27 +/- 8 years) and in IDDM patients with normoalbuminuria (116 men, 74...

  15. Association between angiotensin II receptor gene polymorphism and serum angiotensin converting enzyme (SACE) activity in patients with sarcoidosis.

    Science.gov (United States)

    Takemoto, Y; Sakatani, M; Takami, S; Tachibana, T; Higaki, J; Ogihara, T; Miki, T; Katsuya, T; Tsuchiyama, T; Yoshida, A; Yu, H; Tanio, Y; Ueda, E

    1998-06-01

    Serum angiotensin converting enzyme (SACE) is considered to reflect disease activity in sarcoidosis. SACE activity is increased in many patients with active sarcoid lesions. The mechanism for the increased SACE activity in this disease has not been clarified. ACE insertion/deletion (I/D) gene polymorphism has been reported to have an association with SACE levels in sarcoidosis, but no evidence of an association between angiotensin II receptor gene polymorphism and SACE in this disease has been found. A study of the association of angiotensin II receptor gene polymorphisms with sarcoidosis was therefore undertaken. ACE (I/D), angiotensin II type 1 receptor (AGTR1), and angiotensin II type 2 receptor (AGTR2) gene polymorphisms were investigated by polymerase chain reaction (PCR) and SACE levels were measured in three groups of patients: those with sarcoidosis or tuberculosis and normal controls. There was no difference in allele frequency of AGTR1 and AGTR2 polymorphism among the three groups. Neither AGTR1 nor AGTR2 polymorphisms were associated with sarcoidosis. SACE activity was higher in patients with sarcoidosis with the AGTR1 A/C genotype than in others. However, this tendency was not detected in patients with tuberculosis. The AGTR1 allele C is associated with high activity of SACE in patients with sarcoidosis. It is another predisposing factor for high levels of SACE in patients with sarcoidosis and is considered to be an independent factor from the ACE D allele for high levels of SACE in sarcoidosis. This fact could be one of the explanations for the increased SACE activity in sarcoidosis.

  16. Angiotensin receptors in Dupuytren's disease: a target for pharmacological treatment?

    Science.gov (United States)

    Stephen, Christopher; Touil, Leila; Vaiude, Partha; Singh, Jaipaul; McKirdy, Stuart

    2018-02-01

    Attempts at the pharmacological treatment of Dupuytren's disease have so far been unsuccessful, and the disease is not yet fully understood on a cellular level. The Renin-Angiotensin System has long been understood to play a circulating hormonal role. However, there is much evidence showing Angiotensin II to play a local role in wound healing and fibrosis, with ACE inhibitors being widely used as an anti-fibrotic agent in renal and cardiac disease. This study was designed to investigate the presence of Angiotensin II receptors 1 (AT1) and 2 (AT2) in Dupuytren's tissue to form a basis for further study into the pharmacological treatment of this condition. Tissue was harvested from 11 patients undergoing surgery for Dupuytren's disease. Each specimen was processed into frozen sections and immunostaining was employed to identify AT1 and AT2 receptors. Immunostaining for AT1 receptors was mildly positive in one patient and negative in all the remaining patients. However, all specimens stained extensively for AT2 receptors. This suggests that the expression of AT2 receptors is more prominent than AT1 receptors in Dupuytren's disease. These findings have opened a new avenue for future research involving ACE inhibitors, AT2 agonists, and AT2 antagonists in Dupuytren's disease.

  17. Angiotensin II, hypertension and angiotensin II receptor antagonism: Roles in the behavioural and brain pathology of a mouse model of Alzheimer's disease

    NARCIS (Netherlands)

    Wiesmann, M.; Roelofs, M.; Lugt, R. Van Der; Heerschap, A.; Kiliaan, A.J.; Claassen, J.A.H.R.

    2017-01-01

    Elevated angiotensin II causes hypertension and contributes to Alzheimer's disease by affecting cerebral blood flow. Angiotensin II receptor blockers may provide candidates to reduce (vascular) risk factors for Alzheimer's disease. We studied effects of two months of angiotensin II-induced

  18. A virus-binding hot spot on human angiotensin-converting enzyme 2 is critical for binding of two different coronaviruses.

    Science.gov (United States)

    Wu, Kailang; Chen, Lang; Peng, Guiqing; Zhou, Wenbo; Pennell, Christopher A; Mansky, Louis M; Geraghty, Robert J; Li, Fang

    2011-06-01

    How viruses evolve to select their receptor proteins for host cell entry is puzzling. We recently determined the crystal structures of NL63 coronavirus (NL63-CoV) and SARS coronavirus (SARS-CoV) receptor-binding domains (RBDs), each complexed with their common receptor, human angiotensin-converting enzyme 2 (hACE2), and proposed the existence of a virus-binding hot spot on hACE2. Here we investigated the function of this hypothetical hot spot using structure-guided biochemical and functional assays. The hot spot consists of a salt bridge surrounded by hydrophobic tunnel walls. Mutations that disturb the hot spot structure have significant effects on virus/receptor interactions, revealing critical energy contributions from the hot spot structure. The tunnel structure at the NL63-CoV/hACE2 interface is more compact than that at the SARS-CoV/hACE2 interface, and hence RBD/hACE2 binding affinities are decreased either by NL63-CoV mutations decreasing the tunnel space or by SARS-CoV mutations increasing the tunnel space. Furthermore, NL63-CoV RBD inhibits hACE2-dependent transduction by SARS-CoV spike protein, a successful application of the hot spot theory that has the potential to become a new antiviral strategy against SARS-CoV infections. These results suggest that the structural features of the hot spot on hACE2 were among the driving forces for the convergent evolution of NL63-CoV and SARS-CoV.

  19. No effect of angiotensin II AT(2)-receptor antagonist PD 123319 on cerebral blood flow autoregulation

    DEFF Research Database (Denmark)

    Estrup, T M; Paulson, O B; Strandgaard, S

    2001-01-01

    Blockade of the renin-angiotensin system with angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin AT1-receptor antagonists shift the limits of autoregulation of cerebral blood flow (CBF) towards lower blood pressure (BP). The role of AT2-receptors in the regulation of the cerebral cir...

  20. Study of signal transduction mechanism of angiotensin 2 receptor by means of site-directed mutagenesis; Bui totsuzen hen'iho wo mochiita anjiotenshin 2 reseputa no joho dentatsu kiko no kaimei

    Energy Technology Data Exchange (ETDEWEB)

    Yamano, Yoshiaki [Tottori University, Tottori (Japan). Faculty of Agriculture

    1998-12-16

    The renin-angiotensin system (RAS) plays an important role in the regulation of blood pressure. In order to clarify the signaling mechanism mediated by angiotensin 2 receptor, Gq-protein binding amino acid residues of this receptor were clarified by site-directed mutagenesis study. Amino acid residues in the carboxyl tail region were changed by alanines, individually. These mutated receptors were expressed stably in CHO cells, and GTP effect and second messenger molecules were determined, and three residues (Y 312, F313 and L 314) in this region were determined to be concerned for the binding of Gq protein. The other signaling systems, Gi, MAP kinase, JAK-STAT mediated, were reported to be concerned for this receptor. Novel drags for high blood pressure therapy would be explored by clarifying these signaling mechanisms. (author)

  1. Design, synthesis and biological activity of 6-substituted carbamoyl benzimidazoles as new nonpeptidic angiotensin II AT₁ receptor antagonists.

    Science.gov (United States)

    Zhang, Jun; Wang, Jin-Liang; Zhou, Zhi-Ming; Li, Zhi-Huai; Xue, Wei-Zhe; Xu, Di; Hao, Li-Ping; Han, Xiao-Feng; Fei, Fan; Liu, Ting; Liang, Ai-Hua

    2012-07-15

    A series of 6-substituted carbamoyl benzimidazoles were designed and synthesised as new nonpeptidic angiotensin II AT(1) receptor antagonists. The preliminary pharmacological evaluation revealed a nanomolar AT(1) receptor binding affinity for all compounds in the series, and a potent antagonistic activity in an isolated rabbit aortic strip functional assay for compounds 6f, 6g, 6h and 6k was also demonstrated. Furthermore, evaluation in spontaneous hypertensive rats and a preliminary toxicity evaluation showed that compound 6g is an orally active AT(1) receptor antagonist with low toxicity. Copyright © 2012 Elsevier Ltd. All rights reserved.

  2. Glucocorticoid-induced fetal programming alters the functional complement of angiotensin receptor subtypes within the kidney.

    Science.gov (United States)

    Gwathmey, TanYa M; Shaltout, Hossam A; Rose, James C; Diz, Debra I; Chappell, Mark C

    2011-03-01

    We examined the impact of fetal programming on the functional responses of renal angiotensin receptors. Fetal sheep were exposed in utero to betamethasone (BMX; 0.17 mg/kg) or control (CON) at 80 to 81 days gestation with full-term delivery. Renal nuclear and plasma membrane fractions were isolated from sheep age 1.0 to 1.5 years for receptor binding and fluorescence detection of reactive oxygen species (ROS) or nitric oxide (NO). Mean arterial blood pressure and blood pressure variability were significantly higher in the BMX-exposed adult offspring versus CON sheep. The proportion of nuclear AT(1) receptors sensitive to losartan was 2-fold higher (67 ± 6% vs 27 ± 9%; Psheep (16 ± 3% vs 6 ± 4%; Pfetal programming.

  3. Angiotensin AT1-receptor blockers and cerebrovascular protection: do they actually have a cutting edge over angiotensin-converting enzyme inhibitors?

    DEFF Research Database (Denmark)

    Oprisiu-Fournier, Roxana; Faure, Sébastien; Mazouz, Hakim

    2009-01-01

    First, an update of the vascular systemic and tissue renin-angiotensin-aldosterone system is provided to explain how it is regulated at the systemic and tissue levels, and how many angiotensin peptides and receptors can be modulated by the various antihypertensive drugs. Second, experimental data...... stroke prevention trial PRoFESS, most trials support the hypothesis that angiotensin II-increasing drugs confer specific blood pressure-independent brain ischemia protection when compared with angiotensin II-decreasing drugs or placebo. A careful analysis of the PRoFESS trial, however, reveals study...

  4. Discovery of a Series of Imidazo[4,5-b]pyridines with Dual Activity at Angiotensin II Type 1 Receptor and Peroxisome Proliferator-Activated Receptor-[gamma

    Energy Technology Data Exchange (ETDEWEB)

    Casimiro-Garcia, Agustin; Filzen, Gary F.; Flynn, Declan; Bigge, Christopher F.; Chen, Jing; Davis, Jo Ann; Dudley, Danette A.; Edmunds, Jeremy J.; Esmaeil, Nadia; Geyer, Andrew; Heemstra, Ronald J.; Jalaie, Mehran; Ohren, Jeffrey F.; Ostroski, Robert; Ellis, Teresa; Schaum, Robert P.; Stoner, Chad (Pfizer)

    2013-03-07

    Mining of an in-house collection of angiotensin II type 1 receptor antagonists to identify compounds with activity at the peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}) revealed a new series of imidazo[4,5-b]pyridines 2 possessing activity at these two receptors. Early availability of the crystal structure of the lead compound 2a bound to the ligand binding domain of human PPAR{gamma} confirmed the mode of interaction of this scaffold to the nuclear receptor and assisted in the optimization of PPAR{gamma} activity. Among the new compounds, (S)-3-(5-(2-(1H-tetrazol-5-yl)phenyl)-2,3-dihydro-1H-inden-1-yl)-2-ethyl-5-isobutyl-7-methyl-3H-imidazo[4,5-b]pyridine (2l) was identified as a potent angiotensin II type I receptor blocker (IC{sub 50} = 1.6 nM) with partial PPAR{gamma} agonism (EC{sub 50} = 212 nM, 31% max) and oral bioavailability in rat. The dual pharmacology of 2l was demonstrated in animal models of hypertension (SHR) and insulin resistance (ZDF rat). In the SHR, 2l was highly efficacious in lowering blood pressure, while robust lowering of glucose and triglycerides was observed in the male ZDF rat.

  5. Angiotensin receptor blockers & endothelial dysfunction: Possible correlation & therapeutic implications

    Directory of Open Access Journals (Sweden)

    Miroslav Radenkovic

    2016-01-01

    Full Text Available The endothelium is one of the most important constituents of vascular homeostasis, which is achieved through continual and balanced production of different relaxing and contractile factors. When there is a pathological disturbance in release of these products, endothelial dysfunction (ED will probably occur. ED is considered to be the initial step in the development of atherosclerosis. This pathological activation and inadequate functioning of endothelial cells was shown to be to some extent a reversible process, which all together resulted in increased interest in investigation of different beneficial treatment options. To this point, the pharmacological approach, including for example, the use of angiotensin-converting enzyme inhibitors or statins, was clearly shown to be effective in the improvement of ED. One of many critical issues underlying ED represents instability in the balance between nitric oxide and angiotensin II (Ang II production. Considering that Ang II was confirmed to be important for the development of ED, the aim of this review article was to summarize the findings of up to date clinical studies associated with therapeutic application of angiotensin receptor blockers and improvement in ED. In addition, it was of interest to review the pleiotropic actions of angiotensin receptor blockers linked to the improvement of ED. The prospective, randomized, double-blind, placebo or active-controlled clinical trials were identified and selected for the final evaluation.

  6. Evidence that the angiotensin at 2-receptor agonist compound 21 is also a low affinity thromboxane TXA2-receptor antagonist

    DEFF Research Database (Denmark)

    Fredgart, M.; Leurgans, T.; Stenelo, M.

    2015-01-01

    Objective: The objective of this study was to test whether Compound 21 (C21), a high-affinity, non-peptide angiotensinAT2-receptor agonist, is also an antagonist of thromboxane A2 (TXA2) receptors thus reducing both vasoconstriction and platelet aggregation. Design and method: Binding of C21...... to the TXA2 receptor was determined by TBXA2R Arrestin Biosensor Assay. Mouse mesenteric arteries were mounted in wire myographs, and responses to increasing concentrations of C21 (1nM- 10muM) were recorded during submaximal contractions with 0.1muM U46619 (TXA2 analogue) or 1muMphenylephrine. To control for......AT2-receptor specificity, arteries were pre-incubated with the AT2-receptor antagonist PD123319 (10muM), or mesenteric arteries from AT2-receptor knock-out (AT2R-/y) mice were used. An inhibitory effect of C21 (100nM - 10muM) on U46619 (0,3muM) induced platelet aggregation was examined in whole human...

  7. Angiotensin receptor-neprilysin inhibitors: clinical potential in heart failure and beyond

    Directory of Open Access Journals (Sweden)

    Singh JSS

    2015-06-01

    Full Text Available Jagdeep SS Singh, Chim C Lang Division of Cardiovascular and Diabetes Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, UK Abstract: Heart failure remains a major concern across the globe as life expectancies and delivery of health care continue to improve. There has been a dearth of new developments in heart failure therapies in the last decade until last year, with the release of the results from the PARADIGM-HF Trial heralding the arrival of a promising new class of drug, ie, the angiotensin receptor-neprilysin inhibitor. In this review, we discuss the evolution of our incremental understanding of the neurohormonal mechanisms involved in the pathophysiology of heart failure, which has led to our success in modulating its various pathways. We start by examining the renin-angiotensin-aldosterone system, followed by the challenges of modulating the natriuretic peptide system. We then delve deeper into the pharmacology and mechanisms by which angiotensin receptor-neprilysin inhibitors achieve their significant cardiovascular benefits. Finally, we also consider the potential application of this new class of drug in other areas, such as heart failure with preserved ejection fraction, hypertension, patients with renal impairment, and following myocardial infarction. Keywords: heart failure, angiotensin receptor-neprilysin inhibitor, heart failure with preserved ejection fraction, nesiritide, candoxatril, omapatrilat, hypertension, renal impairment, myocardial infarction

  8. The mechanisms behind decreased internalization of angiotensin II type 1 receptor.

    Science.gov (United States)

    Bian, Jingwei; Zhang, Suli; Yi, Ming; Yue, Mingming; Liu, Huirong

    2018-04-01

    The internalization of angiotensin II type 1 receptor (AT 1 R) plays an important role in maintaining cardiovascular homeostasis. Decreased receptor internalization is closely related to cardiovascular diseases induced by the abnormal activation of AT 1 R, such as hypertension. However, the mechanism behind reduced AT 1 R internalization is not fully understood. This review focuses on four parts of the receptor internalization process (the combination of agonists and receptors, receptor phosphorylation, endocytosis, and recycling) and summarizes the possible mechanisms by which AT 1 R internalization is reduced based on these four parts of the process. (1) The agonist has a large molecular weight or a stronger ability to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5) P 2 ), which can increase the consumption of PtdIns (4,5) P 2 . (2) AT 1 R phosphorylation is weakened because of an abnormal function of phosphorylated kinase or changes in phospho-barcoding and GPCR-β-arrestin complex conformation. (3) The abnormal formation of vesicles or AT 1 R heterodimers with fewer endocytic receptors results in less AT 1 R endocytosis. (4) The enhanced activity and upregulated expression of small GTP-binding protein 4 (Rab4) and 11 (Rab11), which regulate receptor recycling, and phosphatidylinositol 3-kinase increase AT 1 R recycling. In addition, lower expression of AT 1 R-associated protein (ATRAP) or higher expression of AT 1 R-associated protein 1 (ARAP1) can reduce receptor internalization. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Evidence for Heterodimerization and Functional Interaction of the Angiotensin Type 2 Receptor and the Receptor MAS.

    Science.gov (United States)

    Leonhardt, Julia; Villela, Daniel C; Teichmann, Anke; Münter, Lisa-Marie; Mayer, Magnus C; Mardahl, Maibritt; Kirsch, Sebastian; Namsolleck, Pawel; Lucht, Kristin; Benz, Verena; Alenina, Natalia; Daniell, Nicholas; Horiuchi, Masatsugu; Iwai, Masaru; Multhaup, Gerhard; Schülein, Ralf; Bader, Michael; Santos, Robson A; Unger, Thomas; Steckelings, Ulrike Muscha

    2017-06-01

    The angiotensin type 2 receptor (AT2R) and the receptor MAS are receptors of the protective arm of the renin-angiotensin system. They mediate strikingly similar actions. Moreover, in various studies, AT2R antagonists blocked the effects of MAS agonists and vice versa. Such cross-inhibition may indicate heterodimerization of these receptors. Therefore, this study investigated the molecular and functional interplay between MAS and the AT2R. Molecular interactions were assessed by fluorescence resonance energy transfer and by cross correlation spectroscopy in human embryonic kidney-293 cells transfected with vectors encoding fluorophore-tagged MAS or AT2R. Functional interaction of AT2R and MAS was studied in astrocytes with CX3C chemokine receptor-1 messenger RNA expression as readout. Coexpression of fluorophore-tagged AT2R and MAS resulted in a fluorescence resonance energy transfer efficiency of 10.8 ± 0.8%, indicating that AT2R and MAS are capable to form heterodimers. Heterodimerization was verified by competition experiments using untagged AT2R and MAS. Specificity of dimerization of AT2R and MAS was supported by lack of dimerization with the transient receptor potential cation channel, subfamily C-member 6. Dimerization of the AT2R was abolished when it was mutated at cysteine residue 35. AT2R and MAS stimulation with the respective agonists, Compound 21 or angiotensin-(1-7), significantly induced CX3C chemokine receptor-1 messenger RNA expression. Effects of each agonist were blocked by an AT2R antagonist (PD123319) and also by a MAS antagonist (A-779). Knockout of a single of these receptors made astrocytes unresponsive for both agonists. Our results suggest that MAS and the AT2R form heterodimers and that-at least in astrocytes-both receptors functionally depend on each other. © 2017 American Heart Association, Inc.

  10. Association between angiotensin II receptor gene polymorphism and serum angiotensin converting enzyme (SACE) activity in patients with sarcoidosis

    OpenAIRE

    Takemoto, Y.; Sakatani, M.; Takami, S.; Tachibana, T.; Higaki, J.; Ogihara, T.; Miki, T.; Katsuya, T.; Tsuchiyama, T.; Yoshida, A.; Yu, H.; Tanio, Y.; Ueda, E.

    1998-01-01

    BACKGROUND—Serum angiotensin converting enzyme (SACE) is considered to reflect disease activity in sarcoidosis. SACE activity is increased in many patients with active sarcoid lesions. The mechanism for the increased SACE activity in this disease has not been clarified. ACE insertion/deletion (I/D) gene polymorphism has been reported to have an association with SACE levels in sarcoidosis, but no evidence of an association between angiotensin II receptor gene polymorphism and SA...

  11. Pharmacological characterization of BR-A-657, a highly potent nonpeptide angiotensin II receptor antagonist.

    Science.gov (United States)

    Chi, Yong Ha; Lee, Joo Han; Kim, Je Hak; Tan, Hyun Kwang; Kim, Sang Lin; Lee, Jae Yeol; Rim, Hong-Kun; Paik, Soo Heui; Lee, Kyung-Tae

    2013-01-01

    The pharmacological profile of BR-A-657, 2-n-butyl-5-dimethylamino-thiocarbonyl-methyl-6-methyl-3-{[2-(1H-tetrazole-5-yl)biphenyl-4-yl]methyl}-pyrimidin-4(3H)-one, a new nonpeptide AT1-selective angiotensin receptor antagonist, has been investigated in a variety of in vitro and in vivo experimental models. In the present study, BR-A-657 displaced [(125)I][Sar(1)-Ile(8)]angiotensin II (Ang II) from its specific binding sites to AT1 subtype receptors in membrane fractions of HEK-293 cells with an IC50 of 0.16 nM. In a functional assay using isolated rabbit thoracic aorta, BR-A-657 inhibited the contractile response to Ang II (pD'2: 9.15) with a significant reduction in the maximum. In conscious rats, BR-A-657 (0.01, 0.1, 1 mg/kg; intravenously (i.v.)) dose-dependently antagonized Ang II-induced pressor responses. In addition, BR-A-657 dose-dependently decreased mean arterial pressure in furosemide-treated rats and renal hypertensive rats. Moreover, BR-A-657 given orally at 1 and 3 mg/kg reduced blood pressure in conscious renal hypertensive rats. Taken together, these findings indicate that BR-A-657 is a potent and specific antagonist of Ang II at the AT1 receptor subtype, and reveal the molecular basis responsible for the marked lowering of blood pressure in conscious rats.

  12. Functional interactions between 7TM receptors in the renin-angiotensin system--dimerization or crosstalk?

    DEFF Research Database (Denmark)

    Lyngsø, Christina; Erikstrup, Niels; Hansen, Jakob L

    2008-01-01

    . The importance of the RAS is clearly emphasised by the widespread use of drugs targeting this system in clinical practice. These include, renin inhibitors, angiotensin II receptor type I blockers, and inhibitors of the angiotensin converting enzyme. Some of the important effectors within the system are 7...... be important for receptor function, and in the development of cardiovascular diseases. This is very significant, since "dimers" may provide pharmacologists with novel targets for improved drug therapy. However, we know that 7TM receptors can mediate signals as monomeric units, and so far it has been very......The Renin-Angiotensin System (RAS) is important for the regulation of cardiovascular physiology, where it controls blood pressure, and salt- and water homeostasis. Dysregulation of RAS can lead to severe diseases including hypertension, diabetic nephropathy, and cardiac arrhythmia, and -failure...

  13. Direct stimulation of angiotensin II type 2 receptor enhances spatial memory

    DEFF Research Database (Denmark)

    Jing, Fei; Mogi, Masaki; Sakata, Akiko

    2012-01-01

    We examined the possibility that direct stimulation of the angiotensin II type 2 (AT(2)) receptor by a newly generated direct AT(2) receptor agonist, Compound 21 (C21), enhances cognitive function. Treatment with C21 intraperitoneal injection for 2 weeks significantly enhanced cognitive function...

  14. Distortion of maternal-fetal angiotensin II type 1 receptor allele transmission in pre-eclampsia.

    Science.gov (United States)

    Morgan, L; Crawshaw, S; Baker, P N; Brookfield, J F; Broughton Pipkin, F; Kalsheker, N

    1998-01-01

    OBJECTIVE: To investigate the fetal angiotensin II type 1 receptor genotype in pre-eclampsia. DESIGN: Case-control study. POPULATION: Forty-one maternal-fetal pairs from pre-eclamptic pregnancies and 80 maternal-fetal pairs from normotensive pregnancies. METHODS: Maternal and fetal DNA was genotyped at three diallelic polymorphisms, at nucleotides 573, 1062, and 1166, in the coding exon of the angiotensin II type 1 receptor gene, and at a dinucleotide repeat polymorphism in its 3' flanking region. RESULTS: Allele and genotype frequencies at the four polymorphic regions investigated did not differ between pre-eclamptic and normotensive groups, in either fetal or maternal samples. Mothers heterozygous for the dinucleotide repeat allele designated A4 transmitted this allele to the fetus in 15 of 18 informative pre-eclamptic pregnancies and in eight of 26 normotensive pregnancies. This was greater than the expected probability in pre-eclamptic pregnancies (p=0.04) and less than expected in normotensive pregnancies (p<0.005). The 573T variant, which is in partial linkage disequilibrium with the A4 allele, showed a similar distortion of maternal-fetal transmission. CONCLUSION: Angiotensin II type 1 receptor gene expression in the fetus may contribute to the aetiology of pre-eclampsia. It is unclear whether susceptibility is conferred by the fetal genotype acting alone, or by allele sharing by mother and fetus. Possible mechanisms for the effect of the angiotensin II type 1 receptor gene are suggested by the association of the 573T variant with low levels of surface receptor expression on platelets. If receptor expression is similarly genetically determined in the placenta, responsiveness to angiotensin II may be affected, with the potential to influence placentation or placental prostaglandin secretion. PMID:9719367

  15. Heart Failure Therapeutics on the Basis of a Biased Ligand of the Angiotensin-2 Type 1 Receptor Rationale and Design of the BLAST-AHF Study (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure)

    NARCIS (Netherlands)

    Felker, G. Michael; Butler, Javed; Collins, Sean P.; Cotter, Gad; Davison, Beth A.; Ezekowitz, Justin A.; Filippatos, Gerasimos; Levy, Phillip D.; Metra, Marco; Ponikowski, Piotr; Soergel, David G.; Teerlink, John R.; Violin, Jonathan D.; Voors, Adriaan A.; Pang, Peter S.

    The BLAST-AHF (Biased Ligand of the Angiotensin Receptor Study in Acute Heart Failure) study is designed to test the efficacy and safety of TRV027, a novel biased ligand of the angiotensin-2 type 1 receptor, in patients with acute heart failure (AHF). AHF remains a major public health problem, and

  16. Activation of Central PPAR-γ Attenuates Angiotensin II-Induced Hypertension

    Science.gov (United States)

    Yu, Yang; Xue, Bao-Jian; Wei, Shun-Guang; Zhang, Zhi-Hua; Beltz, Terry G; Guo, Fang; Johnson, Alan Kim; Felder, Robert B

    2015-01-01

    Inflammation and renin-angiotensin system activity in the brain contribute to hypertension through effects on fluid intake, vasopressin release, and sympathetic nerve activity. We recently reported that activation of brain peroxisome proliferator-activated receptor (PPAR)-γ in heart failure rats reduced inflammation and renin-angiotensin system activity in the hypothalamic paraventricular nucleus and ameliorated the peripheral manifestations of heart failure. We hypothesized that activation of brain PPAR-γ might have beneficial effects in angiotensin II-induced hypertension. Sprague-Dawley rats received a 2-week subcutaneous infusion of angiotensin II (120 ng/kg/min) combined with a continuous intracerebroventricular infusion of vehicle, the PPAR-γ agonist pioglitazone (3 nmol/h) or the PPAR-γ antagonist GW9662 (7 nmol/h). Angiotensin II+vehicle rats had increased mean blood pressure, increased sympathetic drive as indicated by the mean blood pressure response to ganglionic blockade, and increased water consumption. PPAR-γ mRNA in subfornical organ and hypothalamic paraventricular nucleus was unchanged, but PPAR-γ DNA binding activity was reduced. mRNA for interleukin-1β, tumor necrosis factor-α, cyclooxygenase-2 and angiotensin II type-1 receptor was augmented in both nuclei, and hypothalamic paraventricular nucleus neuronal activity was increased. The plasma vasopressin response to a 6-hour water restriction also increased. These responses to angiotensin II were exacerbated by GW9662 and ameliorated by pioglitazone, which increased PPAR-γ mRNA and PPAR-γ DNA binding activity in subfornical organ and hypothalamic paraventricular nucleus. Pioglitazone and GW9662 had no effects on control rats. The results suggest that activating brain PPAR-γ to reduce central inflammation and brain renin-angiotensin system activity may be a useful adjunct in the treatment of angiotensin II-dependent hypertension. PMID:26101342

  17. The Angiotensin AT2 Receptor

    DEFF Research Database (Denmark)

    Unger, Thomas; Steckelings, Ulrike M.; Dzau, Victor J.

    2015-01-01

    Since its discovery, 25 years ago, the angiotensin AT2 receptor (AT2R) has puzzled the scientific community because of its distinct -localization, regulation, signaling pathways, and biological effects separating it clearly from the classical features of the renin...... programs that can counterbalance pathological processes and enable recovery from disease. The AT2R has thus mutated from an "-enigmatic" receptor to a significant member of the "protective arm" of the RAS. The recent development of novel, small molecule- and peptide-derived AT2

  18. Analysis of angiotensin II binding to human platelets: Differences in young and old subjects

    International Nuclear Information System (INIS)

    Siebers, M.J.; Goodfriend, T.L.; Ball, D.; Elliott, M.E.

    1990-01-01

    We examined the binding of radiolabeled angiotensin II (AII) to human platelets to characterize the apparent increase in AII receptors observed in older subjects. At 22 degrees C, the amount of radioactivity associated with platelets from older subjects increased continuously for more than 2 hours. The same amount of radioactivity was displaced by addition of unlabeled AII at 30 min and 60 min. In the presence of phenylarsine oxide, in the cold, or when labeled antagonist was the ligand, binding came to equilibrium by 30 min. High pressure liquid chromatography demonstrated that 125 I-AII was the major radioactive compound in the supernatant and platelets after incubation, but the platelets also contained radiolabeled AII fragments. Thus, some degradation accompanied interaction of AII and platelets. Phenylarsine oxide did not prevent degradation of bound AII, suggesting that degradation precedes internalization. On average, maximum binding was greater in older subjects whether platelets were incubated with 125 I-AII alone, with 125 I-AII and phenylarsine oxide to prevent internalization, or when the competitive inhibitor 125 I-sar1,ile8-AII was the radioligand. Variability of binding among subjects also increased with age. Thus, platelets bind, degrade, and internalize AII, and the three processes occur to a greater extent in platelets from some, but not all older subjects

  19. Effects of the angiotensin-receptor blocker telmisartan on cardiovascular events in high-risk patients intolerant to angiotensin-converting enzyme inhibitors: a randomised controlled trial

    DEFF Research Database (Denmark)

    NN, NN; Yusuf, S; Teo, K

    2008-01-01

    BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors reduce major cardiovascular events, but are not tolerated by about 20% of patients. We therefore assessed whether the angiotensin-receptor blocker telmisartan would be effective in patients intolerant to ACE inhibitors with cardiovascular...

  20. No effect of angiotensin II AT(2)-receptor antagonist PD 123319 on cerebral blood flow autoregulation

    DEFF Research Database (Denmark)

    Estrup, T M; Paulson, O B; Strandgaard, S

    2001-01-01

    Blockade of the renin-angiotensin system with angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin AT1-receptor antagonists shift the limits of autoregulation of cerebral blood flow (CBF) towards lower blood pressure (BP). The role of AT2-receptors in the regulation of the cerebral...... group. CBF was measured by the intracarotid 133xenon injection method and BP was raised by noradrenaline infusion and lowered by controlled haemorrhage in separate groups of rats. The limits of autoregulation were determined by computed least-sum-of-squares analysis. PD 123319 did not influence baseline...

  1. Angiotensin Converting Enzyme Regulates Cell Proliferation and Migration.

    Directory of Open Access Journals (Sweden)

    Erika Costa de Alvarenga

    Full Text Available The angiotensin-I converting enzyme (ACE plays a central role in the renin-angiotensin system, acting by converting the hormone angiotensin-I to the active peptide angiotensin-II (Ang-II. More recently, ACE was shown to act as a receptor for Ang-II, and its expression level was demonstrated to be higher in melanoma cells compared to their normal counterparts. However, the function that ACE plays as an Ang-II receptor in melanoma cells has not been defined yet.Therefore, our aim was to examine the role of ACE in tumor cell proliferation and migration.We found that upon binding to ACE, Ang-II internalizes with a faster onset compared to the binding of Ang-II to its classical AT1 receptor. We also found that the complex Ang-II/ACE translocates to the nucleus, through a clathrin-mediated process, triggering a transient nuclear Ca2+ signal. In silico studies revealed a possible interaction site between ACE and phospholipase C (PLC, and experimental results in CHO cells, demonstrated that the β3 isoform of PLC is the one involved in the Ca2+ signals induced by Ang-II/ACE interaction. Further studies in melanoma cells (TM-5 showed that Ang-II induced cell proliferation through ACE activation, an event that could be inhibited either by ACE inhibitor (Lisinopril or by the silencing of ACE. In addition, we found that stimulation of ACE by Ang-II caused the melanoma cells to migrate, at least in part due to decreased vinculin expression, a focal adhesion structural protein.ACE activation regulates melanoma cell proliferation and migration.

  2. Focus on Brain Angiotensin III and Aminopeptidase A in the Control of Hypertension

    Directory of Open Access Journals (Sweden)

    John W. Wright

    2012-01-01

    Full Text Available The classic renin-angiotensin system (RAS was initially described as a hormone system designed to mediate cardiovascular and body water regulation. The discovery of a brain RAS composed of the necessary functional components (angiotensinogen, peptidases, angiotensins, and specific receptor proteins independent of the peripheral system significantly expanded the possible physiological and pharmacological functions of this system. This paper first describes the enzymatic pathways resulting in active angiotensin ligands and their interaction with AT1, AT2, and mas receptor subtypes. Recent evidence points to important contributions by brain angiotensin III (AngIII and aminopeptidases A (APA and N (APN in sustaining hypertension. Next, we discuss current approaches to the treatment of hypertension followed by novel strategies that focus on limiting the binding of AngII and AngIII to the AT1 receptor subtype by influencing the activity of APA and APN. We conclude with thoughts concerning future treatment approaches to controlling hypertension and hypotension.

  3. Severe hepatic encephalopathy in a patient with liver cirrhosis after administration of angiotensin-converting enzyme inhibitor/angiotensin II receptor blocker combination therapy: a case report

    Directory of Open Access Journals (Sweden)

    Podda Mauro

    2010-05-01

    Full Text Available Abstract Introduction A combination therapy of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers has been used to control proteinuria, following initial demonstration of its efficacy. However, recently concerns about the safety of this therapy have emerged, prompting several authors to urge for caution in its use. In the following case report, we describe the occurrence of a serious and unexpected adverse drug reaction after administration of a combination of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers to a patient with nephrotic syndrome and liver cirrhosis with severe portal hypertension. Case presentation We administered this combination therapy to a 40-year-old Caucasian man with liver cirrhosis in our Hepatology Clinic, given the concomitant presence of glomerulopathy associated with severe proteinuria. While the administration of one single drug appeared to be well-tolerated, our patient developed severe acute encephalopathy after the addition of the second one. Discontinuation of the therapy led to the disappearance of the side-effect. A tentative rechallenge with the same drug combination led to a second episode of acute severe encephalopathy. Conclusion We speculate that this adverse reaction may be directly related to the effect of angiotensin II on the excretion of blood ammonia. Therefore, we suggest that patients with liver cirrhosis and portal hypertension are at risk of developing clinically relevant encephalopathy when angiotensin-converting enzyme inhibitor and angiotensin II receptor blocker combination therapy is administered, thus indicating the need for a careful clinical follow-up. In addition, the incidence of this serious side-effect should be rigorously evaluated in all patients with liver cirrhosis administered with this common treatment combination.

  4. Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy

    NARCIS (Netherlands)

    Hoffmann, S.; Krause, T.; van Geel, P. P.; Willenbrock, R.; Pagel, I.; Pinto, Y. M.; Buikema, H.; van Gilst, W. H.; Lindschau, C.; Paul, M.; Inagami, T.; Ganten, D.; Urata, H.

    2001-01-01

    Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT1 receptors. However, the role of myocardial AT1 receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that

  5. Overexpression of the human angiotensin II type 1 receptor in the rat heart augments load induced cardiac hypertrophy

    NARCIS (Netherlands)

    Hoffmann, S; van Geel, PP; Willenbrock, R; Pagel, [No Value; Pinto, YM; Buikema, H; van Gilst, WH; Lindschau, C; Paul, M; Inagami, T; Ganten, D; Urata, H

    2001-01-01

    Angiotensin II is known to stimulate cardiac hypertrophy and contractility. Most angiotensin II effects are mediated via membrane bound AT(1) receptors. However, the role of myocardial AT(1) receptors in cardiac hypertrophy and contractility is still rarely defined. To address the hypothesis that

  6. Macrophage-to-sensory neuron crosstalk mediated by Angiotensin II type-2 receptor elicits neuropathic pain

    OpenAIRE

    Krause, Eric; Shepherd, Andrew; Mickle, Aaron; Copits, Bryan; Karlsson, Pall; Kadunganattil, Suraj; Golden, Judith; Tadinada, Satya; Mack, Madison; Haroutounian, Simon; De Kloet, Annette; Samineni, Vijay; Valtcheva, Manouela; Mcilvried, Lisa; Sheahan, Tayler

    2017-01-01

    Peripheral nerve damage initiates a complex series of cellular and structural processes that culminate in chronic neuropathic pain. Our study defines local angiotensin signaling via activation of the Angiotensin II (Ang II) type-2 receptor (AT2R) on macrophages as the critical trigger of neuropathic pain. An AT2R-selective antagonist attenuates neuropathic, but not inflammatory pain hypersensitivity in mice, and requires the cell damage-sensing ion channel transient receptor potential family-...

  7. The angiotensin II type 2 receptor agonist Compound 21 is protective in experimental diabetes-associated atherosclerosis

    DEFF Research Database (Denmark)

    Chow, Bryna S M; Koulis, Christine; Krishnaswamy, Pooja

    2016-01-01

    AIMS/HYPOTHESIS: Angiotensin II is well-recognised to be a key mediator in driving the pathological events of diabetes-associated atherosclerosis via signalling through its angiotensin II type 1 receptor (AT1R) subtype. However, its actions via the angiotensin II type 2 receptor (AT2R) subtype...... are still poorly understood. This study is the first to investigate the role of the novel selective AT2R agonist, Compound 21 (C21) in an experimental model of diabetes-associated atherosclerosis (DAA). METHODS: Streptozotocin-induced diabetic Apoe-knockout mice were treated with vehicle (0.1 mol/l citrate...

  8. ANGIOTENSIN RECEPTOR BLOCKERS WITH PLEIOTROPIC PROPERTIES: A NEW STANDARD IN CARDIOVASCULAR RISK MANAGEMENT AND TREATMENT OF HYPERTENSION

    Directory of Open Access Journals (Sweden)

    V. I. Podzolkov

    2017-01-01

    Full Text Available An increase in the activity of the renin-angiotensin-aldosterone system is one of the most important mechanisms for the realization of the cardiovascular continuum. The role that angiotensin receptor blockers play in achieving target figures of blood pressure and reducing cardiovascular risk is discussed. The importance of pleiotropic properties of angiotensin receptor blockers (in particular, activation of peroxisome proliferator-activated receptors gamma – PPAR-γ in the management of patients with insulin resistance, obesity, dyslipidemia is also covered. The evidence base for the use of telmisartan as a drug with pleiotropic effect in patients with arterial hypertension and associated diseases (diabetes mellitus, obesity, renal dysfunction is discussed. 

  9. Combined Angiotensin Receptor Modulation in the Management of Cardio-Metabolic Disorders

    DEFF Research Database (Denmark)

    Paulis, Ludovit; Foulquier, Sébastien; Namsolleck, Pawel

    2016-01-01

    Cardiovascular and metabolic disorders, such as hypertension, insulin resistance, dyslipidemia or obesity are linked with chronic low-grade inflammation and dysregulation of the renin-angiotensin system (RAS). Consequently, RAS inhibition by ACE inhibitors or angiotensin AT1 receptor (AT1R...... blockade abolishes the AT1R-linked RAS almost completely with subsequent risk of hypotension and hypotension-related events, i.e. syncope or renal dysfunction. Such complications might be especially prominent in patients with renal impairment or patients with isolated systolic hypertension and normal...

  10. Use of angiotensin II receptor blockers in children- a review of ...

    African Journals Online (AJOL)

    2015-05-19

    May 19, 2015 ... sex and height, whereas hypertension is defined as SBP and/or DBP persistently ... strated the efficacy of angiotensin receptor blockers in ... An open-label, multicenter, single-dose study was ..... sure among school children of.

  11. Angiotensin type 1 receptors in the subfornical organ mediate the drinking and hypothalamic-pituitary-adrenal response to systemic isoproterenol.

    Science.gov (United States)

    Krause, Eric G; Melhorn, Susan J; Davis, Jon F; Scott, Karen A; Ma, Li Y; de Kloet, Annette D; Benoit, Stephen C; Woods, Stephen C; Sakai, Randall R

    2008-12-01

    Circulating angiotensin II (ANGII) elicits water intake and activates the hypothalamic-pituitary-adrenal (HPA) axis by stimulating angiotensin type 1 receptors (AT1Rs) within circumventricular organs. The subfornical organ (SFO) and the organum vasculosum of the lamina terminalis (OVLT) are circumventricular organs that express AT1Rs that bind blood-borne ANGII and stimulate integrative and effector regions of the brain. The goal of these studies was to determine the contribution of AT1Rs within the SFO and OVLT to the water intake and HPA response to increased circulating ANGII. Antisense oligonucleotides directed against the AT1R [AT1R antisense (AT1R AS)] were administered into the OVLT or SFO. Quantitative receptor autoradiography confirmed that AT1R AS decreased ANGII binding in the SFO and OVLT compared with the scrambled sequence control but did not affect AT1R binding in other nuclei. Subsequently, water intake, ACTH, and corticosterone (CORT) were assessed after administration of isoproterenol, a beta-adrenergic agonist that decreases blood pressure and elevates circulating ANGII. Delivery of AT1R AS into the SFO attenuated water intake, ACTH, and CORT after isoproterenol, whereas similar treatment in the OVLT had no effect. To determine the specificity of this blunted drinking and HPA response, the same parameters were measured after treatment with hypertonic saline, a stimulus that induces drinking independently of ANGII. Delivery of AT1R AS into the SFO or OVLT had no effect on water intake, ACTH, or CORT after hypertonic saline. The results imply that AT1R within the SFO mediate drinking and HPA responses to stimuli that increase circulating ANGII.

  12. Reappraisal of role of angiotensin receptor blockers in cardiovascular protection

    Directory of Open Access Journals (Sweden)

    Ram CV

    2011-05-01

    Full Text Available C Venkata S RamTexas Blood Pressure Institute, Clinical Research Institute of Dallas Nephrology Associates; and Department of Internal Medicine, University of Texas Southwestern Medical Center at Dallas, Dallas, TX, USAAbstract: Angiotensin-converting enzyme inhibitors (ACEIs and angiotensin receptor blockers (ARBs have shown cardioprotective and renoprotective properties. These agents are recommended as first-line therapy for the treatment of hypertension and the reduction of cardiovascular risk. Early studies pointed to the cardioprotective and renoprotective effects of ARBs in high-risk patients. The ONgoing Telmisartan Alone and in combination with Ramipril Global Endpoint Trial (ONTARGET established the clinical equivalence of the cardioprotective and renoprotective effects of telmisartan and ramipril, but did not find an added benefit of the combination over ramipril alone. Similar findings were observed in the Telmisartan Randomized AssessmeNt Study in aCE INtolerant subjects with cardiovascular Disease (TRANSCEND trial conducted in ACEI-intolerant patients. In ONTARGET, telmisartan had a better tolerability profile with similar renoprotective properties compared with ramipril, suggesting a potential clinical benefit over ramipril. The recently completed Olmesartan Reducing Incidence of Endstage Renal Disease in Diabetic Nephropathy Trial (ORIENT and Olmesartan and Calcium Antagonists Randomized (OSCAR studies will further define the role of ARBs in cardioprotection and renoprotection for high-risk patients.Keywords: angiotensin receptor blockers, hypertension, outcomes, clinical trials

  13. C4d-negative antibody-mediated rejection with high anti-angiotensin II type I receptor antibodies in absence of donor-specific antibodies.

    Science.gov (United States)

    Fuss, Alexander; Hope, Christopher M; Deayton, Susan; Bennett, Greg Donald; Holdsworth, Rhonda; Carroll, Robert P; Coates, P Toby H

    2015-07-01

    Acute antibody-mediated rejection can occur in absence of circulating donor-specific antibodies. Agonistic antibodies targeting the anti-angiotensin II type 1 receptor (anti-AT1 R) are emerging as important non-human leucocyte antigen (HLA) antibodies. Elevated levels of anti-angiotensin II receptor antibodies were first observed in kidney transplant recipients with malignant hypertension and allograft rejection. They have now been studied in three separate kidney transplant populations and associate to frequency of rejection, severity of rejection and graft failure. We report 11 cases of biopsy-proven, Complement 4 fragment d (C4d)-negative, acute rejection occurring without circulating donor-specific anti-HLA antibodies. In eight cases, anti-angiotensin receptor antibodies were retrospectively examined. The remaining three subjects were identified from our centre's newly instituted routine anti-angiotensin receptor antibody screening. All subjects fulfilled Banff 2013 criteria for antibody-mediated rejection and all responded to anti-rejection therapy, which included plasma exchange and angiotensin receptor blocker therapy. These cases support the routine assessment of anti-AT1 R antibodies in kidney transplant recipients to identify subjects at risk. Further studies will need to determine optimal assessment protocol and the effectiveness of pre-emptive treatment with angiotensin receptor blockers. © 2015 Asian Pacific Society of Nephrology.

  14. Addition of vitamin D reverses the decline in GFR following treatment with ACE inhibitors/angiotensin receptor blockers in patients with chronic kidney disease.

    Science.gov (United States)

    Soares, Abel Esteves; Maes, Michael; Godeny, Paula; Matsumoto, Andressa Keiko; Barbosa, Décio Sabbatini; da Silva, Taysa Antonia F; Souza, Flávio Henrique M O; Delfino, Vinicius Daher Alvares

    2017-12-15

    Vitamin D has anti-inflammatory, anti-fibrotic effect, and may block the intrarenal renin-angiotensin system. Adequate vitamin D levels in conjunction with the use of Angiotensin-converting Enzyme Inhibitors/Angiotensin Receptor Blockers may help to slow down chronic kidney disease progression. To study a possible beneficial effect of vitamin D supplementation in chronic kidney disease patients using angiotensin-converting enzyme inhibitors/angiotensin receptor blockers on chronic kidney disease progression we performed a clinical study involving vitamin D supplementation in patients with deficiency of this vitamin. This study was conducted in two chronic kidney disease clinics in the city of Londrina, Brazil, from October 2010 to December 2012. It was involved stage 3 and 4 chronic kidney disease (estimated glomerular filtration rate between 60 and 15mL/min/1.73m 2 ) patients with and without vitamin D deficiency. The patients ingested six-month cholecalciferol 50,000IU oral supplementation to chronic kidney disease patients with vitamin D deficiency. We hypothesize changes in estimated glomerular filtration rate over study period. Our data demonstrate reservation of estimated glomerular filtration with cholecalciferol supplementation to chronic kidney disease patients taking angiotensin-converting enzyme inhibitors/angiotensin receptor blockers. The combination treatment of angiotensin converting enzyme inhibitors/angiotensin receptor blockers with cholecalciferol prevents the decline in estimated glomerular filtration in patients with chronic kidney disease following treatment with angiotensin-converting enzyme inhibitors/angiotensin receptor blockers and may represent a valid approach to reduce renal disease progression in chronic kidney disease patients with vitamin D deficiency. This result needs confirmation in prospective controlled clinical trials. Copyright © 2017. Published by Elsevier Inc.

  15. Angiotensin II type 1a receptor-deficient mice develop angiotensin II-induced oxidative stress and DNA damage without blood pressure increase.

    Science.gov (United States)

    Zimnol, Anna; Amann, Kerstin; Mandel, Philipp; Hartmann, Christina; Schupp, Nicole

    2017-12-01

    Hypertensive patients have an increased risk of developing kidney cancer. We have shown in vivo that besides elevating blood pressure, angiotensin II causes DNA damage dose dependently. Here, the role of blood pressure in the formation of DNA damage is studied. Mice lacking one of the two murine angiotensin II type 1 receptor (AT1R) subtypes, AT1aR, were equipped with osmotic minipumps, delivering angiotensin II during 28 days. Parameters of oxidative stress and DNA damage of kidneys and hearts of AT1aR-knockout mice were compared with wild-type (C57BL/6) mice receiving angiotensin II, and additionally, with wild-type mice treated with candesartan, an antagonist of both AT1R subtypes. In wild-type mice, angiotensin II induced hypertension, reduced kidney function, and led to a significant formation of reactive oxygen species (ROS). Furthermore, genomic damage was markedly increased in this group. All these responses to angiotensin II could be attenuated by concurrent administration of candesartan. In AT1aR-deficient mice treated with angiotensin II, systolic pressure was not increased, and renal function was not affected. However, angiotensin II still led to an increase of ROS in kidneys and hearts of these animals. Additionally, genomic damage in the form of double-strand breaks was significantly induced in kidneys of AT1aR-deficient mice. Our results show that angiotensin II induced ROS production and DNA damage even without the presence of AT1aR and independently of blood pressure changes. Copyright © 2017 the American Physiological Society.

  16. The evolution of renin-angiotensin blockade: angiotensin-converting enzyme inhibitors as the starting point.

    Science.gov (United States)

    Sica, Domenic A

    2010-04-01

    The renin-angiotensin system has been a target in the treatment of hypertension for close to three decades. Several medication classes that block specific aspects of this system have emerged as useful therapies, including angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and, most recently, direct renin inhibitors. There has been a natural history to the development of each of these three drug classes, starting with their use as antihypertensive agents; thereafter, in each case they have been employed as end-organ protective agents. To date, there has been scant evidence to favor angiotensin receptor blockers or direct renin inhibitors over angiotensin-converting enzyme inhibitors in treating hypertension or in affording end-organ protection; thus, angiotensin-converting enzyme inhibitors remain the standard of care when renin-angiotensin system blockade is warranted.

  17. Clathrin-dependent internalization of the angiotensin II AT₁A receptor links receptor internalization to COX-2 protein expression in rat aortic vascular smooth muscle cells.

    Science.gov (United States)

    Morinelli, Thomas A; Walker, Linda P; Velez, Juan Carlos Q; Ullian, Michael E

    2015-02-05

    The major effects of Angiotensin II (AngII) in vascular tissue are mediated by AngII AT1A receptor activation. Certain effects initiated by AT1A receptor activation require receptor internalization. In rat aortic vascular smooth muscle cells (RASMC), AngII stimulates cyclooxygenase 2 protein expression. We have previously shown this is mediated by β-arrestin-dependent receptor internalization and NF-κB activation. In this study, a specific inhibitor of clathrin-mediated endocytosis (CME), pitstop-2, was used to test the hypothesis that clathrin-dependent internalization of activated AT1A receptor mediates NF-κB activation and subsequent cyclooxygenase 2 expression. Radioligand binding assays, real time qt-PCR and immunoblotting were used to document the effects of pitstop-2 on AngII binding and signaling in RASMC. Laser scanning confocal microscopy (LSCM) was used to image pitstop-2׳s effects on AT1 receptor/GFP internalization in HEK-293 cells and p65 NF-κB nuclear localization in RASMC. Pitstop-2 significantly inhibited internalization of AT1A receptor (44.7% ± 3.1% Control vs. 13.2% ± 8.3% Pitstop-2; n=3) as determined by radioligand binding studies in RASMC. Studies utilizing AT1A receptor/GFP expressed in HEK 293 cells and LSCM confirmed these findings. Pitstop-2 significantly inhibited AngII-induced p65 NF-κB phosphorylation and nuclear localization, COX-2 message and protein expression in RASMC without altering activation of p42/44 ERK or TNFα signaling. Pitstop-2, a specific inhibitor of clathrin-mediated endocytosis, confirms that internalization of activated AT1A receptor mediates AngII activation of cyclooxygenase 2 expression in RASMC. These data provide support for additional intracellular signaling pathways activated through β-arrestin mediated internalization of G protein-coupled receptors, such as AT1A receptors. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. Angiotensin receptors and norepinephrine neuromodulation: implications of functional coupling.

    Science.gov (United States)

    Gelband, C H; Sumners, C; Lu, D; Raizada, M K

    1998-02-27

    The objective of this review is to examine the role of neuronal angiotensin II (Ang II) receptors in vitro. Two types of G protein-coupled Ang II receptors have been identified in cardiovascularly relevant areas of the brain: the AT1 and the AT2. We have utilized neurons in culture to study the signaling mechanisms of AT1 and AT2 receptors. Neuronal AT1 receptors are involved in norepinephrine (NE) neuromodulation. NE neuromodulation can be either evoked or enhanced. Evoked NE neuromodulation involves AT1 receptor-mediated, losartan-dependent, rapid NE release, inhibition of K+ channels and stimulation of Ca2+ channels. AT1 receptor-mediated enhanced NE neuromodulation involves the Ras-Raf-MAP kinase cascade and ultimately leads to an increase in NE transporter, tyrosine hydroxylase and dopamine beta-hydroxylase mRNA transcription. Neuronal AT2 receptors signal via a Gi protein and are coupled to activation of PP2A and PLA2 and stimulation of K+ channels. Finally, putative cross-talk pathways between AT1 and AT2 receptors will be discussed.

  19. Angiotensin II type 1 and 2 receptors and lymphatic vessels modulate lung remodeling and fibrosis in systemic sclerosis and idiopathic pulmonary fibrosis.

    Science.gov (United States)

    Parra, Edwin Roger; Ruppert, Aline Domingos Pinto; Capelozzi, Vera Luiza

    2014-01-01

    To validate the importance of the angiotensin II receptor isotypes and the lymphatic vessels in systemic sclerosis and idiopathic pulmonary fibrosis. We examined angiotensin II type 1 and 2 receptors and lymphatic vessels in the pulmonary tissues obtained from open lung biopsies of 30 patients with systemic sclerosis and 28 patients with idiopathic pulmonary fibrosis. Their histologic patterns included cellular and fibrotic non-specific interstitial pneumonia for systemic sclerosis and usual interstitial pneumonia for idiopathic pulmonary fibrosis. We used immunohistochemistry and histomorphometry to evaluate the number of cells in the alveolar septae and the vessels stained by these markers. Survival curves were also used. We found a significantly increased percentage of septal and vessel cells immunostained for the angiotensin type 1 and 2 receptors in the systemic sclerosis and idiopathic pulmonary fibrosis patients compared with the controls. A similar percentage of angiotensin 2 receptor positive vessel cells was observed in fibrotic non-specific interstitial pneumonia and usual interstitial pneumonia. A significantly increased percentage of lymphatic vessels was present in the usual interstitial pneumonia group compared with the non-specific interstitial pneumonia and control groups. A Cox regression analysis showed a high risk of death for the patients with usual interstitial pneumonia and a high percentage of vessel cells immunostained for the angiotensin 2 receptor in the lymphatic vessels. We concluded that angiotensin II receptor expression in the lung parenchyma can potentially control organ remodeling and fibrosis, which suggests that strategies aimed at preventing high angiotensin 2 receptor expression may be used as potential therapeutic target in patients with pulmonary systemic sclerosis and idiopathic pulmonary fibrosis.

  20. Angiotensin receptor blockade improves cardiac mitochondrial activity in response to an acute glucose load in obese insulin resistant rats

    Directory of Open Access Journals (Sweden)

    Max Thorwald

    2018-04-01

    Full Text Available Hyperglycemia increases the risk of oxidant overproduction in the heart through activation of a multitude of pathways. Oxidation of mitochondrial enzymes may impair their function resulting in accumulation of intermediates and reverse electron transfer, contributing to mitochondrial dysfunction. Furthermore, the renin-angiotensin system (RAS becomes inappropriately activated during metabolic syndrome, increasing oxidant production. To combat excess oxidant production, the transcription factor, nuclear factor erythriod-2- related factor 2 (Nrf2, induces expression of many antioxidant genes. We hypothesized that angiotensin II receptor type 1 (AT1 blockade improves mitochondrial function in response to an acute glucose load via upregulation of Nrf2. To address this hypothesis, an oral glucose challenge was performed in three groups prior to dissection (n = 5–8 animals/group/time point of adult male rats: 1 Long Evans Tokushima Otsuka (LETO; lean strain-control, 2 insulin resistant, obese Otsuka Long Evans Tokushima Fatty (OLETF, and 3 OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg/d × 6 weeks. Hearts were collected at T0, T60, and T120 minutes post-glucose infusion. ARB increased Nrf2 binding 32% compared to OLETF at T60. Total superoxide dismutase (SOD and catalase (CAT activities were increased 45% and 66% respectively in ARB treated animals compared to OLETF. Mitochondrial enzyme activities of aconitase, complex I, and complex II increased by 135%, 33% and 66%, respectively in ARB compared to OLETF. These data demonstrate the protective effects of AT1 blockade on mitochondrial function during the manifestation of insulin resistance suggesting that the inappropriate activation of AT1 during insulin resistance may impair Nrf2 translocation and subsequent antioxidant activities and mitochondrial function. Keywords: Angiotensin II, Mitochondria, Cardiac, Antioxidant enzymes, TCA cycle

  1. Potentiation of the actions of bradykinin by angiotensin I-converting enzyme inhibitors. The role of expressed human bradykinin B2 receptors and angiotensin I-converting enzyme in CHO cells.

    Science.gov (United States)

    Minshall, R D; Tan, F; Nakamura, F; Rabito, S F; Becker, R P; Marcic, B; Erdös, E G

    1997-11-01

    Part of the beneficial effects of angiotensin I-converting enzyme (ACE) inhibitors are due to augmenting the actions of bradykinin (BK). We studied this effect of enalaprilat on the binding of [3H]BK to Chinese hamster ovary (CHO) cells stably transfected to express the human BK B2 receptor alone (CHO-3B) or in combination with ACE (CHO-15AB). In CHO-15AB cells, enalaprilat (1 mumol/L) increased the total number of low-affinity [3H]BK binding sites on the cells at 37 degrees C, but not at 4 degrees C, from 18.4 +/- 4.3 to 40.3 +/- 11.9 fmol/10(6) cells (P potentiated the release of [3H]arachidonic acid and the liberation of inositol 1,4,5-trisphosphate (IP3) induced by BK and [Hyp3-Tyr(Me)8]BK. Moreover, enalaprilat (1 mumol/L) completely and immediately restored the response of the B2 receptor, desensitized by the agonist (1 mumol/L [Hyp3-Tyr(Me)8]BK); this effect was blocked by the antagonist, HOE 140. Finally, enalaprilat, but not the prodrug enalapril, decreased internalization of the receptor from 70 +/- 9% to 45 +/- 9% (P desensitization, and decrease internalization, thereby potentiating BK beyond blocking its hydrolysis.

  2. Erratum Aldosterone synthase C-344T, angiotensin II type 1 receptor ...

    Indian Academy of Sciences (India)

    Aldosterone synthase C-344T, angiotensin II type 1 receptor A1166C and 11-β hydroxysteroid dehydrogenase G534A gene polymorphisms and essential hypertension in the population of Odisha, India. Manisha Patnaik, Pallabi Pati, Surendra N. Swain, Manoj K. Mohapatra, Bhagirathi Dwibedi, Shantanu K. Kar.

  3. Centrally Mediated Cardiovascular Actions of the Angiotensin II Type 2 Receptor

    DEFF Research Database (Denmark)

    Steckelings, U Muscha; Kloet, Annette de; Sumners, Colin

    2017-01-01

    Sustained increases in the activity of the sympathetic neural pathways that exit the brain and which increase blood pressure (BP) are a major underlying factor in resistant hypertension. Recently available information on the occurrence of angiotensin II type 2 receptors (AT2Rs) within or adjacent...

  4. Renoprotective effects of angiotensin II receptor blockade in type 1 diabetic patients with diabetic nephropathy

    DEFF Research Database (Denmark)

    Andersen, S; Tarnow, L; Rossing, P

    2000-01-01

    BACKGROUND: Angiotensin I-converting enzyme (ACE) inhibitors reduce angiotensin II formation and induce bradykinin accumulation. Animal studies suggest that bradykinin may play a role for the effects of ACE inhibition on blood pressure and kidney function. Therefore, we compared the renal and hem...... inhibition is primarily caused by interference in the renin-angiotensin system. Our study suggest that losartan represents a valuable new drug in the treatment of hypertension and proteinuria in type 1 diabetic patients with diabetic nephropathy....... and hemodynamic effects of specific intervention in the renin-angiotensin system by blockade of the angiotensin II subtype-1 receptor to the effect of ACE inhibition. METHODS: A randomized, double-blind, cross-over trial was performed in 16 type 1 diabetic patients (10 men), age 42 +/- 2 years (mean +/- SEM...

  5. The Protective Arm of the Renin Angiotensin System (RAS)

    DEFF Research Database (Denmark)

    understanding of the protective side of the Renin Angiotensin System (RAS) involving angiotensin AT2 receptor, ACE2, and Ang(1-7)/Mas receptor Combines the knowledge of editors who pioneered research on the protective renin angiotensin system including; Dr. Thomas Unger, one of the founders of AT2 receptor......The Protective Arm of the Renin Angiotensin System: Functional Aspects and Therapeutic Implications is the first comprehensive publication to signal the protective role of a distinct part of the renin-angiotensin system (RAS), providing readers with early insight into a complex system which...... will become of major medical importance in the near future. Focusing on recent research, The Protective Arm of the Renin Angiotensin System presents a host of new experimental studies on specific components of the RAS, namely angiotensin AT2 receptors (AT2R), the angiotensin (1-7) peptide with its receptor...

  6. Characterization of Angiotensin II Molecular Determinants Involved in AT1 Receptor Functional Selectivity.

    Science.gov (United States)

    Domazet, Ivana; Holleran, Brian J; Richard, Alexandra; Vandenberghe, Camille; Lavigne, Pierre; Escher, Emanuel; Leduc, Richard; Guillemette, Gaétan

    2015-06-01

    The octapeptide angiotensin II (AngII) exerts a variety of cardiovascular effects through the activation of the AngII type 1 receptor (AT1), a G protein-coupled receptor. The AT1 receptor engages and activates several signaling pathways, including heterotrimeric G proteins Gq and G12, as well as the extracellular signal-regulated kinases (ERK) 1/2 pathway. Additionally, following stimulation, βarrestin is recruited to the AT1 receptor, leading to receptor desensitization. It is increasingly recognized that specific ligands selectively bind and favor the activation of some signaling pathways over others, a concept termed ligand bias or functional selectivity. A better understanding of the molecular basis of functional selectivity may lead to the development of better therapeutics with fewer adverse effects. In the present study, we developed assays allowing the measurement of six different signaling modalities of the AT1 receptor. Using a series of AngII peptide analogs that were modified in positions 1, 4, and 8, we sought to better characterize the molecular determinants of AngII that underlie functional selectivity of the AT1 receptor in human embryonic kidney 293 cells. The results reveal that position 1 of AngII does not confer functional selectivity, whereas position 4 confers a bias toward ERK signaling over Gq signaling, and position 8 confers a bias toward βarrestin recruitment over ERK activation and Gq signaling. Interestingly, the analogs modified in position 8 were also partial agonists of the protein kinase C (PKC)-dependent ERK pathway via atypical PKC isoforms PKCζ and PKCι. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

  7. Angiotensin receptor blockade in acute stroke. The Scandinavian Candesartan Acute Stroke Trial

    DEFF Research Database (Denmark)

    Sandset, Else Charlotte; Murray, Gordon; Boysen, Gudrun

    2010-01-01

    BACKGROUND: Elevated blood pressure following acute stroke is common, and yet early antihypertensive treatment is controversial. ACCESS suggested a beneficial effect of the angiotensin receptor blocker candesartan in the acute phase of stroke, but these findings need to be confirmed in new, large...

  8. Receptor-binding domain of SARS-CoV spike protein induces highly potent neutralizing antibodies: implication for developing subunit vaccine

    International Nuclear Information System (INIS)

    He Yuxian; Zhou Yusen; Liu Shuwen; Kou Zhihua; Li Wenhui; Farzan, Michael; Jiang Shibo

    2004-01-01

    The spike (S) protein of severe acute respiratory syndrome (SARS) coronavirus (CoV), a type I transmembrane envelope glycoprotein, consists of S1 and S2 domains responsible for virus binding and fusion, respectively. The S1 contains a receptor-binding domain (RBD) that can specifically bind to angiotensin-converting enzyme 2 (ACE2), the receptor on target cells. Here we show that a recombinant fusion protein (designated RBD-Fc) containing 193-amino acid RBD (residues 318-510) and a human IgG1 Fc fragment can induce highly potent antibody responses in the immunized rabbits. The antibodies recognized RBD on S1 domain and completely inhibited SARS-CoV infection at a serum dilution of 1:10,240. Rabbit antisera effectively blocked binding of S1, which contains RBD, to ACE2. This suggests that RBD can induce highly potent neutralizing antibody responses and has potential to be developed as an effective and safe subunit vaccine for prevention of SARS

  9. Remission of post-transplant focal segmental glomerulosclerosis with angiotensin receptor blockers

    Directory of Open Access Journals (Sweden)

    S B Bansal

    2017-01-01

    Full Text Available Recurrence of focal segmental glomerulosclerosis (FSGS is common after kidney transplantation. Plasmapheresis (PP is considered to be the most effective treatment; however, results are variable and relapse is common after stopping plasmapheresis. Here, we report an unusual case of recurrent FSGS, who achieved complete remission with angiotensin receptor blocker therapy.

  10. Effects of a novel bradykinin B1 receptor antagonist and angiotensin II receptor blockade on experimental myocardial infarction in rats.

    Directory of Open Access Journals (Sweden)

    Dongmei Wu

    Full Text Available The aim of the present study was to evaluate the cardiovascular effects of the novel bradykinin B1 receptor antagonist BI-113823 following myocardial infarction (MI and to determine whether B1 receptor blockade alters the cardiovascular effects of an angiotensin II type 1 (AT1 receptor antagonist after MI in rats.Sprague Dawley rats were subjected to permanent occlusion of the left descending coronary artery. Cardiovascular function was determined at 7 days post MI. Treatment with either B1 receptor antagonist (BI-113823 or AT1 receptor antagonist (irbesartan alone or in combination improved post-MI cardiac function as evidenced by attenuation of elevated left ventricular end diastolic pressure (LVEDP; greater first derivative of left ventricular pressure (± dp/dt max, left ventricle ejection fraction, fractional shorting, and better wall motion; as we as reductions in post-MI up-regulation of matrix metalloproteinases 2 (MMP-2 and collagen III. In addition, the cardiac up-regulation of B1 receptor and AT1 receptor mRNA were markedly reduced in animals treated with BI 113823, although bradykinin B2 receptor and angiotensin 1 converting enzyme (ACE1 mRNA expression were not significantly affected by B1 receptor blockade.The present study demonstrates that treatment with the novel B1 receptor antagonist, BI-113823 improves post-MI cardiac function and does not influence the cardiovascular effects of AT1 receptor antagonist following MI.

  11. New basic science initiatives with the angiotensin II receptor blocker valsartan

    Directory of Open Access Journals (Sweden)

    Marc de Gasparo

    2000-06-01

    Full Text Available Summary Angiotensin II (Ang II plays a key role in the regulation of blood pressure and fluid homeostasis. Valsartan is a highly selective Ang II receptor blocker that specifically and selectively blocks Ang II at the AT1-receptor. In animal models, valsartan has shown positive effects on vasoconstriction, proliferation, remodelling, endothelial function and thrombogenesis, inflammation and atherosclerosis. These data are likely to be confirmed by the results of current clinical trials and valsartan is set to provide improved cardiovascular therapy in the future.

  12. Dual angiotensin receptor and neprilysin inhibition as an alternative to angiotensin-converting enzyme inhibition in patients with chronic systolic heart failure

    DEFF Research Database (Denmark)

    McMurray, John J V; Packer, Milton; Desai, Akshay S

    2013-01-01

    and natriuresis, inhibit abnormal growth, suppress the RAAS and sympathetic nervous system, and augment parasympathetic activity. The best understood of these mediators are the natriuretic peptides which are metabolized by the enzyme neprilysin. LCZ696 belongs to a new class of drugs, the angiotensin receptor...

  13. CARBOHYDRATE-CONTAINING COMPOUNDS WHICH BIND TO CARBOHYDRATE BINDING RECEPTORS

    DEFF Research Database (Denmark)

    1995-01-01

    Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases.......Carbohydrate-containing compounds which contain saccharides or derivatives thereof and which bind to carbohydrate binding receptors are useful in pharmaceutical products for treatment of inflammatory diseases and other diseases....

  14. beta-Arrestin 1 and 2 stabilize the angiotensin II type I receptor in distinct high-affinity conformations

    DEFF Research Database (Denmark)

    Sanni, S J; Hansen, J T; Bonde, M M

    2010-01-01

    The angiotensin II type 1 (AT(1)) receptor belongs to family A of 7 transmembrane (7TM) receptors. The receptor has important roles in the cardiovascular system and is commonly used as a drug target in cardiovascular diseases. Interaction of 7TM receptors with G proteins or beta-arrestins often...

  15. [Assessment of the utilization of angiotensin receptor blockers in hypertension].

    Science.gov (United States)

    Peña Cabia, S; Ricote Lobera, I; Santos Mena, B; Hidalgo Correas, F J; Climent Florez, B; García Díaz, B

    2013-01-01

    To assess the degree in which the utilization of angiotensin receptor blockers (ARBs) in our Healthcare Area fits the criteria proposed by the Autonomous Community of Madrid (CAM) before setting «Plan de Actuación de ARA-II» («Action Plan ARA-II»). To study the indications for which are prescribed and to identify those factors that can show influence in prescription. Drug utilization study of the type indication-prescription, descriptive and transversal, for which ARBs-treated and hypertensive patients admitted to a University General Hospital for a study period of 3 months were selected. Based on the clinical situations summarized in the CAM Document «Criterios para establecer el lugar en la terapéutica de los antagonistas de los receptores de la angiotensina II» («Criteria for the place of angiotensin receptor blockers in the therapeutic»), a percentage of patients with «appropriate prescription» and «inadequate prescription« of ARBs was calculated and analyzed in order to determine if the age and the sex were related to the type of prescription or the main indications for which they had been prescribed. Out of the 153 patients included in the study, 67.3% had a «inadequate prescription«, 47.6% of them due to an ARBs prescription as the first drug inhibitor of the reninangiotensin- aldosterone system and 34.0% owing to a poor control of blood pressure with angiotensin-converting enzyme inhibitors (ACEi). There were no statistically significant differences found either by age or sex in the type of prescription or in the main indications for which they were prescribed. The adequacy of the criteria for the utilisation of ARBs Document occurred in 32.7% of cases. In addition, factors such as age and sex did not seem to affect the type of prescription. Misconceptions of superiority of ARBs versus ACEi were evidenced as well. Copyright © 2013 SEFH. Published by AULA MEDICA. All rights reserved.

  16. Common angiotensin receptor blockers may directly modulate the immune system via VDR, PPAR and CCR2b

    Directory of Open Access Journals (Sweden)

    Lee Robert E

    2006-01-01

    Full Text Available Abstract Background There have been indications that common Angiotensin Receptor Blockers (ARBs may be exerting anti-inflammatory actions by directly modulating the immune system. We decided to use molecular modelling to rapidly assess which of the potential targets might justify the expense of detailed laboratory validation. We first studied the VDR nuclear receptor, which is activated by the secosteroid hormone 1,25-dihydroxyvitamin-D. This receptor mediates the expression of regulators as ubiquitous as GnRH (Gonadatrophin hormone releasing hormone and the Parathyroid Hormone (PTH. Additionally we examined Peroxisome Proliferator-Activated Receptor Gamma (PPARgamma, which affects the function of phagocytic cells, and the C-CChemokine Receptor, type 2b, (CCR2b, which recruits monocytes to the site of inflammatory immune challenge. Results Telmisartan was predicted to strongly antagonize (Ki≈0.04nmol the VDR. The ARBs Olmesartan, Irbesartan and Valsartan (Ki≈10 nmol are likely to be useful VDR antagonists at typical in-vivo concentrations. Candesartan (Ki≈30 nmol and Losartan (Ki≈70 nmol may also usefully inhibit the VDR. Telmisartan is a strong modulator of PPARgamma (Ki≈0.3 nmol, while Losartan (Ki≈3 nmol, Irbesartan (Ki≈6 nmol, Olmesartan and Valsartan (Ki≈12 nmol also seem likely to have significant PPAR modulatory activity. Olmesartan andIrbesartan (Ki≈9 nmol additionally act as antagonists of a theoretical modelof CCR2b. Initial validation of this CCR2b model was performed, and a proposed model for the AngiotensinII Type1 receptor (AT2R1 has been presented. Conclusion Molecular modeling has proven valuable to generate testable hypotheses concerning receptor/ligand binding and is an important tool in drug design. ARBs were designed to act as antagonists for AT2R1, and it was not surprising to discover their affinity for the structurally similar CCR2b. However, this study also found evidence that ARBs modulate the

  17. Expression of Angiotensin II Types 1 and 2 Receptors in Endometriotic Lesions.

    Science.gov (United States)

    Nakao, Takehiro; Chishima, Fumihisa; Sugitani, Masahiko; Tsujimura, Ryusuke; Hayashi, Chuyu; Yamamoto, Tatsuo

    2017-01-01

    The aim of this study was to evaluate the gene and protein expression of angiotensin type (AT) 1, AT2 receptors in endometriotic lesions and its relation to prostaglandin (PG) synthases. Endometriosis samples were obtained from 32 patients with endometriotic cysts. Endometrial tissues were obtained during operations for benign gynecological conditions. The expression of the AT1 and AT2 receptor mRNA and that of PG-endoperoxide synthase 2 and microsomal PGE2 synthase-1 (mPGES-1) was examined by quantitative RT-PCR. Immunohistochemical staining was performed for these receptors. AT1 and AT2 receptor proteins were mostly located in endometrial glandular epithelium and some stromal cells. Immunoreactivity of the receptor proteins was observed in both the eutopic endometrium and endometriotic lesions. The AT1/AT2 ratio in endometriotic cysts (median 7.29, range 1.88-187.60) was significantly increased compared with that in the eutopic endometrium in the proliferative-phase in controls (median 1.01, range 0.37-2.09, p < 0.001). There was a relationship between the AT1 mRNA expression and that of mPGES-1 mRNA in the endometriotic cysts (r = 0.394089, p < 0.05). There was a significant relationship between the mRNA expression of the AT2 receptor and that of mPGES-1 in eutopic endometrium of non-endometriotic control (r = 0.610714, p < 0.05). Renin-angiotensin system may play an important role in the pathophysiology of endometriosis. © 2016 S. Karger AG, Basel.

  18. Combination inhibition of the renin-angiotensin system: is more better?

    Science.gov (United States)

    Krause, Michelle W; Fonseca, Vivian A; Shah, Sudhir V

    2011-08-01

    Angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers are considered the standard of care for treatment of cardiovascular disease and chronic kidney disease. Combination therapy with both angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers effectively inhibits the renin-angiotensin system as well as potentiates the vasodilatory effects of bradykinin. It has been advocated that this dual blockade approach theoretically should result in improved clinical outcomes in both cardiovascular disease and chronic kidney disease. Clinical trial evidence for the use of combination therapy with angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers in cardiovascular disease has provided conflicting results in hypertension, congestive heart failure, and ischemic heart disease. Clinical trial evidence to support combination therapy with angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers in chronic kidney disease has largely been based on proteinuria reduction as a surrogate marker for clinically meaningful outcomes. Recent large-scale randomized clinical trials have not been able to validate protection in halting progression in chronic kidney disease with a dual blockade approach. This review serves as an appraisal on the clinical evidence of combination angiotensin-converting enzyme inhibition and angiotensin II receptor blockade in both cardiovascular disease and chronic kidney disease.

  19. Angiotensin-2-mediated Ca2+ signaling in the retinal pigment epithelium: role of angiotensin-receptor-associated-protein and TRPV2 channel.

    Directory of Open Access Journals (Sweden)

    Rene Barro-Soria

    Full Text Available Angiotensin II (AngII receptor (ATR is involved in pathologic local events such as neovascularisation and inflammation including in the brain and retina. The retinal pigment epithelium (RPE expresses ATR in its AT1R form, angiotensin-receptor-associated protein (Atrap, and transient-receptor-potential channel-V2 (TRPV2. AT1R and Atrap co-localize to the basolateral membrane of the RPE, as shown by immunostaining. Stimulation of porcine RPE (pRPE cells by AngII results in biphasic increases in intracellular free Ca(2+inhibited by losartan. Xestospongin C (xest C and U-73122, blockers of IP3R and PLC respectively, reduced AngII-evoked Ca(2+response. RPE cells from Atrap(-/- mice showed smaller AngII-evoked Ca(2+peak (by 22% and loss of sustained Ca(2+elevation compared to wild-type. The TRPV channel activator cannabidiol (CBD at 15 µM stimulates intracellular Ca(2+-rise suggesting that porcine RPE cells express TRPV2 channels. Further evidence supporting the functional expression of TRPV2 channels comes from experiments in which 100 µM SKF96365 (a TRPV channel inhibitor reduced the cannabidiol-induced Ca(2+-rise. Application of SKF96365 or reduction of TRPV2 expression by siRNA reduced the sustained phase of AngII-mediated Ca(2+transients by 53%. Thus systemic AngII, an effector of the local renin-angiotensin system stimulates biphasic Ca(2+transients in the RPE by releasing Ca(2+from cytosolic IP3-dependent stores and activating ATR/Atrap and TRPV2 channels to generate a sustained Ca(2+elevation.

  20. The ACE-2/Ang1-7/Mas cascade enhances bone structure and metabolism following angiotensin-II type 1 receptor blockade.

    Science.gov (United States)

    Abuohashish, Hatem M; Ahmed, Mohammed M; Sabry, Dina; Khattab, Mahmoud M; Al-Rejaie, Salim S

    2017-07-15

    The renin angiotensin system (RAS) regulates numerous systemic functions and is expressed locally in skeletal tissues. Angiotensin1-7 (Ang1-7) is a beneficial member of the RAS, and the therapeutic effects of a large number of angiotensin receptors blockers (ARBs) are mediated by an Ang1-7-dependent cascade. This study examines whether the reported osteo-preservative effects of losartan are mediated through the angiotensin converting enzyme2 (ACE-2)/Ang1-7/Mas pathway in ovariectomized (OVX) rats. Sham and OVX animals received losartan (10mg/kg/d p.o.) for 6 weeks. A specific Mas receptor blocker (A-779) was delivered via mini-osmotic pumps during the losartan treatment period. Serum and urine bone metabolism biomarker levels were measured. Bone trabecular and cortical morphometry were quantified in distal femurs, whereas mineral contents were estimated in ashed bones, serum and urine. Finally, the expression of RAS components, the receptor activator of NF-κB ligand (RANKL) and osteoprotegerin (OPG) was determined. Losartan significantly improved the elevated bone metabolism marker levels and altered trabecular and cortical structures in OVX animals, and restored normal urinary and skeletal mineral levels. Mas receptor inhibition significantly abolished all osteo-protective effects of losartan and enhanced the deleterious effects of OVX. Losartan enhanced OVX-induced up-regulation of ACE-1, AngII, angiotensin type 1 (AT 1 ) receptor and RANKL expression, and increased ACE-2, Ang1-7, Mas and OPG expression in OVX animals. However, A-779 significantly eradicated the effects of losartan on RAS components and RANKL/OPG expression. Thus, Ang1-7 are involved in the osteo-preservative effects of losartan via Mas receptor, which may add therapeutic value to this well-known antihypertensive agent. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. CORRELATION BETWEEN ANGIOTENSIN-CONVERTING ENZYME INHIBITORS LIPOPHILICITY AND PROTEIN BINDING DATA

    Directory of Open Access Journals (Sweden)

    Jasna Trbojević-Stanković

    2012-01-01

    Full Text Available Angiotensin-converting enzyme (ACE inhibitors represent a significant group of drugs primarily used in the treatment of hypertension and congestive heart failure. In this research, seven ACE inhibitors (enalapril, quinapril, fosinopril, lisinopril, cilazapril, ramipril, benazepril were studied to evaluate the relationship between their protein binding and calculated (logP values or ultra-high performance liquid chromatographytandem mass spectrometry (UHPLC-MS and reversed-phase thin-layer chromatography (RP-TLC lipophilicity data (ϕ0, CHI or C0 parameters, respectively. Their protein binding data varied from negligible (lisinopril to 99% (fosinopril, while calculated logPKOWWINvalues ranged from -0.94 (lisinopril to 6.61 (fosinopril. The good correlations were established between protein binding values and logPKOWWIN data (R2=0.7520 as well as between protein binding and chromatographic hydrophobicity data, ϕ0, CHI or C0parameters (R2 were 0.6160, 0.6242 and 0.6547, respectively. The possible application of hydrophobicity data in drugs protein binding evaluation can be of great importance in drug bioavailability.

  2. Angiotensin II-AT1–receptor signaling is necessary for cyclooxygenase-2–dependent postnatal nephron generation

    DEFF Research Database (Denmark)

    Frölich, Stefanie; Slattery, Patrick; Thomas, Dominique

    2017-01-01

    was achievable. Plasma renin concentration in mouse pups showed a birth peak and a second peak around day P8 during the first 10 days post birth. Administration of the angiotensin II receptor AT1 antagonist telmisartan from day P1 to P3 did not result in cortical damage. However, telmisartan treatment from day P...... development. Inhibition of the renin angiotensin system by aliskiren and enalapril caused similar glomerular defects as telmisartan. Administration of the AT1 receptor agonist L162313 to COX-2-/- pups improved kidney growth, ameliorated renal defects, but had no beneficial effect on reduced cortical mass. L...

  3. Attenuation of the pressor response to exogenous angiotensin by angiotensin receptor blockers and benazepril hydrochloride in clinically normal cats.

    Science.gov (United States)

    Jenkins, Tiffany L; Coleman, Amanda E; Schmiedt, Chad W; Brown, Scott A

    2015-09-01

    To compare the attenuation of the angiotensin I-induced blood pressure response by once-daily oral administration of various doses of angiotensin receptor blockers (irbesartan, telmisartan, and losartan), benazepril hydrochloride, or lactose monohydrate (placebo) for 8 days in clinically normal cats. 6 healthy cats (approx 17 months old) with surgically implanted arterial telemetric blood pressure-measuring catheters. Cats were administered orally the placebo or each of the drug treatments (benazepril [2.5 mg/cat], irbesartan [6 and 10 mg/kg], telmisartan [0.5, 1, and 3 mg/kg], and losartan [2.5 mg/kg]) once daily for 8 days in a crossover study. Approximately 90 minutes after capsule administration on day 8, each cat was anesthetized and arterial blood pressure measurements were recorded before and after IV administration of each of 4 boluses of angiotensin I (20, 100, 500, and 1,000 ng/kg). This protocol was repeated 24 hours after benazepril treatment and telmisartan (3 mg/kg) treatment. Differences in the angiotensin I-induced change in systolic arterial blood pressure (ΔSBP) among treatments were determined. At 90 minutes after capsule administration, only losartan did not significantly reduce ΔSBP in response to the 3 higher angiotensin doses, compared with placebo. Among drug treatments, telmisartan (3 mg/kg dosage) attenuated ΔSBP to a significantly greater degree than benazepril and all other treatments. At 24 hours, telmisartan was more effective than benazepril (mean ± SEM ΔSBP, 15.7 ± 1.9 mm Hg vs 55.9 ± 12.42 mm Hg, respectively). Results indicated that telmisartan administration may have advantages over benazepril administration for cats with renal or cardiovascular disease.

  4. Cognitive enhancing effect of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers on learning and memory

    Science.gov (United States)

    Nade, V. S.; Kawale, L. A.; Valte, K. D.; Shendye, N. V.

    2015-01-01

    Objective: The present study was designed to investigate cognitive enhancing property of angiotensin-converting enzymes inhibitors (ACEI) and angiotensin receptor blockers (ARBs) in rats. Materials and Methods: The elevated plus maze (EPM), passive avoidance test (PAT), and water maze test (WMT) were used to assess cognitive enhancing activity in young and aged rats. Ramipril (10 mg/kg, p.o.), perindopril (10 mg/kg, i.p), losartan (20 mg/kg, i.p), and valsartan (20 mg/kg, p.o) were administered to assess their effect on learning and memory. Scopolamine (1 mg/kg, i.p) was used to impair cognitive function. Piracetam (200 mg/kg, i.p) was used as reference drug. Results: All the treatments significantly attenuated amnesia induced by aging and scopolamine. In EPM, aged and scopolamine-treated rats showed an increase in transfer latency (TL) whereas, ACEI and ARBs showed a significant decrease in TL. Treatment with ACEI and ARBs significantly increased step down latencies and decreased latency to reach the platform in target quadrant in young, aged and scopolamine-treated animals in PAT and WMT, respectively. The treatments inhibited acetylcholinesterase (AChE) enzyme in the brain. Similarly, all the treatments attenuated scopolamine-induced lipid peroxidation and normalize antioxidant enzymes. Conclusion: The results suggest that the cognitive enhancing effect of ACEI and ARBs may be due to inhibition of AChE or by regulation of antioxidant system or increase in formation of angiotensin IV. PMID:26069362

  5. Introductory statement: Of receptors and analogs in renin-angiotensin-aldosterone and adrenergic systems

    International Nuclear Information System (INIS)

    Eliahou, H.E.; Iaina, A.

    1980-01-01

    This article reports on the role of the octapeptides angiotensin II (A II)-effector and its receptor on hypertensive patients and in animal experiments. By applying the A-II-receptor blockers, vasodilates drugs, β-receptor blockers and by sodium depletion, the behaviour of the blood pressure, the plasmareninactivity, and of the aldosteron were investigated; a comparative investigation between the A II and A III effectors was also carried out. The iodo-hippurate uptake was reduced with an artificially produced renal arterial ischemia. In general, this investigation provided new viewpoints in the understanding of the possible pathogenetic mechanism of essential hypertonism. (APR) [de

  6. A comparative study of the prevalence of hyperkalemia with the use of angiotensin-converting enzyme inhibitors versus angiotensin receptor blockers

    Directory of Open Access Journals (Sweden)

    Seyed Ali Sadjadi

    2009-07-01

    Full Text Available Seyed Ali Sadjadi1, James I McMillan1, Navin Jaipaul1, Patricia Blakely1, Su Su Hline21Section of Nephrology (111N, Jerry L Pettis Memorial Veterans Medical Center, Loma Linda, CA, USA; 2Divison of Nephrology, Loma Linda University Medical Center, Loma Linda, CA, USABackground and objectives: Angiotensin-converting enzyme inhibitors (ACEI and angiotensin receptor blockers (ARB are increasingly used in a variety of settings including heart failure, renal failure, arterial hypertension, and diabetic nephropathy. The objective of this study was to investigate the prevalence of hyperkalemia with ACEI and ARB use, in a population of the United States veterans.Design, settings, material, and measurements: Retrospective observational cohort study of 1163 patients on ACEIs and 1168 patients on ARBs in a single Veterans Affairs Medical Center. Electronic medical records were reviewed over a 12-month period with data collected on various demographic, laboratory, comorbidity, and medication related variables. Results: Hyperkalemia (>5 mEq/L was observed in 20.4% of patients on ACEIs and 31.0% on ARBs. Severe hyperkalemia (6 mEq/L or higher, was observed in 0.8% of ACEI and 2.8% of ARB users. In univariate logistic regression analyses, diabetes mellitus; serum glucose, total carbon dioxide content, creatinine, and estimated glomerular filtration rate (GFR were significantly associated with hyperkalemia. ARB use, when compared to ACEI, was associated with a 42% increase in odds of hyperkalemia (odds ratio [OR] = 1.42; p = 0.001 in a model including adjustment for GFR and a 56% increase in odds of hyperkalemia (OR = 1.56; p < 0.001 in a model including adjustment for serum creatinine.Conclusions: Hyperkalemia, associated with the use of ACEIs and ARBs, is usually mild and severe hyperkalemia is rare. Hyperkalemia is more common with ARBs than ACEIs. ARB use, when compared to ACEI use, may significantly and independently be associated with increased odds of

  7. Estimation of the number of angiotensin II AT1 receptors in rat kidney afferent and efferent arterioles

    DEFF Research Database (Denmark)

    Razga, Zsolt; Nyengaard, Jens Randel

    2007-01-01

    of angiotensin II AT1 receptors along the length of the arterioles and per arteriole, we combined immunoelectron microscopy with stereology. RESULTS: The number of AT1 receptor molecules was significantly lower in the renin-positive smooth muscle cells (SMCs) than in the renin-negative SMCs of the afferent...

  8. Troglitazone stimulates β-arrestin-dependent cardiomyocyte contractility via the angiotensin II type 1A receptor

    International Nuclear Information System (INIS)

    Tilley, Douglas G.; Nguyen, Anny D.; Rockman, Howard A.

    2010-01-01

    Peroxisome proliferator-activated receptor γ (PPARγ) agonists are commonly used to treat cardiovascular diseases, and are reported to have several effects on cardiovascular function that may be due to PPARγ-independent signaling events. Select angiotensin receptor blockers (ARBs) interact with and modulate PPARγ activity, thus we hypothesized that a PPARγ agonist may exert physiologic effects via the angiotensin II type 1 A receptor (AT1 A R). In AT1 A R-overexpressing HEK 293 cells, both angiotensin II (Ang II) and the PPARγ agonist troglitazone (Trog) enhanced AT1 A R internalization and recruitment of endogenous β-arrestin1/2 (βarr1/2) to the AT1 A R. A fluorescence assay to measure diacylglycerol (DAG) accumulation showed that although Ang II induced AT1 A R-G q protein-mediated DAG accumulation, Trog had no impact on DAG generation. Trog-mediated recruitment of βarr1/2 was selective to AT1 A R as the response was prevented by an ARB- and Trog-mediated βarr1/2 recruitment to β1-adrenergic receptor (β1AR) was not observed. In isolated mouse cardiomyocytes, Trog increased both % and rate of cell shortening to a similar extent as Ang II, effects which were blocked with an ARB. Additionally, these effects were found to be βarr2-dependent, as cardiomyocytes isolated from βarr2-KO mice showed blunted contractile responses to Trog. These findings show for the first time that the PPARγ agonist Trog acts at the AT1 A R to simultaneously block G q protein activation and induce the recruitment of βarr1/2, which leads to an increase in cardiomyocyte contractility.

  9. Angiotensin-(1-7) augments endothelium-dependent relaxations of porcine coronary arteries to bradykinin by inhibiting angiotensin-converting enzyme 1.

    Science.gov (United States)

    Raffai, Gábor; Khang, Gilson; Vanhoutte, Paul M

    2014-05-01

    Angiotensin-converting enzyme 2 (ACE2) converts angiotensin II to angiotensin-(1-7) that activates Mas receptors, inhibits ACE1, and modulates bradykinin receptor sensitivity. This in vitro study compared the direct and indirect effects of angiotensin-(1-7), the ACE1 inhibitor captopril, and diminazene aceturate (DIZE) an alleged ACE2 activator in rings of porcine coronary arteries, by measuring changes of isometric tension. Angiotensin-(1-7), captopril, and DIZE did not cause significant changes in tension before or after desensitization of bradykinin receptors in preparations contracted with U46619. Bradykinin caused concentration-dependent and endothelium-dependent relaxations that were not affected by DIZE but were potentiated to a similar extent by angiotensin-(1-7) and captopril, given alone or in combination. Bradykinin responses potentiated by angiotensin-(1-7) and captopril were not affected by the BK1 antagonist SSR240612 and remained augmented in the presence of either N-nitro-L-arginine methyl ester hydrochloride plus indomethacin or TRAM-34 plus UCL-1684. ACE2 was identified in the coronary endothelium by immunofluorescence, but its basal activity was not influenced by DIZE. These results suggest that in coronary arteries, angiotensin-(1-7) and captopril both improves NO bioavailability and enhances endothelium-dependent hyperpolarization to bradykinin solely by ACE1 inhibition. Endothelial ACE2 activity cannot be increased by DIZE to produce local adequate amounts of angiotensin-(1-7) to influence vascular tone.

  10. Angiotensin II Receptor Blockers

    Science.gov (United States)

    ... 2010. Mann JFE, et al. Renin-angiotensin system inhibition in the treatment of hypertension. http://www.uptodate. ... profit organization and proceeds from Web advertising help support our mission. Mayo Clinic does not endorse any ...

  11. The non-biphenyl-tetrazole angiotensin AT1 receptor antagonist eprosartan is a unique and robust inverse agonist of the active state of the AT1 receptor.

    Science.gov (United States)

    Takezako, Takanobu; Unal, Hamiyet; Karnik, Sadashiva S; Node, Koichi

    2018-03-23

    Conditions such as hypertension and renal allograft rejection are accompanied by chronic, agonist-independent, signalling by angiotensin II AT 1 receptors. The current treatment paradigm for these diseases entails the preferred use of inverse agonist AT 1 receptor blockers (ARBs). However, variability in the inverse agonist activities of common biphenyl-tetrazole ARBs for the active state of AT 1 receptors often leads to treatment failure. Therefore, characterization of robust inverse agonist ARBs for the active state of AT 1 receptors is necessary. To identify the robust inverse agonist for active state of AT 1 receptors and its molecular mechanism, we performed site-directed mutagenesis, competition binding assay, inositol phosphate production assay and molecular modelling for both ground-state wild-type AT 1 receptors and active-state N111G mutant AT 1 receptors. Although candesartan and telmisartan exhibited weaker inverse agonist activity for N111G- compared with WT-AT 1 receptors, only eprosartan exhibited robust inverse agonist activity for both N111G- and WT- AT 1 receptors. Specific ligand-receptor contacts for candesartan and telmisartan are altered in the active-state N111G- AT 1 receptors compared with the ground-state WT-AT 1 receptors, suggesting an explanation of their attenuated inverse agonist activity for the active state of AT 1 receptors. In contrast, interactions between eprosartan and N111G-AT 1 receptors were not significantly altered, and the inverse agonist activity of eprosartan was robust. Eprosartan may be a better therapeutic option than other ARBs. Comparative studies investigating eprosartan and other ARBs for the treatment of diseases caused by chronic, agonist-independent, AT 1 receptor activation are warranted. © 2018 The British Pharmacological Society.

  12. Expression and role of the angiotensin II AT2 receptor in human prostate tissue: in search of a new therapeutic option for prostate cancer.

    Science.gov (United States)

    Guimond, Marie-Odile; Battista, Marie-Claude; Nikjouitavabi, Fatemeh; Carmel, Maude; Barres, Véronique; Doueik, Alexandre A; Fazli, Ladan; Gleave, Martin; Sabbagh, Robert; Gallo-Payet, Nicole

    2013-07-01

    Evidence shows that angiotensin II type 1 receptor (AT1R) blockers may be associated with improved outcome in prostate cancer patients. It has been proposed that part of this effect could be due to angiotensin II type 2 receptor (AT2R) activation, the only active angiotensin II receptor in this situation. This study aimed to characterize the localization and expression of AT2R in prostate tissues and to assess its role on cell morphology and number in prostatic epithelial cells in primary culture. AT2R and its AT2R-interacting protein (ATIP) expression were assessed on non-tumoral and tumoral human prostate using tissue microarray immunohistochemistry, binding assay, and Western blotting. AT2R effect on cell number was measured in primary cultures of epithelial cells from non-tumoral human prostate. AT2R was localized at the level of the acinar epithelial layer and its expression decreased in cancers with a Gleason score 6 or higher. In contrast, ATIP expression increased with cancer progression. Treatment of primary cell cultures from non-tumoral prostate tissues with C21/M024, a selective AT2R agonist, alone or in co-incubation with losartan, an AT1R antagonist, significantly decreased cell number compared to untreated cells. AT2R and ATIP are present in non-tumoral human prostate tissues and differentially regulated according to Gleason score. The decrease in non-tumoral prostate cell number upon selective AT2R stimulation suggests that AT2R may have a protective role against prostate cancer development. Treatment with a selective AT2R agonist could represent a new approach for prostate cancer prevention or for patients on active surveillance. Copyright © 2013 Wiley Periodicals, Inc.

  13. Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

    Science.gov (United States)

    Torres-Tirado, David; Knabb, Maureen; Castaño, Irene; Patrón-Soberano, Araceli; De Las Peñas, Alejandro; Rubio, Rafael

    2016-01-01

    Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses. Copyright © 2016 the American Physiological Society.

  14. Effects of angiotensin II receptor blockade on cerebral, cardiovascular, counter-regulatory, and symptomatic responses during hypoglycaemia in patients with type 1 diabetes

    DEFF Research Database (Denmark)

    Færch, Louise H; Thorsteinsson, Birger; Tarnow, Lise

    2015-01-01

    INTRODUCTION: High spontaneous activity of the renin-angiotensin system (RAS) results in more pronounced cognitive impairment and more prolonged QTc interval during hypoglycaemia in type 1 diabetes. We tested whether angiotensin II receptor blockade improves cerebral and cardiovascular function d...

  15. Gonadal cell surface receptor for plasma retinol-binding protein

    International Nuclear Information System (INIS)

    Krishna Bhat, M.; Cama, H.R.

    1979-01-01

    A specific membrane receptor for plasma retinol-binding protein has been demonstrated in testicular cells. Prealbumin-2 did not show any specific binding to the membrane. The affinity of retinol-binding protein for receptor drastically decreases upon delivery of retinol and the retinol-binding protein does not enter the cell. The mechanism of delivery of retinol to the target cell by plasma retinol-binding protein has been investigated. The process involves two steps; direct binding of retinol-binding protein to the receptor and uptake of retinol by the target cell with a concomitant drastic reduction in the affinity of the retinol-binding protein to the receptor. Probably the second step of the process needs a cytosolic factor, possibly the cellular retinol-binding protein or an enzyme. The binding of retinol-binding protein to the receptor is saturable and reversible. The interaction shows a Ksub(d) value of 2.1x10 -10 . The specific binding of a retinol-binding protein with great affinity has been employed in the development of a method for radioassay of the receptor. The receptor level of the gonadal cell has been found to vary with the stage of differentiation. The receptor concentrations in 11-week-old birds and adult birds are comparable. Testosterone treatment of 11-week-old birds produced a substantial increase in the receptor concentration over control, while the protein content increased marginally, indicating that, probably, synthesis of the receptor is specifcally induced by testosterone during spermatogenesis, and the concentration of receptor is relatively higher before the formation of the acrosome. (Auth.)

  16. Conformational Profiling of the AT1 Angiotensin II Receptor Reflects Biased Agonism, G Protein Coupling, and Cellular Context.

    Science.gov (United States)

    Devost, Dominic; Sleno, Rory; Pétrin, Darlaine; Zhang, Alice; Shinjo, Yuji; Okde, Rakan; Aoki, Junken; Inoue, Asuka; Hébert, Terence E

    2017-03-31

    Here, we report the design and use of G protein-coupled receptor-based biosensors to monitor ligand-mediated conformational changes in receptors in intact cells. These biosensors use bioluminescence resonance energy transfer with Renilla luciferase (RlucII) as an energy donor, placed at the distal end of the receptor C-tail, and the small fluorescent molecule FlAsH as an energy acceptor, its binding site inserted at different positions throughout the intracellular loops and C-terminal tail of the angiotensin II type I receptor. We verified that the modifications did not compromise receptor localization or function before proceeding further. Our biosensors were able to capture effects of both canonical and biased ligands, even to the extent of discriminating between different biased ligands. Using a combination of G protein inhibitors and HEK 293 cell lines that were CRISPR/Cas9-engineered to delete Gα q , Gα 11 , Gα 12 , and Gα 13 or β-arrestins, we showed that Gα q and Gα 11 are required for functional responses in conformational sensors in ICL3 but not ICL2. Loss of β-arrestin did not alter biased ligand effects on ICL2P2. We also demonstrate that such biosensors are portable between different cell types and yield context-dependent readouts of G protein-coupled receptor conformation. Our study provides mechanistic insights into signaling events that depend on either G proteins or β-arrestin. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Angiotensin II Type 1 receptor (AT1) signaling in astrocytes regulates synaptic degeneration-induced leukocyte entry to the central nervous system

    DEFF Research Database (Denmark)

    Füchtbauer, L; Groth-Rasmussen, Maria; Holm, Thomas Hellesøe

    2011-01-01

    Astrocytes are the major cellular component of the blood-brain barrier glia limitans and act as regulators of leukocyte infiltration via chemokine expression. We have studied angiotensin-II receptor Type 1 (AT1) and related NF-κB signaling in astrocytes. Angiotensin II derives from cleavage of an...

  18. Moderation of dietary sodium potentiates the renal and cardiovascular protective effects of angiotensin receptor blockers

    DEFF Research Database (Denmark)

    Lambers Heerspink, Hiddo J; Holtkamp, Frank A; Parving, Hans-Henrik

    2012-01-01

    Dietary sodium restriction has been shown to enhance the short-term response of blood pressure and albuminuria to angiotensin receptor blockers (ARBs). Whether this also enhances the long-term renal and cardiovascular protective effects of ARBs is unknown. Here we conducted a post-hoc analysis of...

  19. Structural Analysis of Botulinum Neurotoxin Type G Receptor Binding

    Energy Technology Data Exchange (ETDEWEB)

    Schmitt, John; Karalewitz, Andrew; Benefield, Desire A.; Mushrush, Darren J.; Pruitt, Rory N.; Spiller, Benjamin W.; Barbieri, Joseph T.; Lacy, D. Borden (Vanderbilt); (MCW)

    2010-10-19

    Botulinum neurotoxin (BoNT) binds peripheral neurons at the neuromuscular junction through a dual-receptor mechanism that includes interactions with ganglioside and protein receptors. The receptor identities vary depending on BoNT serotype (A-G). BoNT/B and BoNT/G bind the luminal domains of synaptotagmin I and II, homologous synaptic vesicle proteins. We observe conditions under which BoNT/B binds both Syt isoforms, but BoNT/G binds only SytI. Both serotypes bind ganglioside G{sub T1b}. The BoNT/G receptor-binding domain crystal structure provides a context for examining these binding interactions and a platform for understanding the physiological relevance of different Syt receptor isoforms in vivo.

  20. Evidence for a cyclic AMP-dependent pathway in angiotensin AT1-receptor activation of human omental arteries

    Directory of Open Access Journals (Sweden)

    Hoa Ytterberg

    2001-03-01

    Full Text Available Enhanced responses to vasoconstriction induced by neuropeptide Y and α2-adrenoceptor agonists have been seen following pharmacological activation of the adenylyl cyclase (AC system. Since preliminary studies revealed only minor responses to angiotensin II (Ang II in human omental arteries, we have investigated whether enhanced activity of AC may unravel further functional Ang II receptors. Human omental arteries were obtained in conjunction with elective gut surgery. After dissection of the vessel, the endothelium was removed by 10 sec of Triton X-100 treatment. Ring segments (1—2 mm long were mounted on a myograph and studied. Ang II produced small contractions, 27±5% relative to the response elicited by 60 mM K+. However, enhanced Ang II (105±10%, p<0.001 responses were seen during AC activation by forskolin (0.1—1 µM. This enhanced contractile response to Ang II was not inhibited by the angiotensin II type 2 (AT2-receptor antagonist PD 123319 (0.1 µM, but was blocked in an insurmountable way by the angiotensin II type 1 (AT1-receptor antagonist candesartan (1 nM and in a surmountable manner by losartan (0.1 µM and irbesartan (0.1 µM. Pertussis toxin (a Gi-protein blocker and the protein kinase C inhibitor, RO31—8220 (0.01, 0.1 and 1 µM, markedly reduced this response, while the protein kinase A inhibitor, H89 (1, 10 µM, had no effect. RT-PCR provided evidence for the presence of mRNA for both AT1- and AT2-receptors. The results suggest that both a cAMP-dependent and a cAMP-independent mechanism are involved in the contractile responses to Ang II in human omental arteries and that both responses are mediated via the AT1-receptor.

  1. Receptor binding studies of the living heart

    International Nuclear Information System (INIS)

    Syrota, A.

    1988-01-01

    Receptors form a class of intrinsic membrane proteins (or glycoproteins) defined by the high affinity and specificity with which they bind ligands. Many receptors are associated directly or indirectly with membrane ion channels that open or close after a conformational change of the receptor induced by the binding of the neurotransmitter. Changes in number and/or affinity of cardiac neurotransmitter receptors have been associated with myocardial ischemia and infarction, congestive heart failure, and cardiomyopathy as well as diabetes or thyroid-induced heart muscle disease. These alterations of cardiac receptors have been demonstrated in vitro on membrane homogenates from samples collected mainly during surgery or postmortem. The disadvantage of these in vitro binding techniques is that receptors lose their natural environment and their relationships with the other components of the tissue

  2. Angiotensin II type 1 receptor blockade by telmisartan prevents stress-induced impairment of memory via HPA axis deactivation and up-regulation of brain-derived neurotrophic factor gene expression.

    Science.gov (United States)

    Wincewicz, D; Juchniewicz, A; Waszkiewicz, N; Braszko, J J

    2016-09-01

    Physical and psychological aspects of chronic stress continue to be a persistent clinical problem for which new pharmacological treatment strategies are aggressively sought. By the results of our previous work it has been demonstrated that telmisartan (TLM), an angiotensin type 1 receptor (AT1) blocker (ARB) and partial agonist of peroxisome proliferator-activated receptor gamma (PPARγ), alleviates stress-induced cognitive decline. Understanding of mechanistic background of this phenomenon is hampered by both dual binding sites of TLM and limited data on the consequences of central AT1 blockade and PPARγ activation. Therefore, a critical need exists for progress in the characterization of this target for pro-cognitive drug discovery. An unusual ability of novel ARBs to exert various PPARγ binding activities is commonly being viewed as predominant over angiotensin blockade in terms of neuroprotection. Here we aimed to verify this hypothesis using an animal model of chronic psychological stress (Wistar rats restrained 2.5h daily for 21days) with simultaneous oral administration of TLM (1mg/kg), GW9662 - PPARγ receptor antagonist (0.5mg/kg), or both in combination, followed by a battery of behavioral tests (open field, elevated plus maze, inhibitory avoidance - IA, object recognition - OR), quantitative determination of serum corticosterone (CORT) and evaluation of brain-derived neurotrophic factor (BDNF) gene expression in the medial prefrontal cortex (mPFC) and hippocampus (HIP). Stressed animals displayed decreased recall of the IA behavior (pBDNF in the mPFC (paxis deactivation associated with changes in primarily cortical gene expression. This study confirms the dual activities of TLM that controls hypertension and cognition through AT1 blockade. Copyright © 2016 Elsevier Inc. All rights reserved.

  3. Addition of a Nitric Oxide Donor to an Angiotensin II Type 1 Receptor Blocker May Cancel Its Blood Pressure-Lowering Effects.

    Science.gov (United States)

    Yahiro, Eiji; Miura, Shin-Ichiro; Suematsu, Yasunori; Matsuo, Yoshino; Arimura, Tadaaki; Kuwano, Takashi; Imaizumi, Satoshi; Iwata, Atsushi; Uehara, Yoshinari; Saku, Keijiro

    2015-01-01

    While physiological levels of nitric oxide (NO) protect the endothelium and have vasodilatory effects, excessive NO has adverse effects on the cardiovascular system. Recently, new NO-releasing pharmacodynamic hybrids of angiotensin II (Ang II) type 1 (AT1) receptor blockers (ARBs) have been developed.We analyzed whether olmesartan with NO-donor side chains (Olm-NO) was superior to olmesartan (Olm) for the control of blood pressure (BP). Although there was no significant difference in binding affinity to AT1 wild-type (WT) receptor between Olm and Olm-NO in a cell-based binding assay, the suppressive effect of Olm-NO on Ang II-induced inositol phosphate (IP) production was significantly weaker than that of Olm in AT1 WT receptor-expressing cells. While Olm had a strong inverse agonistic effect on IP production, Olm-NO did not. Next, we divided 18 C57BL mice into 3 groups: Ang II (infusion using an osmotic mini-pump) as a control group, Ang II (n = 6) + Olm, and Ang II (n = 6) + Olm-NO groups (n = 6). Olm-NO did not block Ang II-induced high BP after 10 days, whereas Olm significantly decreased BP. In addition, Olm, but not Olm-NO, significantly reduced the ratio of heart weight to body weight (HW/BW) with downregulation of the mRNA levels of atrial natriuretic peptide.An ARB with a NO-donor may cancel BP-lowering effects probably due to excessive NO and a weak blocking effect by Olm-NO toward AT1 receptor activation.

  4. Attenuation of Immune-Mediated Renal Injury by Telmisartan, an Angiotensin Receptor Blocker and a Selective PPAR-γ Activator

    Directory of Open Access Journals (Sweden)

    Yuki Hamano

    2011-09-01

    Full Text Available Background/Aims: Anti-glomerular basement membrane (GBM nephritis is characterized by activation of the renin-angiotensin system. This study aimed to determine the question of whether a temporary angiotensin II blockade at the initial stage of anti-GBM nephritis is able to attenuate the disease as well as differences in renoprotection among angiotensin II receptor blockers (ARBs with distinct peroxisome proliferator-activated receptor (PPAR-γ-modulating activities. Methods: C57BL/6J mice were immunized with rabbit IgG, followed by intravenous injection of rabbit anti-mouse antibodies. Mice were then treated with telmisartan, losartan, and telmisartan + GW9662 (a PPAR-γ antagonist for 5 days, or hydralazine for 9 days. On days 8 and 13, mice were sacrificed to obtain tissues for histological analysis. Results: The temporary administration of telmisartan significantly suppressed glomerular damage compared to hydralazine. Losartan showed a similar effect but was less effective. Co-administration of GW9662 attenuated the renoprotective effect of telmisartan, almost to levels observed with losartan. In particular, it limited the decreased infiltration of inflammatory cells and preservation of capillaries in the glomeruli induced by telmisartan. Conclusion: Temporary angiotensin II blockade at the initial stage of anti-GBM disease dramatically inhibited its progression. In addition to a class effect of ARBs, telmisartan modified inflammation and endothelial damage in the kidney through its PPAR-γ-agonistic action.

  5. Electroacupuncture improves cerebral blood flow and attenuates moderate ischemic injury via Angiotensin II its receptors-mediated mechanism in rats.

    Science.gov (United States)

    Li, Jing; He, Jiaojun; Du, Yuanhao; Cui, Jingjun; Ma, Ying; Zhang, Xuezhu

    2014-11-11

    To investigate the effects and potential mechanism of electroacupuncture intervention on expressions of Angiotensin II and its receptors-mediated signaling pathway in experimentally induced cerebral ischemia. Totally 126 male Wistar rats were randomly divided into control group, model group and EA group. The latter two were further divided into ten subgroups (n = 6) following Middle Cerebral Artery Occlusion (MCAO). Changes in regional cerebral blood flow (rCBF) and expressions of Angiotensin II and its receptors (AT1R, AT2R), as well as effector proteins in phosphatidyl inositol signal pathway were monitored before and at different times after MCAO. MCAO-induced decline of ipsilateral rCBF was partially suppressed by electroacupuncture, and contralateral blood flow was also superior to that of model group. Angiotensin II level was remarkably elevated immediately after MCAO, while electroacupuncture group exhibited significantly lower levels at 1 to 3 h and the value was significantly increased thereafter. The enhanced expression of AT1R was partially inhibited by electroacupuncture, while increased AT2R level was further induced. Electroacupuncture stimulation attenuated and postponed the upregulated-expressions of Gq and CaM these upregulations. ELISA results showed sharply increased expressions of DAG and IP3, which were remarkably neutralized by electroacupuncture. MCAO induced significant increases in expression of Angiotensin II and its receptor-mediated signal pathway. These enhanced expressions were significantly attenuated by electroacupuncture intervention, followed by reduced vasoconstriction and improved blood supply in ischemic region, and ultimately conferred beneficial effects on cerebral ischemia.

  6. The Angiotensin II Type 1 Receptor Antagonist Losartan Affects NHE1-Dependent Melanoma Cell Behavior

    Directory of Open Access Journals (Sweden)

    Daniel Navin Olschewski

    2018-03-01

    Full Text Available Background/Aims: The peptide hormone angiotensin II (ATII plays a prominent role in regulating vasoconstriction and blood pressure. Its primary target is the angiotensin II receptor type 1 (AT1, the stimulation of which induces an increase in cytosolic [Ca2+] and calmodulin activation. Ca2+-bound (activated calmodulin stimulates the activity of the Na+/ H+ exchanger isoform 1 (NHE1; and increased NHE1 activity is known to promote melanoma cell motility. The competitive AT1 receptor inhibitor losartan is often used to lower blood pressure in hypertensive patients. Since AT1 mediates ATII-stimulated NHE1 activity, we set out to investigate whether ATII and losartan have an impact on NHE1-dependent behavior of human melanoma (MV3 cells. Methods: ATII receptor expression was verified by PCR, F-actin was visualized using fluorescently labeled phalloidin, and cytosolic [Ca2+] and pH were determined ratiometrically using Fura-2 and BCECF, respectively. MV3 cell behavior was analyzed using migration, adhesion, invasion and proliferation assays. Results: MV3 cells express both AT1 and the angiotensin II receptor type 2 (AT2. Stimulation of MV3 cells with ATII increased NHE1 activity which could be counteracted by both losartan and the Ca2+/ calmodulin inhibitor ophiobolin-A. ATII stimulation induced a decrease in MV3 cell migration and a more spherical cell morphology accompanied by an increase in the density of F-actin. Independently of the presence of ATII, both NHE1 and migratory activity were reduced when AT1 was blocked by losartan. On the other hand, losartan clearly increased cell adhesion to, and the invasion of, a collagen type I substrate. The AT2 inhibitor PD123319 did not affect NHE1 activity, proliferation and migration, but increased adhesion and invasion. Conclusion: Losartan inhibits NHE1 activity and the migration of human melanoma cells. At the same time, losartan promotes MV3 cell adhesion and invasion. The therapeutic use of AT1

  7. The Impact of Age-Related Dysregulation of the Angiotensin System on Mitochondrial Redox Balance

    Directory of Open Access Journals (Sweden)

    Ramya eVajapey

    2014-11-01

    Full Text Available Aging is associated with the accumulation of various deleterious changes in cells. According to the free radical and mitochondrial theory of aging, mitochondria initiate most of the deleterious changes in aging and govern life span. The failure of mitochondrial reduction-oxidation (redox homeostasis and the formation of excessive free radicals are tightly linked to dysregulation in the Renin Angiotensin System (RAS. A main rate-controlling step in RAS is renin, an enzyme that hydrolyzes angiotensinogen to generate angiotensin I. Angiotensin I is further converted to Angiotensin II (Ang II by angiotensin-converting enzyme (ACE. Ang II binds with equal affinity to two main angiotensin receptors—type 1 (AT1R and type 2 (AT2R. The binding of Ang II to AT1R activates NADPH oxidase, which leads to increased generation of cytoplasmic reactive oxygen species (ROS. This Ang II-AT1R–NADPH-ROS signal triggers the opening of mitochondrial KATP channels and mitochondrial ROS production in a positive feedback loop. Furthermore, RAS has been implicated in the decrease of many of ROS scavenging enzymes, thereby leading to detrimental levels of free radicals in the cell.AT2R is less understood, but evidence supports an anti-oxidative and mitochondria-protective function for AT2R. The overlap between age related changes in RAS and mitochondria, and the consequences of this overlap on age-related diseases are quite complex. RAS dysregulation has been implicated in many pathological conditions due to its contribution to mitochondrial dysfunction. Decreased age-related, renal and cardiac mitochondrial dysfunction was seen in patients treated with angiotensin receptor blockers. The aim of this review is to: (a report the most recent information elucidating the role of RAS in mitochondrial redox hemostasis and (b discuss the effect of age-related activation of RAS on generation of free radicals.

  8. The Sacubitril/Valsartan, a First-in-Class, Angiotensin Receptor Neprilysin Inhibitor (ARNI): Potential Uses in Hypertension, Heart Failure, and Beyond.

    Science.gov (United States)

    Kario, Kazuomi

    2018-01-27

    Sacubitril/valsartan (LCZ696) is a first-in-class, novel-acting, angiotensin receptor neprilysin inhibitor (ARNI) that provides inhibition of neprilysin and the angiotensin (AT 1 ) receptor. A recent clinical trial PRARDIGM-HF demonstrated that this drug is superior to angiotensin-converting enzyme (ACE) inhibitors for improving the prognosis in the patients with heart failure, and this has resulted in the drug being included in clinical practice guidelines for the management of heart failure with reduced ejection fraction (EF). In addition, sacubitril/valsartan has been developed for the management of hypertension, because it has unique anti-aging properties. However, the clinical evidence of mechanism has not been well validated. A recent mechanistic study PARAMETER demonstrated that sacubitril/valsartan (LCZ696) is superior to angiotensin receptor blocker (ARB) monotherapy for reducing central aortic systolic pressure (primary endpoint) as well as for central aortic pulse pressure (secondary endpoint) and nocturnal BP preferentially. Considering these results, sacubitril/valsartan may be an attractive therapeutic agent to treat the elderly with age-related hypertension phenotypes, such as drug-uncontrolled (resistant) hypertension characterized as systolic (central) hypertension (structural hypertension) and/or nocturnal hypertension (salt-sensitive hypertension). These are the high-risk hypertension phenotypes which are prone to develop heart failure with preserved EF and chronic kidney disease. Sacubitril/valsartan may be effective to suppress the age-related continuum from hypertension to heart failure, and it could be clinically useful not only for secondary prevention, but also as primary prevention of heart failure in uncontrolled elderly hypertensive patients.

  9. The angiotensin type 2 receptor agonist Compound 21 elicits cerebroprotection in endothelin-1 induced ischemic stroke

    DEFF Research Database (Denmark)

    Joseph, Jason P; Mecca, Adam P; Regenhardt, Robert W

    2014-01-01

    Evidence indicates that angiotensin II type 2 receptors (AT2R) exert cerebroprotective actions during stroke. A selective non-peptide AT2R agonist, Compound 21 (C21), has been shown to exert beneficial effects in models of cardiac and renal disease, as well as hemorrhagic stroke. Here, we hypothe...

  10. Effects of angiotensin II type 1 receptor blocker on bones in mice with type 1 diabetes induced by streptozotocin.

    Science.gov (United States)

    Zhang, Yan; Diao, Teng-Yue; Gu, Sa-Sa; Wu, Shu-Yan; Gebru, Yoseph A; Chen, Xi; Wang, Jing-Yu; Ran, Shu; Wong, Man-Sau

    2014-09-01

    This study was performed to address the pathological roles of the skeletal renin-angiotensin system (RAS) in type 1 diabetes-induced osteoporosis and the effects of the angiotensin II type 1 receptor blocker losartan on bones in diabetic mice. Bone histomorphology was detected by H&E staining, Safranin O staining and X-ray radiography. Micro-CT was performed for the analysis of bone parameters. Gene and protein expression were determined by RT-PCR and immunoblotting. Type 1 diabetic mice displayed osteopenia phenotype, and losartan treatment had no osteoprotective effects on diabetic mice as shown by the reduction of bone mineral density and microarchitectural parameters at the proximal metaphysis of the tibia. The mRNA expression of AGT, renin receptor and ACE, and protein expression of renin and AT1R were markedly up-regulated in the bones of vehicle-treated diabetic mice compared to those of non-diabetic mice. The treatment with losartan further significantly increased the expression of AGT, renin, angiotensin II and AT1R, and reduced the expression of AT2R receptor as compared to those of diabetic mice. Local bone RAS functionally played a role in the development of type 1 diabetic osteoporosis, and losartan had no bone-sparing function in diabetes mice because of enhance skeletal RAS activity. © The Author(s) 2013.

  11. Angiotensin II Type 2 Receptor and Receptor Mas Are Colocalized and Functionally Interdependent in Obese Zucker Rat Kidney

    DEFF Research Database (Denmark)

    Patel, Sanket N; Ali, Quaisar; Samuel, Preethi

    2017-01-01

    The actions of angiotensin II type 2 receptor (AT2R) and the receptor Mas (MasR) are complex but show similar pronatriuretic function; particularly, AT2R expression and natriuretic function are enhanced in obese/diabetic rat kidney. In light of some reports suggesting a potential positive...... interaction between these receptors, we tested hypothesis that renal AT2R and MasR physically interact and are interdependent to stimulate cell signaling and promote natriuresis in obese rats. We found that infusion of AT2R agonist C21 in obese Zucker rats (OZR) increased urine flow and urinary Na excretion...... coimmunoprecipitated with MasR in cortical homogenate of OZR. Immunoblotting of cortical homogenate cross-linked with zero-length oxidative (sulfhydryl groups) cross-linker cupric-phenanthroline revealed a shift of AT2R and MasR bands upward with overlapping migration for their complexes which were sensitive...

  12. Anthology of the renin-angiotensin system: a one hundred reference approach to angiotensin II antagonists.

    Science.gov (United States)

    Ménard, J

    1993-04-01

    To provide a historical overview of the renin-angiotensin system as a guide to the introduction of a new therapeutic pathway, non-peptide inhibition of a angiotensin II. One hundred references were selected as a personal preference, for their originality or for their potential impact on medicine. This review raises the following questions for future research. (1) Will the long-term cardiovascular effects of angiotensin converting enzyme (ACE) inhibition, angiotensin II antagonism and renin inhibition be similar or not, and dependent or independent of blood pressure levels? (2) What are the local-regional interactions between vasoconstrictor and vasodilator systems, and does the renin-angiotensin system synchronize these regional hemodynamic regulatory mechanisms? (3) If hypertension is the result of an interaction between genetic and environmental factors, do proteins secreted through constitutive pathways contribute to the genetic abnormality (prorenin, angiotensinogen, ACE) while regulated secretion (renin) and other regulatory mechanisms (angiotensin II receptors) provide biological support for the environmental effects?

  13. A combined role of calcium channel blockers and angiotensin receptor blockers in stroke prevention

    Directory of Open Access Journals (Sweden)

    Ji-Guang Wang

    2009-07-01

    Full Text Available Ji-Guang WangCentre for Epidemiological Studies and Clinical Trials, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, ChinaAbstract: Stroke is a leading cause of death and disability worldwide. The importance of lowering blood pressure for reducing the risk of stroke is well established. However, not all the benefits of antihypertensive treatments in stroke can be accounted for by reductions in BP and there may be differences between antihypertensive classes as to which provides optimal protection. Dihydropyridine calcium channel blockers, such as amlodipine, and angiotensin receptor blockers, such as valsartan, represent the two antihypertensive drug classes with the strongest supportive data for the prevention of stroke. Therefore, when combination therapy is required, a combination of these two antihypertensive classes represents a logical approach.Keywords: stroke, angiotensin, calcium channel, cerebrovascular, hypertension, blood pressure

  14. Effects of angiotensin converting enzyme inhibitor and angiotensin II antagonist receptor on neointima hyperplasia after vascular balloon injury

    International Nuclear Information System (INIS)

    Wang Yeling; Zhao Lihua

    2004-01-01

    Objective: To study the effects of angiotensin converting enzyme inhibitor (captopril) and angiotensin II antagonist receptor (valsartan) on neointima hyperplasia after vascular balloon injury. Methods: Thirty-six rabbit models were randomly divided into three groups: injuried group, captopril group and valsartan group. Captopril (2 mg·kg -1 ·d -1 po) and valsartan (10 mg·kg -1 ·d -1 po) were given to twelve rabbits respectively from 1 day before the right carotidarteries were injuried by 2.0 mm ballon cathether to 14 days after injury in captopil group and valsartan group. The medicine was not administered in the injuried group. The tissue plasminogen activator (tPA), plaminogen activor inhibitor-1 (PAI-1) antigen level and plasma endothelin (ET) levels were measured before injury, and 7, 14 days after vascular injury. The pathomorphoiogical examination were carried out 14 days after angioplasty. Results: The levels of plasma PAI-1 and ET in captopril group and valsartan group were significantly lower than those in the injuried group (P<0.05). The intimal thickness and extent of lumen stenosis in captopril and valsartan groups were significantly lower than those in the injuried group (P<0.05). Conclusion: Captopril and valsartan can inhibit neointima hyperplasia after vascular ballon injury. (authors)

  15. Angiotensin II Type 2 Receptor Agonist Experts Sustained Neuroprotective Effects In Aged Rats

    DEFF Research Database (Denmark)

    Sumners, Colin; Isenberg, Jacob; Harmel, Allison

    2016-01-01

    OBJECTIVE: The renin angiotensin system is a promising target for stroke neuroprotection and therapy through activation of angiotensin type II receptors (AT2R). The selective non-peptide AT2R agonist, Compound 21 (C21), has been shown to exhibit neuroprotection and improve stroke outcomes...... in preclinical studies, effects that likely involve neurotropic actions. However, these beneficial actions of C21 have not been demonstrated to occur beyond 1 week post stroke. The objective of this study was to determine if systemic administration of C21 would exert sustained neuroprotective effects in aged...... min), 24 h, and 48 h after stroke. Infarct size was assessed by magnetic resonance imaging at 21 days post MCAO. Animals received blinded neurological exams at 4 h, 24 h, 72 h, 7d, 14d, and 21d post-MCAO. RESULTS: Systemic treatment with C21 after stroke significantly improved neurological function...

  16. Radioimmunoassay - renin - angiotensin. Principles of radioimmunoassay and their application in measuring renin and angiotensin

    Energy Technology Data Exchange (ETDEWEB)

    Krause, D K; Hummerich, W; Poulsen, K [eds.

    1978-01-01

    Typical pitfalls such as impurity of 'standard', tracer damage, crossreactivity of antiserum, unspecific binding of protecting proteins, blank effects with negative results, charcoal stripping, invisible coprecipitate or uncertainty in the analysis of the calibration curve (graph, logit-log, polynormal or spline function) can occur in any type of radioimmunoassay; they are detailed in the general part of this book. The special position occupied by radioimmunological quantification of parameters of the renin-angiotensin system creates additional, even more serious problems. While the radioimmunological determination of the decapeptide angiotensin I no longer causes major obstacles, measurement of the biologically active octapeptide angiotensin II is still only possible in a few centers. The (indirect) determination of plasma renin is characterized by a situation where the enzyme renin may be clearly defined in theory as a specific 10-11-leucine-leucine-endopeptidase cleaving only a decapeptide, but the actual renin assay, however, measures various forms of renin and other angiotensin-forming (or angiotensin-destroying) enzymes at the same time.

  17. Sodium butyrate suppresses angiotensin II-induced hypertension by inhibition of renal (pro)renin receptor and intrarenal renin-angiotensin system.

    Science.gov (United States)

    Wang, Lei; Zhu, Qing; Lu, Aihua; Liu, Xiaofen; Zhang, Linlin; Xu, Chuanming; Liu, Xiyang; Li, Haobo; Yang, Tianxin

    2017-09-01

    Butyrate, a short-chain fatty acid, is the end product of the fermentation of complex carbohydrates by the gut microbiota. Recently, sodium butyrate (NaBu) has been found to play a protective role in a number of chronic diseases. However, it is still unclear whether NaBu has a therapeutic potential in hypertension. The present study was aimed to investigate the role of NaBu in angiotensin II (Ang II)-induced hypertension and to further explore the underlying mechanism. Ang II was infused into uninephrectomized Sprague-Dawley rats with or without intramedullary infusion of NaBu for 14 days. Mean arterial blood pressure was recorded by the telemetry system. Renal tissues, serum samples, and 24-h urine samples were collected to examine renal injury and the regulation of the (pro)renin receptor (PRR) and renin. Intramedullary infusion of NaBu in Sprague-Dawley rats lowered the Ang II-induced mean arterial pressure from 129 ± 6 mmHg to 108 ± 4 mmHg (P renal injury, including urinary albumin, glomerulosclerosis, and renal fibrosis, as well as the expression of inflammatory mediators tumor necrosis factor α, interleukin 6. The renal expression of PRR, angiotensinogen, angiotensin I-converting enzyme and the urinary excretion of soluble PRR, renin, and angiotensinogen were all increased by Ang II infusion but decreased by NaBu treatment. In cultured innermedullary collecting duct cells, NaBu treatment attenuated Ang II-induced expression of PRR and renin. These results demonstrate that NaBu exerts an antihypertensive action, likely by suppressing the PRR-mediated intrarenal renin-angiotensin system.

  18. Exercise training modulates the hepatic renin-angiotensin system in fructose-fed rats.

    Science.gov (United States)

    Frantz, Eliete Dalla Corte; Medeiros, Renata Frauches; Giori, Isabele Gomes; Lima, Juliana Bittencourt Silveira; Bento-Bernardes, Thais; Gaique, Thaiane Gadioli; Fernandes-Santos, Caroline; Fernandes, Tiago; Oliveira, Edilamar Menezes; Vieira, Carla Paulo; Conte-Junior, Carlos Adam; Oliveira, Karen Jesus; Nobrega, Antonio Claudio Lucas

    2017-09-01

    What is the central question of this study? What are the effects of exercise training on the hepatic renin-angiotensin system and their contribution to damage resulting from fructose overload in rats? What is the main finding and its importance? Exercise training attenuated the deleterious actions of the angiotensin-converting enzyme/angiotensin II/angiotensin II type 1 receptor axis and increased expression of the counter-regulatory (angiotensin-converting enzyme 2/angiotensin (1-7)/Mas receptor) axis in the liver. Therefore, our study provides evidence that exercise training modulates the hepatic renin-angiotensin system, which contributes to reducing the progression of metabolic dysfunction and non-alcoholic fatty liver disease in fructose-fed rats. The renin-angiotensin system (RAS) has been implicated in the development of metabolic syndrome. We investigated whether the hepatic RAS is modulated by exercise training and whether this modulation improves the deleterious effects of fructose overload in rats. Male Wistar rats were divided into (n = 8 each) control (CT), exercise control (CT-Ex), high-fructose (HFr) and exercise high-fructose (HFr-Ex) groups. Fructose-drinking rats received d-fructose (100 g l -1 ). After 2 weeks, CT-Ex and HFr-Ex rats were assigned to a treadmill training protocol at moderate intensity for 8 weeks (60 min day -1 , 4 days per week). We assessed body mass, glucose and lipid metabolism, hepatic histopathology, angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) activity, the angiotensin concentration and the expression profile of proteins affecting the hepatic RAS, gluconeogenesis and inflammation. Neither fructose overload nor exercise training influenced body mass gain and serum ACE and ACE2 activity. The HFr group showed hyperinsulinaemia, but exercise training normalized this parameter. Exercise training was effective in preventing hepatic steatosis and in preventing triacylglycerol and

  19. Angiotensin II induces apoptosis in intestinal epithelial cells through the AT2 receptor, GATA-6 and the Bax pathway

    International Nuclear Information System (INIS)

    Sun, Lihua; Wang, Wensheng; Xiao, Weidong; Liang, Hongyin; Yang, Yang; Yang, Hua

    2012-01-01

    Highlights: ► Ang II-induced apoptosis in intestinal epithelial cell through AT2 receptor. ► The apoptosis process involves in the Bax/Bcl-2 intrinsic pathway. ► GATA-6 short hairpin RNA reduced Bax expression, but not Bcl-2. ► GATA-6 may play a critical role in apoptosis in response to the Ang II challenge. -- Abstract: Angiotensin II (Ang II) has been shown to play an important role in cell apoptosis. However, the mechanisms of Ang-II-induced apoptosis in intestinal epithelial cells are not fully understood. GATA-6 is a zinc finger transcription factor expressed in the colorectal epithelium, which directs cell proliferation, differentiation and apoptosis. In the present study we investigated the underlying mechanism of which GATA-6 affects Ang-II induced apoptosis in intestinal epithelial cells. The in vitro intestinal epithelial cell apoptosis model was established by co-culturing Caco-2 cells with Ang II. Pretreatment with Angiotensin type 2 (AT2) receptor antagonist, PD123319, significantly reduced the expression of Bax and prevented the Caco-2 cells apoptosis induced by Ang II. In addition, Ang II up-regulated the expression of GATA-6. Interestingly, GATA-6 short hairpin RNA prevented Ang II-induced intestinal epithelial cells apoptosis and reduced the expression of Bax, but not Bcl-2. Taken together, the present study suggests that Angiotensin II promotes apoptosis in intestinal epithelial cells through GATA-6 and the Bax pathway in an AT2 receptor-dependent manner.

  20. The angiotensin-receptor blocker candesartan for treatment of acute stroke (SCAST): a randomised, placebo-controlled, double-blind trial

    DEFF Research Database (Denmark)

    Sandset, Else Charlotte; Bath, Philip M W; Boysen, Gudrun

    2011-01-01

    BACKGROUND: Raised blood pressure is common in acute stroke, and is associated with an increased risk of poor outcomes. We aimed to examine whether careful blood-pressure lowering treatment with the angiotensin-receptor blocker candesartan is beneficial in patients with acute stroke and raised bl...

  1. No significant effect of angiotensin II receptor blockade on intermediate cardiovascular end points in hemodialysis patients

    DEFF Research Database (Denmark)

    Peters, Christian Daugaard; Kjaergaard, Krista D; Jensen, Jens D

    2014-01-01

    Agents blocking the renin-angiotensin-aldosterone system are frequently used in patients with end-stage renal disease, but whether they exert beneficial cardiovascular effects is unclear. Here the long-term effects of the angiotensin II receptor blocker, irbesartan, were studied in hemodialysis......, and residual renal function. Brachial blood pressure decreased significantly in both groups, but there was no significant difference between placebo and irbesartan. Use of additional antihypertensive medication, ultrafiltration volume, and dialysis dosage were not different. Intermediate cardiovascular end...... points such as central aortic blood pressure, carotid-femoral pulse wave velocity, left ventricular mass index, N-terminal brain natriuretic prohormone, heart rate variability, and plasma catecholamines were not significantly affected by irbesartan treatment. Changes in systolic blood pressure during...

  2. Blockade of AT1 type receptors for angiotensin II prevents cardiac microvascular fibrosis induced by chronic stress in Sprague-Dawley rats.

    Science.gov (United States)

    Firoozmand, Lília Taddeo; Sanches, Andrea; Damaceno-Rodrigues, Nilsa Regina; Perez, Juliana Dinéia; Aragão, Danielle Sanches; Rosa, Rodolfo Mattar; Marcondes, Fernanda Klein; Casarini, Dulce Elena; Caldini, Elia Garcia; Cunha, Tatiana Sousa

    2018-04-20

    To test the effects of chronic-stress on the cardiovascular system, the model of chronic mild unpredictable stress (CMS) has been widely used. The CMS protocol consists of the random, intermittent, and unpredictable exposure of laboratory animals to a variety of stressors, during 3 consecutive weeks. In this study, we tested the hypothesis that exposure to the CMS protocol leads to left ventricle microcirculatory remodeling that can be attenuated by angiotensin II receptor blockade. Male Sprague-Dawley rats were randomly assigned into four groups: Control, Stress, Control + losartan, and Stress + losartan (N = 6, each group, losartan: 20 mg/kg/day). The rats were euthanized 15 days after CMS exposure, and blood samples and left ventricle were collected. Rats submitted to CMS presented increased glycemia, corticosterone, noradrenaline and adrenaline concentration, and losartan reduced the concentration of the circulating amines. Cardiac angiotensin II, measured by high-performance liquid chromatography (HPLC), was significantly increased in the CMS group, and losartan treatment reduced it, while angiotensin 1-7 was significantly higher in the CMS losartan-treated group as compared with CMS. Histological analysis, verified by transmission electron microscopy, showed that rats exposed to CMS presented increased perivascular collagen and losartan effectively prevented the development of this process. Hence, CMS induced a state of microvascular disease, with increased perivascular collagen deposition, that may be the trigger for further development of cardiovascular disease. In this case, CMS fibrosis is associated with increased production of catecholamines and with a disruption of renin-angiotensin system balance, which can be prevented by angiotensin II receptor blockade.

  3. Specific binding of atrial natriuretic factor in brain microvessels

    International Nuclear Information System (INIS)

    Chabrier, P.E.; Roubert, P.; Braquet, P.

    1987-01-01

    Cerebral capillaries constitute the blood-brain barrier. Studies of specific receptors (neurotransmitters or hormones) located on this structure can be performed by means of radioligand-binding techniques on isolated brain microvessels. The authors examined on pure bovine cerebral microvessel preparations the binding of atrial natriuretic factor (ANF), using 125 I-labeled ANF. Saturation and competition experiments demonstrated the presence of a single class of ANF-binding sites with high affinity and with a binding capacity of 58 fmol/mg of protein. The binding of 125 I-labeled ANF to brain microvessels is specific, reversible, and time dependent, as is shown by association-dissociation experiments. The demonstration of specific ANF-binding sites on brain microvessels supposes a physiological role of ANF on brain microvasculature. The coexistence of ANF and angiotensin II receptors on this cerebrovascular tissue suggests that the two circulating peptides may act as mutual antagonists in the regulation of brain microcirculation and/or blood-brain barrier function

  4. Renin-Angiotensin Inhibitors Decrease Recurrence after Transurethral Resection of Bladder Tumor in Patients with Nonmuscle Invasive Bladder Cancer.

    Science.gov (United States)

    Blute, Michael L; Rushmer, Timothy J; Shi, Fangfang; Fuller, Benjamin J; Abel, E Jason; Jarrard, David F; Downs, Tracy M

    2015-11-01

    Prior reports suggest that renin-angiotensin system inhibition may decrease nonmuscle invasive bladder cancer recurrence. We evaluated whether angiotensin converting enzyme inhibitor or angiotensin receptor blocker treatment at initial surgery was associated with decreased recurrence or progression in patients with nonmuscle invasive bladder cancer. Using an institutional bladder cancer database we identified 340 patients with data available on initial transurethral resection of bladder tumor. Progression was defined as an increase to stage T2. Cox proportional hazards models were used to evaluate associations with recurrence-free and progression-free survival. Median patient age was 69.6 years. During a median followup of 3 years (IQR 1.3-6.1) 200 patients (59%) had recurrence and 14 (4.1%) had stage progression. Of those patients 143 were receiving angiotensin converting enzyme inhibitor/angiotensin receptor blockers at the time of the first transurethral resection. On univariate analysis factors associated with improved recurrence-free survival included carcinoma in situ (p = 0.040), bacillus Calmette-Guérin therapy (p = 0.003) and angiotensin converting enzyme inhibitor/angiotensin receptor blocker therapy (p = 0.009). Multivariate analysis demonstrated that patients treated with bacillus Calmette-Guérin therapy (HR 0.68, 95% CI 0.47-0.87, p = 0.002) or angiotensin converting enzyme inhibitor/angiotensin receptor blocker therapy (HR 0.61, 95% CI 0.45-0.84, p = 0.005) were less likely to experience tumor recurrence. The 5-year recurrence-free survival rate was 45.6% for patients treated with angiotensin converting enzyme inhibitor/angiotensin receptor blockers and 28.1% in those not treated with angiotensin converting enzyme inhibitor/angiotensin receptor blockers (p = 0.009). Subgroup analysis was performed to evaluate nonmuscle invasive bladder cancer pathology (Ta, T1 and carcinoma in situ) in 85 patients on bacillus Calmette-Guérin therapy alone and in

  5. Angiotensin II up-regulates PAX2 oncogene expression and activity in prostate cancer via the angiotensin II type I receptor.

    Science.gov (United States)

    Bose, Sudeep K; Gibson, Willietta; Giri, Shailendra; Nath, Narender; Donald, Carlton D

    2009-09-01

    Paired homeobox 2 gene (PAX2) is a transcriptional regulator, aberrantly expressed in prostate cancer cells and its down-regulation promotes cell death in these cells. The molecular mechanisms of tumor progression by PAX2 over-expression are still unclear. However, it has been reported that angiotensin-II (A-II) induces cell growth in prostate cancer via A-II type 1 receptor (AT1R) and is mediated by the phosphorylation of mitogen activated protein kinase (MAPK) as well as signal transducer and activator of transcription 3 (STAT3). Here we have demonstrated that A-II up-regulates PAX2 expression in prostate epithelial cells and prostate cancer cell lines resulting in increased cell growth. Furthermore, AT1R receptor antagonist losartan was shown to inhibit A-II induced PAX2 expression in prostate cancer. Moreover, analysis using pharmacological inhibitors against MEK1/2, ERK1/2, JAK-II, and phospho-STAT3 demonstrated that AT1R-mediated stimulatory effect of A-II on PAX2 expression was regulated in part by the phosphorylation of ERK1/2, JAK II, and STAT3 pathways. In addition, we have showed that down-regulation of PAX2 by an AT1R antagonist as well as JAK-II and STAT3 inhibitors suppress prostate cancer cell growth. Collectively, these findings show for the first time that the renin-angiotensin system (RAS) may promote prostate tumorigenesis via up-regulation of PAX2 expression. Therefore, PAX2 may be a novel therapeutic target for the treatment of carcinomas such as prostate cancer via the down-regulation of its expression by targeting the AT1R signaling pathways.

  6. Mu opioid receptor binding sites in human brain

    International Nuclear Information System (INIS)

    Pilapil, C.; Welner, S.; Magnan, J.; Zamir, N.; Quirion, R.

    1986-01-01

    Our experiments focused on the examination of the distribution of mu opioid receptor binding sites in normal human brain using the highly selective ligand [ 3 H]DAGO, in both membrane binding assay and in vitro receptor autoradiography. Mu opioid binding sites are very discretely distributed in human brain with high densities of sites found in the posterior amygdala, caudate, putamen, hypothalamus and certain cortical areas. Moreover the autoradiographic distribution of [ 3 H]DAGO binding sites clearly reveals the discrete lamination (layers I and III-IV) of mu sites in cortical areas

  7. Vascular Type 1A Angiotensin II Receptors Control BP by Regulating Renal Blood Flow and Urinary Sodium Excretion.

    Science.gov (United States)

    Sparks, Matthew A; Stegbauer, Johannes; Chen, Daian; Gomez, Jose A; Griffiths, Robert C; Azad, Hooman A; Herrera, Marcela; Gurley, Susan B; Coffman, Thomas M

    2015-12-01

    Inappropriate activation of the type 1A angiotensin (AT1A) receptor contributes to the pathogenesis of hypertension and its associated complications. To define the role for actions of vascular AT1A receptors in BP regulation and hypertension pathogenesis, we generated mice with cell-specific deletion of AT1A receptors in smooth muscle cells (SMKO mice) using Loxp technology and Cre transgenes with robust expression in both conductance and resistance arteries. We found that elimination of AT1A receptors from vascular smooth muscle cells (VSMCs) caused a modest (approximately 7 mmHg) yet significant reduction in baseline BP and exaggerated sodium sensitivity in mice. Additionally, the severity of angiotensin II (Ang II)-dependent hypertension was dramatically attenuated in SMKO mice, and this protection against hypertension was associated with enhanced urinary excretion of sodium. Despite the lower BP, acute vasoconstrictor responses to Ang II in the systemic vasculature were largely preserved (approximately 80% of control levels) in SMKO mice because of exaggerated activity of the sympathetic nervous system rather than residual actions of AT1B receptors. In contrast, Ang II-dependent responses in the renal circulation were almost completely eliminated in SMKO mice (approximately 5%-10% of control levels). These findings suggest that direct actions of AT1A receptors in VSMCs are essential for regulation of renal blood flow by Ang II and highlight the capacity of Ang II-dependent vascular responses in the kidney to effect natriuresis and BP control. Copyright © 2015 by the American Society of Nephrology.

  8. Novel Drosophila receptor that binds multiple growth factors

    International Nuclear Information System (INIS)

    Rosner, M.R.; Thompson, K.L.; Garcia, V.; Decker, S.J.

    1986-01-01

    The authors have recently reported the identification of a novel growth factor receptor from Drosophila cell cultures that has dual binding specificity for both insulin and epidermal growth factor (EGF). This 100 kDa protein is also antigenically related to the cytoplasmic region of the mammalian EGF receptor-tyrosine kinase. They now report that this protein binds to mammalian nerve growth factor and human transforming growth factor alpha as well as insulin and EGF with apparent dissociation constants ranging from 10 -6 to 10 -8 M. The 100 kDa protein can be affinity-labeled with these 125 I-labeled growth factors after immunoprecipitation with anti-EGF receptor antiserum. These four growth factors appear to share a common binding site, as evidenced by their ability to block affinity labelling by 125 I-insulin. No significant binding to the 100 kDa protein was observed with platelet-derived growth factor, transforming growth factor beta, or glucagon. The 100 kDa Drosophila protein has a unique ligand-binding spectrum with no direct counterpart in mammalian cells and may represent an evolutionary precursor of the mammalian receptors for these growth factors

  9. Progress on the application of ligand receptor binding assays in radiopharmaceuticals

    International Nuclear Information System (INIS)

    Zhou Xue; Qian Jinping; Kong Aiying; Zhu Lin

    2010-01-01

    Receptor binding assay is an important drug screening method, which can quickly and inexpensively study the interactions between the targeted receptor and the potential ligands in vitro and provide the information of the relative binding affinity of ligand-receptor. The imaging of many radiopharmaceuticals is based on highly selective radioligand-receptor binding. The technique plays an important role in the design and screening of receptor-targeting radiopharmaceuticals. (authors)

  10. The influence of certain molecular descriptors of fecal elimination of angiotensin II receptor antagonists

    Directory of Open Access Journals (Sweden)

    Trbojević-Stanković Jasna B.

    2015-01-01

    Full Text Available Angiotensin II receptor antagonists (ARBs modulate the function of the renin-angiotensin-aldosterone system and are commonly prescribed antihypertensive drugs, especially in patients with renal failure. In this study, the relationship between several molecular properties of seven ARBs (candesartan, eprosartan, irbesartan, losartan, olmesartan, telmisartan, valsartan and their fecal elimination data obtained from the literature were investigated. The ARB molecular descriptors were calculated using three software packages. Simple linear regression analysis showed the best 2 correlation between fecal elimination data and lipophilicity descriptor, ClogP values (R2 = 0.725. Multiple linear regression was applied to examine the correlation of ARBs’ fecal elimination data with their lipophilicity and one additional, calculated descriptor. The best correlation (R2 = 0.909 with an acceptable probability value, P <0.05 was established between the ARB fecal elimination data and their lipophilicity and aqueous solubility data. Applying computed molecular descriptors for evaluating drug elimination is of great importance in drug research.

  11. [3H]cytisine binding to nicotinic cholinergic receptors in brain

    International Nuclear Information System (INIS)

    Pabreza, L.A.; Dhawan, S.; Kellar, K.J.

    1991-01-01

    Cytisine, a ganglionic agonist, competes with high affinity for brain nicotinic cholinergic receptors labeled by any of several nicotinic 3 H-agonist ligands. Here we have examined the binding of [ 3 H]cytisine in rat brain homogenates. [ 3 H]Cytisine binds with high affinity (Kd less than 1 nM), and specific binding represented 60-90% of total binding at all concentrations examined up to 15 nM. The nicotinic cholinergic agonists nicotine, acetylcholine, and carbachol compete with high affinity for [ 3 H]cytisine binding sites, whereas among nicotinic receptor antagonists only dihydro-beta-erythroidine competes with high affinity (in the nanomolar range). Comparison of binding in several brain regions showed that [ 3 H]cytisine binding is higher in the thalamus, striatum, and cortex than in the hippocampus, cerebellum, or hypothalamus. The pharmacology and brain regional distribution of [ 3 H]cytisine binding sites are those predicted for neuronal nicotinic receptor agonist recognition sites. The high affinity and low nonspecific binding of [ 3 H]cytisine should make it a very useful ligand for studying neuronal nicotinic receptors

  12. Synthesis of isotopically labelled angiotensin II receptor antagonist GR138950X

    International Nuclear Information System (INIS)

    Carr, R.M.; Cable, K.M.; Newman, J.J.; Sutherland, D.R.

    1996-01-01

    Syntheses of [ 13 C] and [ 14 C]-labelled versions of angiotensin II receptor antagonist GR138950X, labelled in the imidazole carboxamide residue, are described. These involved preparation of an iodoimidazole substrate by a novel iododecarboxylation procedure, followed by cyanation with a mixture of carbon-labelled potassium cyanide and copper (l) iodide in DMF at high temperature. The preparation of a mass-labelled (M+5) version of GR138950X is also described. This involved the synthesis of an [ 13 C 3 , 15 N 2 ]-labelled imidazole from a 1,2,3-tricarbonyl compound, [ 13 C 3 ]propionaldehyde and [ 15 N]ammonia. The labelled imidazole was further elaborated into multiply-labelled GR138950X. (Author)

  13. Angiotensin and bradykinin interactions with phospholipids

    International Nuclear Information System (INIS)

    Elliott, M.E.; Goodfriend, T.L.

    1979-01-01

    Reversible interactions were demonstrated between some phospholipids and some polypeptides related to angiotensin and bradykinin. The extent of the interaction was dependent on the structures of the lipid and peptide. The naturally occurring compounds that interacted most avidly were cardiolipin and (des-Asp 1 )-angiotensins. The apparent dissociation constant of this complex in chloroform was 10 -5 M. The complex contained more than one cardiolipin molecule/molecule of peptide. Kinins interacted most strongly with lecithin. The phospholipids altered the chromatographic behaviour of radioiodinated derivatives of the polypeptides, and solubilized radioactive and unlabeled polypeptides in chloroform. In aqueous media, cardiolipin suspensions preferentially bound (des-Asp 1 )-angiotensin II, and inhibited its binding by antibody. The interactions were sensitive to pH and cations in the aqueous phase, and were reversed by some reagents added to the organic phase. These interactions have direct implications for binding reactions of peptides in vitro, and may bear upon the actions of the hormones in vivo. (Auth.)

  14. Human myometrial adrenergic receptors: identification of the beta-adrenergic receptor by [3H]dihydroalprenolol binding

    International Nuclear Information System (INIS)

    Hayashida, D.N.; Leung, R.; Goldfien, A.; Roberts, J.M.

    1982-01-01

    The radioactive beta-adrenergic antagonist [ 3 H] dihydroalprenolol (DHA) binds to particulate preparations of human myometrium in a manner compatible with binding to the beta-adrenergic receptor. The binding of DHA is rapid (attaining equilibrium in 12 minutes), readily reversible (half time = 16 minutes), high affinity (K/sub D/ = 0.50 nM), low capacity (Bmax = 70 fmoles/mg of protein), and stereoselective ([-]-propranolol is 100 times as potent as [+] -propranolol in inhibiting DHA binding). Adrenergic agonists competed for DHA binding sites in a manner compatible with beta-adrenergic interactions and mirrored β 2 pharmacologic potencies: isoproterenol > epinephrine >> norepinephrine. Studies in which zinterol, a β 2 -adrenergic agonist, competed for DHA binding sites in human myometrial particulate indicated that at least 87% of the beta-adrenergic receptors present are β 2 -adrenergic receptors. Binding of DHA to human myometrial beta-adrenergic receptors provides a tool which may be used in the examination of gonadal hormonal modification of adrenergic response in human uterus as well as in the analysis of beta-adrenergic agents as potentially useful tocolytic agents

  15. Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to "Biased Opioids"?

    Science.gov (United States)

    Root-Bernstein, Robert; Turke, Miah; Subhramanyam, Udaya K Tiruttani; Churchill, Beth; Labahn, Joerg

    2018-01-17

    Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

  16. Keeping pace with ACE: are ACE inhibitors and angiotensin II type 1 receptor antagonists potential doping agents?

    Science.gov (United States)

    Wang, Pei; Fedoruk, Matthew N; Rupert, Jim L

    2008-01-01

    In the decade since the angiotensin-converting enzyme (ACE) gene was first proposed to be a 'human gene for physical performance', there have been numerous studies examining the effects of ACE genotype on physical performance phenotypes such as aerobic capacity, muscle function, trainability, and athletic status. While the results are variable and sometimes inconsistent, and corroborating phenotypic data limited, carriers of the ACE 'insertion' allele (the presence of an alu repeat element in intron 16 of the gene) have been reported to have higher maximum oxygen uptake (VO2max), greater response to training, and increased muscle efficiency when compared with individuals carrying the 'deletion' allele (absence of the alu repeat). Furthermore, the insertion allele has been reported to be over-represented in elite athletes from a variety of populations representing a number of endurance sports. The mechanism by which the ACE insertion genotype could potentiate physical performance is unknown. The presence of the ACE insertion allele has been associated with lower ACE activity (ACEplasma) in number of studies, suggesting that individuals with an innate tendency to have lower ACE levels respond better to training and are at an advantage in endurance sporting events. This could be due to lower levels of angiotensin II (the vasoconstrictor converted to active form by ACE), higher levels of bradykinin (a vasodilator degraded by ACE) or some combination of the two phenotypes. Observations that individuals carrying the ACE insertion allele (and presumably lower ACEplasma) have an enhanced response to training or are over-represented amongst elite athletes raises the intriguing question: would individuals with artificially lowered ACEplasma have similar training or performance potential? As there are a number of drugs (i.e. ACE inhibitors and angiotensin II type 1 receptor antagonists [angiotensin receptor blockers--ARBs]) that have the ability to either reduce ACEplasma

  17. The renin–angiotensin system and diabetes: An update

    Directory of Open Access Journals (Sweden)

    Antônio Ribeiro-Oliveira Jr

    2008-08-01

    Full Text Available Antônio Ribeiro-Oliveira Jr1, Anelise Impeliziere Nogueira1, Regina Maria Pereira2, Walkiria Wingester Vilas Boas3, Robson Augusto Souza dos Santos4, Ana Cristina Simões e Silva51Laboratório de Endocrinologia, Departamento de Clínica Médica, 2Departamento de Ciências Biológicas, Centro Universitário de Belo Horizonte, UNIBH, Belo Horizonte, MG, Brazil; 3Hospital Life Center, Belo Horizonte, MG, Brazil; 4Laboratório de Hipertensão, Departamento de Fisiologia e Biofísica, Instituto de Ciências Biológicas, UFMG, Belo Horizonte, MG, Brazil; 5Departamento de Pediatria, Faculdade de Medicina, Universidade Federal de Minas Gerais (UFMG, Belo Horizonte, MG, BrazilAbstract: In the past few years the classical concept of the renin–angiotensin system (RAS has experienced substantial conceptual changes. The identification of the renin/prorenin receptor, the angiotensin-converting enzyme homologue ACE2 as an angiotensin peptide processing enzyme, Mas as a receptor for Ang-(1-7 and the possibility of signaling through ACE, have contributed to switch our understanding of the RAS from the classical limited-proteolysis linear cascade to a cascade with multiple mediators, multiple receptors, and multi-functional enzymes. In this review we will focus on the recent findings related to RAS and, in particular, on its role in diabetes by discussing possible interactions between RAS mediators, endothelium function, and insulin signaling transduction pathways as well as the putative role of ACE2-Ang-(1-7-Mas axis in disease pathogenesis.Keywords: renin–angiotensin system, diabetes, angiotensin II, angiotensin-(1-7, insulin, endothelium

  18. Angiotensin (1-7) ameliorates the structural and biochemical alterations of ovariectomy-induced osteoporosis in rats via activation of ACE-2/Mas receptor axis.

    Science.gov (United States)

    Abuohashish, Hatem M; Ahmed, Mohammed M; Sabry, Dina; Khattab, Mahmoud M; Al-Rejaie, Salim S

    2017-05-23

    The local and systemic renin angiotensin system (RAS) influences the skeletal system micro-structure and metabolism. Studies suggested angiotensin 1-7 (Ang(1-7)) as the beneficial RAS molecule via Mas receptor activation. This study examines the function of Ang(1-7) in bone micro-architecture and metabolism in an ovariectomized (OVX) rodent model of osteoporosis. OVX rats showed structural and bone metabolic degeneration in parallel with suppressed expressions of the angiotensin converting enzyme-2 (ACE-2)/Ang(1-7)/Mas components. The infusion of Ang(1-7) markedly alleviated the altered bone metabolism and significantly enhanced both trabecular (metaphyseal) and cortical (metaphyseal-diaphyseal) morphometry. Urinary and bones minerals were also improved in OVX rats by Ang(1-7). The infusion of the heptapeptide enhanced ACE-2/Mas receptor expressions, while down-regulated AngII, ACE, and AngII type-1 receptor (AT1R) in OVX animals. Moreover, Ang(1-7) markedly improved osteoprotegerin (OPG) and lowered receptor activator NF-κB ligand (RANKL) expressions. The defensive properties of Ang(1-7) on bone metabolism, structure and minerals were considerably eradicated after blockage of Mas receptor with A-779. Ang(1-7)-induced up-regulated ACE-2/Ang(1-7)/Mas cascade and OPG expressions were abolished and the expressions of ACE/AngII/AT1R and RANKL were provoked by A-779. These findings shows for the first time the novel valuable therapeutic role of Ang(1-7) on bone health and metabolism through the ACE-2/Mas cascade.

  19. A bioluminescence resonance energy transfer 2 (BRET2) assay for monitoring seven transmembrane receptor and insulin receptor crosstalk

    DEFF Research Database (Denmark)

    Sanni, Samra Joke; Kulahin, Nikolaj; Jorgensen, Rasmus

    2017-01-01

    The angiotensin AT1 receptor is a seven transmembrane (7TM) receptor, which mediates the regulation of blood pressure. Activation of angiotensin AT1 receptor may lead to impaired insulin signaling indicating crosstalk between angiotensin AT1 receptor and insulin receptor signaling pathways....... To elucidate the molecular mechanisms behind this crosstalk, we applied the BRET2 technique to monitor the effect of angiotensin II on the interaction between Rluc8 tagged insulin receptor and GFP2 tagged insulin receptor substrates 1, 4, 5 (IRS1, IRS4, IRS5) and Src homology 2 domain-containing protein (Shc......). We demonstrate that angiotensin II reduces the interaction between insulin receptor and IRS1 and IRS4, respectively, while the interaction with Shc is unaffected, and this effect is dependent on Gαq activation. Activation of other Gαq-coupled 7TM receptors led to a similar reduction in insulin...

  20. Central administration of angiotensin IV rapidly enhances novel object recognition among mice.

    Science.gov (United States)

    Paris, Jason J; Eans, Shainnel O; Mizrachi, Elisa; Reilley, Kate J; Ganno, Michelle L; McLaughlin, Jay P

    2013-07-01

    Angiotensin IV (Val(1)-Tyr(2)-Ile(3)-His(4)-Pro(5)-Phe(6)) has demonstrated potential cognitive-enhancing effects. The present investigation assessed and characterized: (1) dose-dependency of angiotensin IV's cognitive enhancement in a C57BL/6J mouse model of novel object recognition, (2) the time-course for these effects, (3) the identity of residues in the hexapeptide important to these effects and (4) the necessity of actions at angiotensin IV receptors for procognitive activity. Assessment of C57BL/6J mice in a novel object recognition task demonstrated that prior administration of angiotensin IV (0.1, 1.0, or 10.0, but not 0.01 nmol, i.c.v.) significantly enhanced novel object recognition in a dose-dependent manner. These effects were time dependent, with improved novel object recognition observed when angiotensin IV (0.1 nmol, i.c.v.) was administered 10 or 20, but not 30 min prior to the onset of the novel object recognition testing. An alanine scan of the angiotensin IV peptide revealed that replacement of the Val(1), Ile(3), His(4), or Phe(6) residues with Ala attenuated peptide-induced improvements in novel object recognition, whereas Tyr(2) or Pro(5) replacement did not significantly affect performance. Administration of the angiotensin IV receptor antagonist, divalinal-Ang IV (20 nmol, i.c.v.), reduced (but did not abolish) novel object recognition; however, this antagonist completely blocked the procognitive effects of angiotensin IV (0.1 nmol, i.c.v.) in this task. Rotorod testing demonstrated no locomotor effects with any angiotensin IV or divalinal-Ang IV dose tested. These data demonstrate that angiotensin IV produces a rapid enhancement of associative learning and memory performance in a mouse model that was dependent on the angiotensin IV receptor. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Sodium restriction potentiates the renoprotective effects of combined vitamin D receptor activation and angiotensin-converting enzyme inhibition in established proteinuric nephropathy.

    NARCIS (Netherlands)

    Mirkovic, K.; Frenay, A.S.; Born, J. van den; Goor, H van; Navis, G.; Borst, M.H. de; Bindels, R.J.M.; Hoenderop, J.G.J.; Hillebrands, J.L.

    2017-01-01

    BACKGROUND: Renin-angiotensin-aldosterone system (RAAS) blockade provides renoprotective effects in chronic kidney disease (CKD); yet progressive renal function loss remains common. Dietary sodium restriction potentiates the renoprotective effects of RAAS blockade. Vitamin D receptor activator

  2. Estrogen receptor-independent catechol estrogen binding activity: protein binding studies in wild-type, Estrogen receptor-alpha KO, and aromatase KO mice tissues.

    Science.gov (United States)

    Philips, Brian J; Ansell, Pete J; Newton, Leslie G; Harada, Nobuhiro; Honda, Shin-Ichiro; Ganjam, Venkataseshu K; Rottinghaus, George E; Welshons, Wade V; Lubahn, Dennis B

    2004-06-01

    Primary evidence for novel estrogen signaling pathways is based upon well-documented estrogenic responses not inhibited by estrogen receptor antagonists. In addition to 17beta-E2, the catechol estrogen 4-hydroxyestradiol (4OHE2) has been shown to elicit biological responses independent of classical estrogen receptors in estrogen receptor-alpha knockout (ERalphaKO) mice. Consequently, our research was designed to biochemically characterize the protein(s) that could be mediating the biological effects of catechol estrogens using enzymatically synthesized, radiolabeled 4-hydroxyestrone (4OHE1) and 4OHE2. Scatchard analyses identified a single class of high-affinity (K(d) approximately 1.6 nM), saturable cytosolic binding sites in several ERalphaKO estrogen-responsive tissues. Specific catechol estrogen binding was competitively inhibited by unlabeled catechol estrogens, but not by 17beta-E2 or the estrogen receptor antagonist ICI 182,780. Tissue distribution studies indicated significant binding differences both within and among various tissues in wild-type, ERalphaKO, and aromatase knockout female mice. Ligand metabolism experiments revealed extensive metabolism of labeled catechol estrogen, suggesting that catechol estrogen metabolites were responsible for the specific binding. Collectively, our data provide compelling evidence for the interaction of catechol estrogen metabolites with a novel binding protein that exhibits high affinity, specificity, and selective tissue distribution. The extensive biochemical characterization of this binding protein indicates that this protein may be a receptor, and thus may mediate ERalpha/beta-independent effects of catechol estrogens and their metabolites.

  3. Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor

    Energy Technology Data Exchange (ETDEWEB)

    Peng, Guiqing; Sun, Dawei; Rajashankar, Kanagalaghatta R.; Qian, Zhaohui; Holmes, Kathryn V.; Li, Fang (Cornell); (UMM-MED); (Colorado)

    2011-09-28

    Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same {beta}-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein-protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the {beta}-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.

  4. Edaravone inhibits pressure overload-induced cardiac fibrosis and dysfunction by reducing expression of angiotensin II AT1 receptor

    Directory of Open Access Journals (Sweden)

    Zhang WW

    2017-10-01

    Full Text Available Wei-Wei Zhang,1,2 Feng Bai,1 Jin Wang,1 Rong-Hua Zheng,1 Li-Wang Yang,1 Erskine A James,3 Zhi-Qing Zhao1,4 1Department of Physiology, Shanxi Medical University, 2Department of Anesthesiology, Shanxi Provincial People’s Hospital, Taiyuan, Shanxi, China; 3Department of Internal Medicine, Navicent Health, Macon, 4Department of Basic Biomedical Sciences, Mercer University School of Medicine, Savannah, GA, USA Abstract: Angiotensin II (Ang II is known to be involved in the progression of ventricular dysfunction and heart failure by eliciting cardiac fibrosis. The purpose of this study was to demonstrate whether treatment with an antioxidant compound, edaravone, reduces cardiac fibrosis and improves ventricular function by inhibiting Ang II AT1 receptor. The study was conducted in a rat model of transverse aortic constriction (TAC. In control, rats were subjected to 8 weeks of TAC. In treated rats, edaravone (10 mg/kg/day or Ang II AT1 receptor blocker, telmisartan (10 mg/kg/day was administered by intraperitoneal injection or gastric gavage, respectively, during TAC. Relative to the animals with TAC, edaravone reduced myocardial malonaldehyde level and increased superoxide dismutase activity. Protein level of the AT1 receptor was reduced and the AT2 receptor was upregulated, as evidenced by the reduced ratio of AT1 over AT2 receptor (0.57±0.2 vs 3.16±0.39, p<0.05 and less locally expressed AT1 receptor in the myocardium. Furthermore, the protein level of angiotensin converting enzyme 2 was upregulated. In coincidence with these changes, edaravone significantly decreased the populations of macrophages and myofibroblasts in the myocardium, which were accompanied by reduced levels of transforming growth factor beta 1 and Smad2/3. Collagen I synthesis was inhibited and collagen-rich fibrosis was attenuated. Relative to the TAC group, cardiac systolic function was preserved, as shown by increased left ventricular systolic pressure (204±51 vs 110±19

  5. DMPD: LPS-binding proteins and receptors. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 9665271 LPS-binding proteins and receptors. Fenton MJ, Golenbock DT. J Leukoc Biol.... 1998 Jul;64(1):25-32. (.png) (.svg) (.html) (.csml) Show LPS-binding proteins and receptors. PubmedID 9665271 Title LPS-binding prot...eins and receptors. Authors Fenton MJ, Golenbock DT. Publication J Leukoc Biol. 199

  6. Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

    Science.gov (United States)

    Turke, Miah; Subhramanyam, Udaya K. Tiruttani; Churchill, Beth; Labahn, Joerg

    2018-01-01

    Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR) were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists. PMID:29342106

  7. Adrenergic Agonists Bind to Adrenergic-Receptor-Like Regions of the Mu Opioid Receptor, Enhancing Morphine and Methionine-Enkephalin Binding: A New Approach to “Biased Opioids”?

    Directory of Open Access Journals (Sweden)

    Robert Root-Bernstein

    2018-01-01

    Full Text Available Extensive evidence demonstrates functional interactions between the adrenergic and opioid systems in a diversity of tissues and organs. While some effects are due to receptor and second messenger cross-talk, recent research has revealed an extracellular, allosteric opioid binding site on adrenergic receptors that enhances adrenergic activity and its duration. The present research addresses whether opioid receptors may have an equivalent extracellular, allosteric adrenergic binding site that has similar enhancing effects on opioid binding. Comparison of adrenergic and opioid receptor sequences revealed that these receptors share very significant regions of similarity, particularly in some of the extracellular and transmembrane regions associated with adrenergic binding in the adrenergic receptors. Five of these shared regions from the mu opioid receptor (muOPR were synthesized as peptides and tested for binding to adrenergic, opioid and control compounds using ultraviolet spectroscopy. Adrenergic compounds bound to several of these muOPR peptides with low micromolar affinity while acetylcholine, histamine and various adrenergic antagonists did not. Similar studies were then conducted with purified, intact muOPR with similar results. Combinations of epinephrine with methionine enkephalin or morphine increased the binding of both by about half a log unit. These results suggest that muOPR may be allosterically enhanced by adrenergic agonists.

  8. ACE inhibition is superior to angiotensin receptor blockade for renography in renal artery stenosis

    International Nuclear Information System (INIS)

    Karanikas, Georgios; Becherer, Alexander; Wiesner, Karoline; Dudczak, Robert; Kletter, Kurt

    2002-01-01

    Angiotensin converting enzyme (ACE) inhibitors as well as angiotensin II receptor antagonists are able to prevent the vasoconstrictive effect of angiotensin II on the efferent renal vessels, which is believed to play an important role in renovascular hypertension. This effect is assumed to be essential for the demonstration of renovascular hypertension by captopril renography. In this study, renographic changes induced by captopril and the AT1 receptor antagonist valsartan were compared in patients with a high probability for renovascular hypertension. Twenty-five patients with 33 stenosed renal arteries (grade of stenosis >50%) and hypertension were studied. Captopril, valsartan and baseline renography were performed within 48 h using technetium-99m mercaptoacetyltriglycine. Blood pressure was monitored, plasma renin concentration before and after intervention was determined and urinary flow was estimated from the urinary output of the hydrated patients. Alterations in renographic curves after intervention were evaluated according to the Santa Fe consensus on ACE inhibitor renography. Captopril renography was positive, indicating renovascular hypertension, in 25 of the 33 stenosed vessels, whereas valsartan renography was positive in only ten. Blood pressure during captopril and valsartan renography was not different; reduction in blood pressure was the same after valsartan and captopril. Plasma renin concentration was comparable for valsartan and captopril studies, showing suppressed values after intervention in as many as 12 of the 25 patients. Urinary flow after valsartan was higher than after captopril (P<0.05). However, this difference could not explain the markedly higher sensitivity of captopril compared with valsartan in demonstrating renal artery stenosis. In 14 of the 25 patients, blood pressure response to revascularisation was monitored, showing a much better predictive value for captopril renography. It is concluded that captopril renography is much

  9. ACE inhibition is superior to angiotensin receptor blockade for renography in renal artery stenosis

    Energy Technology Data Exchange (ETDEWEB)

    Karanikas, Georgios; Becherer, Alexander; Wiesner, Karoline; Dudczak, Robert; Kletter, Kurt [Department of Nuclear Medicine, University of Vienna (Austria)

    2002-03-01

    Angiotensin converting enzyme (ACE) inhibitors as well as angiotensin II receptor antagonists are able to prevent the vasoconstrictive effect of angiotensin II on the efferent renal vessels, which is believed to play an important role in renovascular hypertension. This effect is assumed to be essential for the demonstration of renovascular hypertension by captopril renography. In this study, renographic changes induced by captopril and the AT1 receptor antagonist valsartan were compared in patients with a high probability for renovascular hypertension. Twenty-five patients with 33 stenosed renal arteries (grade of stenosis >50%) and hypertension were studied. Captopril, valsartan and baseline renography were performed within 48 h using technetium-99m mercaptoacetyltriglycine. Blood pressure was monitored, plasma renin concentration before and after intervention was determined and urinary flow was estimated from the urinary output of the hydrated patients. Alterations in renographic curves after intervention were evaluated according to the Santa Fe consensus on ACE inhibitor renography. Captopril renography was positive, indicating renovascular hypertension, in 25 of the 33 stenosed vessels, whereas valsartan renography was positive in only ten. Blood pressure during captopril and valsartan renography was not different; reduction in blood pressure was the same after valsartan and captopril. Plasma renin concentration was comparable for valsartan and captopril studies, showing suppressed values after intervention in as many as 12 of the 25 patients. Urinary flow after valsartan was higher than after captopril (P<0.05). However, this difference could not explain the markedly higher sensitivity of captopril compared with valsartan in demonstrating renal artery stenosis. In 14 of the 25 patients, blood pressure response to revascularisation was monitored, showing a much better predictive value for captopril renography. It is concluded that captopril renography is much

  10. Human myometrial adrenergic receptors during pregnancy: identification of the alpha-adrenergic receptor by [3H] dihydroergocryptine binding

    International Nuclear Information System (INIS)

    Jacobs, M.M.; Hayashida, D.; Roberts, J.M.

    1985-01-01

    The radioactive alpha-adrenergic antagonist [ 3 H] dihydroergocryptine binds to particulate preparations of term pregnant human myometrium in a manner compatible with binding to the alpha-adrenergic receptor (alpha-receptor). [ 3 H] Dihydroergocryptine binds with high affinity (KD = 2 nmol/L and low capacity (receptor concentration = 100 fmol/mg of protein). Adrenergic agonists compete for [ 3 H] dihydroergocryptine binding sites stereo-selectively ([-]-norepinephrine is 100 times as potent as [+]-norepinephrine) and in a manner compatible with alpha-adrenergic potencies (epinephrine approximately equal to norepinephrine much greater than isoproterenol). Studies in which prazosin, an alpha 1-antagonist, and yohimbine, and alpha 2-antagonist, competed for [ 3 H] dihydroergocryptine binding sites in human myometrium indicated that approximately 70% are alpha 2-receptors and that 30% are alpha 1-receptors. [ 3 H] dihydroergocryptine binding to human myometrial membrane particulate provides an important tool with which to study the molecular mechanisms of uterine alpha-adrenergic response

  11. Interaction of a non-peptide agonist with angiotensin II AT1 receptor mutants

    DEFF Research Database (Denmark)

    Costa-Neto, Claudio M; Miyakawa, Ayumi A; Pesquero, João B

    2002-01-01

    and inositol phosphate turnover assays in COS-7 cells transiently transfected with the wild-type and mutant forms of the receptor. Mutant receptors bore modifications in the extracellular region: T88H, Y92H, G1961, G196W, and D278E. Compound L-162,313 displaced [125I]-Sar1,Leu8-AngII from the mutants G196I...... and G196W with IC50 values similar to that of the wild-type. The affinity was, however, slightly affected by the D278E mutation and more significantly by the T88H and Y92H mutations. In inositol phosphate turnover assays, the ability of L-162,313 to trigger the activation cascade was compared...... with that of angiotensin II. These assays showed that the G196W mutant reached a relative maximum activation exceeding that of the wild-type receptor; the efficacy was slightly reduced in the G1961 mutant and further reduced in the T88H, Y92H, and D278E mutants. Our data suggest that residues of the extracellular domain...

  12. Ligand recognition by RAR and RXR receptors: binding and selectivity.

    Science.gov (United States)

    Sussman, Fredy; de Lera, Angel R

    2005-10-06

    Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

  13. Sex Differences in Serotonin 1 Receptor Binding in Rat Brain

    Science.gov (United States)

    Fischette, Christine T.; Biegon, Anat; McEwen, Bruce S.

    1983-10-01

    Male and female rats exhibit sex differences in binding by serotonin 1 receptors in discrete areas of the brain, some of which have been implicated in the control of ovulation and of gonadotropin release. The sex-specific changes in binding, which occur in response to the same hormonal (estrogenic) stimulus, are due to changes in the number of binding sites. Castration alone also affects the number of binding sites in certain areas. The results lead to the conclusion that peripheral hormones modulate binding by serotonin 1 receptors. The status of the serotonin receptor system may affect the reproductive capacity of an organism and may be related to sex-linked emotional disturbances in humans.

  14. Characterization of a second ligand binding site of the insulin receptor

    International Nuclear Information System (INIS)

    Hao Caili; Whittaker, Linda; Whittaker, Jonathan

    2006-01-01

    Insulin binding to its receptor is characterized by high affinity, curvilinear Scatchard plots, and negative cooperativity. These properties may be the consequence of binding of insulin to two receptor binding sites. The N-terminal L1 domain and the C-terminus of the α subunit contain one binding site. To locate a second site, we examined the binding properties of chimeric receptors in which the L1 and L2 domains and the first Fibronectin Type III repeat of the insulin-like growth factor-I receptor were replaced by corresponding regions of the insulin receptor. Substitutions of the L2 domain and the first Fibronectin Type III repeat together with the L1 domain produced 80- and 300-fold increases in affinity for insulin. Fusion of these domains to human immunoglobulin Fc fragment produced a protein which bound insulin with a K d of 2.9 nM. These data strongly suggest that these domains contain an insulin binding site

  15. Structural Insights into Immune Recognition of the Severe Acute Respiratory Syndrome Coronavirus S Protein Receptor Binding Domain

    Energy Technology Data Exchange (ETDEWEB)

    Pak, J.; Sharon, C; Satkunarajah, M; Thierry, C; Cameron, C; Kelvin, D; Seetharaman, J; Cochrane, A; Plummer, F; et. al.

    2009-01-01

    The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) is responsible for host cell attachment and fusion of the viral and host cell membranes. Within S the receptor binding domain (RBD) mediates the interaction with angiotensin-converting enzyme 2 (ACE2), the SARS-CoV host cell receptor. Both S and the RBD are highly immunogenic and both have been found to elicit neutralizing antibodies. Reported here is the X-ray crystal structure of the RBD in complex with the Fab of a neutralizing mouse monoclonal antibody, F26G19, elicited by immunization with chemically inactivated SARS-CoV. The RBD-F26G19 Fab complex represents the first example of the structural characterization of an antibody elicited by an immune response to SARS-CoV or any fragment of it. The structure reveals that the RBD surface recognized by F26G19 overlaps significantly with the surface recognized by ACE2 and, as such, suggests that F26G19 likely neutralizes SARS-CoV by blocking the virus-host cell interaction.

  16. Aldosterone downregulates delayed rectifier potassium currents through an angiotensin type 1 receptor-dependent mechanism.

    Science.gov (United States)

    Lv, Yankun; Wang, Yanjun; Zhu, Xiaoran; Zhang, Hua

    2018-01-01

    We have previously shown that aldosterone downregulates delayed rectifier potassium currents (I Ks ) via activation of the mineralocorticoid receptor (MR) in adult guinea pig cardiomyocytes. Here, we investigate whether angiotensin II/angiotensin type 1 receptor (AngII/AT1R) and intracellular calcium also play a role in these effects. Ventricular cardiomyocytes were isolated from adult guinea pigs and incubated with aldosterone (1 μmol·L -1 ) either alone or in combination with enalapril (1 μmol·L -1 ), losartan (1 μmol·L -1 ), nimodipine (1 μmol·L -1 ), or BAPTA-AM (2.5 μmol·L -1 ) for 24 h. We used the conventional whole cell patch-clamp technique to record the I Ks component. In addition, we evaluated expression of the I Ks subunits KCNQ1 and KCNE1 using Western blotting. Our results showed that both enalapril and losartan, but not nimodipine or BAPTA-AM, completely reversed the aldosterone-induced inhibition of I Ks and its effects on KCNQ1/KCNE1 protein levels. Furthermore, we found that AngII/AT1R mediates the inhibitory effects of aldosterone on I Ks . Finally, the downregulation of I Ks induced by aldosterone did not occur secondarily to a change in intracellular calcium concentrations. Taken together, our findings demonstrate that crosstalk between MR and AT1R underlies the effects of aldosterone, and provide new insights into the mechanism underlying potassium channels.

  17. The human angiotensin AT(1) receptor supports G protein-independent extracellular signal-regulated kinase 1/2 activation and cellular proliferation

    DEFF Research Database (Denmark)

    Hansen, Jakob Lerche; Aplin, Mark; Hansen, Jonas Tind

    2008-01-01

    The angiotensin AT(1) receptor is a key regulator of blood pressure and body fluid homeostasis, and it plays a key role in the pathophysiology of several cardiovascular diseases such as hypertension, cardiac hypertrophy, congestive heart failure, and arrhythmia. The importance of human angiotensi...

  18. Free energy calculations offer insights into the influence of receptor flexibility on ligand-receptor binding affinities.

    Science.gov (United States)

    Dolenc, Jožica; Riniker, Sereina; Gaspari, Roberto; Daura, Xavier; van Gunsteren, Wilfred F

    2011-08-01

    Docking algorithms for computer-aided drug discovery and design often ignore or restrain the flexibility of the receptor, which may lead to a loss of accuracy of the relative free enthalpies of binding. In order to evaluate the contribution of receptor flexibility to relative binding free enthalpies, two host-guest systems have been examined: inclusion complexes of α-cyclodextrin (αCD) with 1-chlorobenzene (ClBn), 1-bromobenzene (BrBn) and toluene (MeBn), and complexes of DNA with the minor-groove binding ligands netropsin (Net) and distamycin (Dist). Molecular dynamics simulations and free energy calculations reveal that restraining of the flexibility of the receptor can have a significant influence on the estimated relative ligand-receptor binding affinities as well as on the predicted structures of the biomolecular complexes. The influence is particularly pronounced in the case of flexible receptors such as DNA, where a 50% contribution of DNA flexibility towards the relative ligand-DNA binding affinities is observed. The differences in the free enthalpy of binding do not arise only from the changes in ligand-DNA interactions but also from changes in ligand-solvent interactions as well as from the loss of DNA configurational entropy upon restraining.

  19. Flavonoids with M1 Muscarinic Acetylcholine Receptor Binding Activity

    Directory of Open Access Journals (Sweden)

    Meyyammai Swaminathan

    2014-06-01

    Full Text Available Muscarinic acetylcholine receptor-active compounds have potential for the treatment of Alzheimer’s disease. In this study, a series of natural and synthetic flavones and flavonols was assayed in vitro for their ability to inhibit radioligand binding at human cloned M1 muscarinic receptors. Several compounds were found to possess competitive binding affinity (Ki = 40–110 µM, comparable to that of acetylcholine (Ki = 59 µM. Despite the fact that these compounds lack a positively-charged ammonium group under physiological conditions, molecular modelling studies suggested that they bind to the orthosteric site of the receptor, mainly through non-polar interactions.

  20. Targeting Renin–Angiotensin System Against Alzheimer’s Disease

    Directory of Open Access Journals (Sweden)

    Abadi Kahsu Gebre

    2018-04-01

    Full Text Available Renin Angiotensin System (RAS is a hormonal system that regulates blood pressure and fluid balance through a coordinated action of renal, cardiovascular, and central nervous systems. In addition to its hemodynamic regulatory role, RAS involves in many brain activities, including memory acquisition and consolidation. This review has summarized the involvement of RAS in the pathology of Alzheimer’s disease (AD, and the outcomes of treatment with RAS inhibitors. We have discussed the effect of brain RAS in the amyloid plaque (Aβ deposition, oxidative stress, neuroinflammation, and vascular pathology which are directly and indirectly associated with AD. Angiotensin II (AngII via AT1 receptor is reported to increase brain Aβ level via different mechanisms including increasing amyloid precursor protein (APP mRNA, β-secretase activity, and presenilin expression. Similarly, it was associated with tau phosphorylation, and reactive oxygen species generation. However, these effects are counterbalanced by Ang II mediated AT2 signaling. The protective effect observed with angiotensin receptor blockers (ARBs and angiotensin converting enzyme inhibitors (ACEIs could be as the result of inhibition of Ang II signaling. ARBs also offer additional benefit by shifting the effect of Ang II toward AT2 receptor. To conclude, targeting RAS in the brain may benefit patients with AD though it still requires further in depth understanding.

  1. Interactive Effectiveness of Angiotensin-Converting Enzyme Inhibitors and Angiotensin Receptor Blockers or Their Combination on Survival of Hemodialysis Patients

    Science.gov (United States)

    Kido, Ryo; Akizawa, Tadao; Fukagawa, Masafumi; Onishi, Yoshihiro; Yamaguchi, Takuhiro; Fukuhara, Shunichi

    2018-01-01

    Background Does the use of angiotensin-converting enzyme inhibitors and angiotensin receptor blockers individually or as a combination confer a survival benefit in hemodialysis patients? The answer to this question is yet unclear. Methods We performed a case-cohort study using data from the Mineral and Bone Disorder Outcomes Study for Japanese CKD stage 5D patients (MBD-5D), a 3-year multicenter prospective case-cohort study, including 8,229 hemodialysis patients registered from 86 facilities in Japan. All patients had secondary hyperparathyroidism, a condition defined as a parathyroid hormone level ≥180 pg/mL and/or receiving vitamin D receptor activators. We compared all-cause mortality rates between those receiving ACEI, ARB, and their combination and non-users with interaction testing. We used marginal structural Poisson regression (causal model) to estimate the causal effect and interaction adjusted for possible time-dependent confounding. Cardiovascular mortality was also evaluated. Results Among 3,762 randomly sampled subcohort patients, those taking ACEI, ARB, and their combination at baseline accounted for 4.0, 31.6, and 3.8%, respectively. Over 3 years, 1,226 all-cause and 462 cardiovascular deaths occurred. Compared to non-users, ARB-alone users had a lower all-cause mortality rate (adjusted incident rate ratio [aIRR] 0.62, 95% CI 0.50–0.76), whereas ACEI-alone users showed a statistically similar rate (aIRR 1.01, 95% CI 0.57–1.77). On the contrary, combination users had a greater mortality rate (aIRR 2.56, 95% CI 1.22–5.37), showing significant interaction (p = 0.03). Analysis for cardiovascular mortality showed similar results. Conclusion Among hemodialysis patients with secondary hyperparathyroidism, unlike ACEI use, ARB use was associated with greater survival than non-use. Conversely, combination use was associated with greater mortality. Controlled trials are warranted to verify the causality factors of these associations. PMID:29161689

  2. Secondary metabolites of Cynodon dactylon as an antagonist to angiotensin II type1 receptor: Novel in silico drug targeting approach for diabetic retinopathy

    Science.gov (United States)

    Jananie, R. K.; Priya, V.; Vijayalakshmi, K.

    2012-01-01

    Objectives: To study the ability of the secondary metabolites of Cynodon dactylon to serve as an antagonist to angiotensin II type 1 receptor (AT1); activation of this receptor plays a vital role in diabetic retinopathy (DR). Materials and Methods: In silico methods are mainly harnessed to reduce time, cost and risk associated with drug discovery. Twenty-four compounds were identified as the secondary metabolites of hydroalcoholic extract of C. dactylon using the GCMS technique. These were considered as the ligands or inhibitors that would serve as an antagonist to the AT1. The ACD/Chemsketch tool was used to generate 3D structures of the ligands. A molecular file format converter tool was used to convert the generated data to the PDB format (Protein Data Bank) and was used for docking studies. The AT1 structure was retrieved from the Swissprot data base and PDB and visualized using the Rasmol tool. Domain analysis was carried from the Pfam data base; following this, the active site of the target protein was identified using a Q-site finder tool. The ability of the ligands to bind with the active site of AT1 was studied using the Autodocking tool. The docking results were analyzed using the WebLab viewer tool. Results: Sixteen ligands showed effective binding with the target protein; diazoprogesteron, didodecyl phthalate, and 9,12-octadecadienoyl chloride (z,z) may be considered as compounds that could be used to bind with the active site sequence of AT1. Conclusions: The present study shows that the metabolites of C. dactylon could serve as a natural antagonist to AT1 that could be used to treat diabetic retinopathy. PMID:22368412

  3. Heterogeneous Downregulation of Angiotensin II AT1-A and AT1-B Receptors in Arterioles in STZ-Induced Diabetic Rat Kidneys

    Directory of Open Access Journals (Sweden)

    Zsolt Razga

    2014-01-01

    Full Text Available Introduction. The renin granulation of kidney arterioles is enhanced in diabetes despite the fact that the level of angiotensin II in the diabetic kidney is elevated. Therefore, the number of angiotensin II AT1-A and AT1-B receptors in afferent and efferent arteriole’s renin-positive and renin-negative smooth muscle cells (SMC was estimated. Method. Immunohistochemistry at the electron microscopic level was combined with 3D stereological sampling techniques. Results. In diabetes the enhanced downregulation of AT1-B receptors in the renin-positive than in the renin-negative SMCs in both arterioles was resulted: the significant difference in the number of AT1 (AT1-A + AT1-B receptors between the two types of SMCs in the normal rats was further increased in diabetes and in contrast with the significant difference observed between the afferent and efferent arterioles in the normal animals, there was no such difference in diabetes. Conclusions. The enhanced downregulation of the AT1-B receptors in the renin-negative SMCs in the efferent arterioles demonstrates that the regulation of the glomerular filtration rate by the pre- and postglomerular arterioles is changed in diabetes. The enhanced downregulation of the AT1-B receptors in the renin-positive SMCs in the arterioles may result in an enhanced level of renin granulation in the arterioles.

  4. Binding-site analysis of opioid receptors using monoclonal anti-idiotypic antibodies

    International Nuclear Information System (INIS)

    Conroy, W.G.

    1988-01-01

    Structural relatedness between the variable region of anti-ligand antibodies and opioid binding sites allowed the generation of anti-idiotypic antibodies which recognized opioid receptors. The IgG 3 k antibodies which bound to opioid receptors were obtained when an anti-morphine antiserum was the idiotype. Both antibodies bound to opioid receptors, but only one of these blocked the binding of [ 3 H]naloxone. The antibody which did not inhibit the binding of [ 3 H]naloxone was itself displaced from the receptor by opioid ligands. The unique binding properties displayed by this antibody indicated that anti-idiotypic antibodies are not always a perfect image of the original ligand, and therefore may be more useful than typical ligands as probes for the receptor. An auto-anti-idiotypic technique was successfully used to obtain anti-opioid receptor antibodies. Another IgG 3 k antibody that blocked the binding of [ 3 H]naloxone to rat brain opioid receptors was obtained when a mouse was immunized with naloxone conjugated to bovine serum albumin. These data confirmed that an idiotype-anti-idiotype network which can generate an anti-receptor antibody normally functions when an opioid ligand is introduced into an animal in an immunogenic form

  5. Angiotensin II receptor one (AT1) mediates dextrose induced endoplasmic reticulum stress and superoxide production in human coronary artery endothelial cells.

    Science.gov (United States)

    Haas, Michael J; Onstead-Haas, Luisa; Lee, Tracey; Torfah, Maisoon; Mooradian, Arshag D

    2016-10-01

    Renin-angiotensin-aldosterone system (RAAS) has been implicated in diabetes-related vascular complications partly through oxidative stress. To determine the role of angiotensin II receptor subtype one (AT1) in dextrose induced endoplasmic reticulum (ER) stress, another cellular stress implicated in vascular disease. Human coronary artery endothelial cells with or without AT1 receptor knock down were treated with 27.5mM dextrose for 24h in the presence of various pharmacologic blockers of RAAS and ER stress and superoxide (SO) production were measured. Transfection of cells with AT1 antisense RNA knocked down cellular AT1 by approximately 80%. The ER stress was measured using the placental alkaline phosphatase (ES-TRAP) assay and western blot analysis of glucose regulated protein 78 (GRP78), c-jun-N-terminal kinase 1 (JNK1), phospho-JNK1, eukaryotic translation initiation factor 2α (eIF2α) and phospho-eIF2α measurements. Superoxide (SO) generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride (MCLA) chemiluminescence. In cells with AT1 knock down, dextrose induced ER stress was significantly blunted and treatment with 27.5mM dextrose resulted in significantly smaller increase in SO production compared to 27.5mM dextrose treated and sham transfected cells. Dextrose induced ER stress was reduced with pharmacologic blockers of AT1 (losartan and candesartan) and mineralocorticoid receptor blocker (spironolactone) but not with angiotensin converting enzyme inhibitors (captopril and lisinopril). The dextrose induced SO generation was inhibited by all pharmacologic blockers of RAAS tested. The results indicate that dextrose induced ER stress and SO production in endothelial cells are mediated at least partly through AT1 receptor activation. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  6. Increased expression of endothelin ET(B) and angiotensin AT(1) receptors in peripheral resistance arteries of patients with suspected acute coronary syndrome

    DEFF Research Database (Denmark)

    Dimitrijevic, Ivan; Ekelund, Ulf; Edvinsson, Lars

    2009-01-01

    of arterial vasoconstrictor endothelin (ET) and angiotensin (AT) receptors. Our aim was to investigate if the arterial expressions of these receptors are changed in patients with suspected but ruled out acute coronary syndrome (ACS). Small subcutaneous arteries (diameter of 100 microm) were surgically removed...... in the regulation of coronary tone and in the development of atherosclerosis, and may be related to increased cardiovascular risk....

  7. Transdermal delivery of angiotensin II receptor blockers (ARBs), angiotensin-converting enzyme inhibitors (ACEIs) and others for management of hypertension.

    Science.gov (United States)

    Ahad, Abdul; Al-Mohizea, Abdullah Mohammed; Al-Jenoobi, Fahad Ibrahim; Aqil, Mohd

    2016-01-01

    Angiotensin II receptor blockers (ARBs), angiotensin-converting enzyme inhibitors (ACEIs) are some of the most commonly prescribed medications for hypertension. Most of all conventional dosage forms of ARBs and ACEIs undergo extensive first-pass metabolism, which significantly reduces bioavailability. Majority of ARBs and ACEIs are inherently short acting due to a rapid elimination half-life. In addition, oral dosage forms of ARBs and ACEIs have many high incidences of adverse effects due to variable absorption profiles, higher frequency of administration and poor patient compliance. Many attempts have been made globally at the laboratory level to investigate the skin permeation and to develop transdermal therapeutic systems of various ARBs, ACEIs and other anti-hypertensives, to circumvent the drawbacks associated with their conventional dosage form. This manuscript presents an outline of the transdermal research specifically in the area of ARBs, ACEIs and other anti-hypertensives reported in various pharmaceutical journals. The transdermal delivery has gained a significant importance for systemic treatment as it is able to avoid first-pass metabolism and major fluctuations of plasma levels typical of repeated oral administration. As we can experience from this review article that transdermal delivery of different ARBs and ACEIs improves bioavailability as well as patient compliance by many folds. In fact, the rationale development of some newer ARBs, ACEIs and other anti-hypertensives transdermal systems will provide new ways of treatment, circumventing current limitations for conventional dosage forms.

  8. Receptor binding properties and antinociceptive effects of chimeric peptides consisting of a micro-opioid receptor agonist and an ORL1 receptor antagonist.

    Science.gov (United States)

    Kawano, Susumu; Ito, Risa; Nishiyama, Miharu; Kubo, Mai; Matsushima, Tomoko; Minamisawa, Motoko; Ambo, Akihiro; Sasaki, Yusuke

    2007-07-01

    Receptor binding properties and antinociceptive activities of chimeric peptides linked by spacers were investigated. The peptides consisted of the micro-opioid receptor ligand dermorphin (Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH(2)) or its analog YRFB (Tyr-D-Arg-Phe-betaAla-NH(2)) linked to the ORL1 receptor ligand Ac-Arg-Tyr-Tyr-Arg-Ile-Lys-NH(2) (Ac-RYYRIK-NH(2)). All chimeric peptides were found to possess high receptor binding affinities for both micro-opioid and ORL1 receptors in mouse brain membranes although their binding affinities for both receptors in spinal membranes were significantly lower. Among them, chimeric peptide 2, which consists of dermorphin and Ac-RYYRIK-NH(2) connected by a long spacer, had the highest binding affinity towards both receptors. In the tail-flick test following intrathecal (i.t.) administration to mice, all chimeric peptides showed potent and dose-dependent antinociceptive activities with an ED(50) of 1.34-4.51 (pmol/mouse), nearly comparable to dermorphin alone (ED(50); 1.08 pmol/mouse). In contrast to their micro-opioid receptor binding profiles, intracerebroventricular (i.c.v.) administration of the chimeric peptides resulted in much less potent antinociceptive activity (ED(50) 5.55-100peptides, and the regulation of mu-opioid receptor-mediated antinociception in brain. The present chimeric peptides may be useful as pharmacological tools for studies on micro-opioid receptor/ORL1 receptor heterodimers.

  9. Dietary sodium deprivation evokes activation of brain regional neurons and down-regulation of angiotensin II type 1 receptor and angiotensin-convertion enzyme mRNA expression.

    Science.gov (United States)

    Lu, B; Yang, X J; Chen, K; Yang, D J; Yan, J Q

    2009-12-15

    Previous studies have indicated that the renin-angiotensin-aldosterone system (RAAS) is implicated in the induction of sodium appetite in rats and that different dietary sodium intakes influence the mRNA expression of central and peripheral RAAS components. To determine whether dietary sodium deprivation activates regional brain neurons related to sodium appetite, and changes their gene expression of RAAS components of rats, the present study examined the c-Fos expression after chronic exposure to low sodium diet, and determined the relationship between plasma and brain angiotensin I (ANG I), angiotensin II (ANG II) and aldosterone (ALD) levels and the sodium ingestive behavior variations, as well as the effects of prolonged dietary sodium deprivation on ANG II type 1 (AT1) and ANG II type 2 (AT2) receptors and angiotensin-convertion enzyme (ACE) mRNA levels in the involved brain regions using the method of real-time polymerase chain reaction (PCR). Results showed that the Fos immunoreactivity (Fos-ir) expression in forebrain areas such as subfornical organ (SFO), paraventricular hypothalamic nuclei (PVN), supraoptic nucleus (SON) and organum vasculosum laminae terminalis (OVLT) all increased significantly and that the levels of ANG I, ANG II and ALD also increased in plasma and forebrain in rats fed with low sodium diet. In contrast, AT1, ACE mRNA in PVN, SON and OVLT decreased significantly in dietary sodium depleted rats, while AT2 mRNA expression did not change in the examined areas. These results suggest that many brain areas are activated by increased levels of plasma and/or brain ANG II and ALD, which underlies the elevated preference for hypertonic salt solution after prolonged exposure to low sodium diet, and that the regional AT1 and ACE mRNA are down-regulated after dietary sodium deprivation, which may be mediated by increased ANG II in plasma and/or brain tissue.

  10. Familial defective apolipoprotein B-100: low density lipoproteins with abnormal receptor binding

    International Nuclear Information System (INIS)

    Innerarity, T.L.; Weisgraber, K.H.; Arnold, K.S.; Mahley, R.W.; Krauss, R.M.; Vega, G.L.; Grundy, S.M.

    1987-01-01

    Previous in vivo turnover studies suggested that retarded clearance of low density lipoproteins (LDL) from the plasma of some hypercholesterolemic patients is due to LDL with defective receptor binding. The present study examined this postulate directly by receptor binding experiments. The LDL from a hypercholesterolemic patient (G.R.) displayed a reduced ability to bind to the LDL receptors on normal human fibroblasts. The G.R. LDL possessed 32% of normal receptor binding activity. Likewise, the G.R. LDL were much less effective than normal LDL in competing with 125 I-labeled normal LDL for cellular uptake and degradation and in stimulating intracellular cholesteryl ester synthesis. The defect in LDL binding appears to be due to a genetic abnormality of apolipoprotein B-100: two brothers of the proband possess LDL defective in receptor binding, whereas a third brother and the proband's son have normally binding LDL. Further, the defect in receptor binding does not appear to be associated wit an abnormal lipid composition or structure of the LDL. Normal and abnormal LDL subpopulations were partially separated from plasma of two subjects by density-gradient ultracentrifugation, a finding consistent with the presence of a normal and a mutant allele. The affected family members appear to be heterozygous for this disorder, which has been designated familial defective apolipoprotein B-100. These studies indicate that the defective receptor binding results in inefficient clearance of LDL and the hypercholesterolemia observed in these patients

  11. Angiotensin-(1-7): A Novel Peptide to Treat Hypertension and Nephropathy in Diabetes?

    Science.gov (United States)

    Padda, Ranjit Singh; Shi, Yixuan; Lo, Chao-Sheng; Zhang, Shao-Ling; Chan, John S D

    2015-10-14

    The renin-angiotensin system (RAS) plays a pivotal role in mammalian homeostasis physiology. The RAS can be delineated into a classical RAS (the pressor arm) including angiotensinogen (Agt), renin, angiotensin-converting enzyme (ACE), angiotensin II (Ang II) and angiotensin type 1 receptor (AT1R), and a counterbalancing novel RAS (the depressor arm) including Agt, renin, angiotensin-converting enzyme-2 (ACE-2), angiotensin-(1-7) (Ang 1-7) and Ang 1-7 receptor (or Mas receptor (MasR)). Hyperglycemia (diabetes) induces severe tissue oxidative stress, which stimulates the pressor arm of the renal RAS axis and leads to an increase in ACE/ACE-2 ratio, with excessive formation of Ang II. There is a growing body of evidence for beneficial effects of the depressor arm of RAS (ACE-2/Ang 1-7/MasR) axis in diabetes, hypertension and several other diseased conditions. Evidence from in vitro, in vivo and clinical studies reflects anti-oxidant, anti-fibrotic, and anti-inflammatory properties of Ang 1-7. Most of the currently available therapies only target suppression of the pressor arm of RAS with angiotensin receptor blockers (ARBs) and ACE inhibitors (ACEi). However, it is time to consider simultaneous activation of the depressor arm for more effective outcomes. This review summarizes the recent updates on the protective role of Ang 1-7 in hypertension and kidney injury in diabetes, as well as the possible underlying mechanism(s) of Ang 1-7 action, suggesting that the ACE-2/Ang 1-7/MasR axis can be developed as a therapeutic target for the treatment of diabetes-induced hypertension and renal damage.

  12. Altered renal expression of angiotensin II receptors, renin receptor, and ACE-2 precede the development of renal fibrosis in aging rats.

    Science.gov (United States)

    Schulman, Ivonne Hernandez; Zhou, Ming-Sheng; Treuer, Adriana V; Chadipiralla, Kiranmai; Hare, Joshua M; Raij, Leopoldo

    2010-01-01

    The susceptibility to fibrosis and progression of renal disease is mitigated by inhibition of the renin-angiotensin system (RAS). We hypothesized that activation of the intrarenal RAS predisposes to renal fibrosis in aging. Intrarenal expression of angiotensin II type 1 (AT(1)R), type 2 (AT(2)R), and (pro)renin receptors, ACE and ACE-2, as well as pro- and antioxidant enzymes were measured in 3-month-old (young), 14-month-old (middle-aged), and 24-month-old (old) male Sprague-Dawley rats. Old rats manifested glomerulosclerosis and severe tubulointerstitial fibrosis with increased fibronectin and TGF-β expression (7-fold). AT(1)R /AT(2)R ratios were increased in middle-aged (cortical 1.6-fold, medullary 5-fold) and old rats (cortical 2-fold, medullary 4-fold). Similarly, (pro)renin receptor expression was increased in middle-aged (cortical 2-fold, medullary 3-fold) and old (cortical 5-fold, medullary 3-fold) rats. Cortical ACE was increased (+35%) in old rats, whereas ACE-2 was decreased (-50%) in middle-aged and old rats. NADPH oxidase activity was increased (2-fold), whereas antioxidant capacity and expression of the mitochondrial enzyme manganese superoxide dismutase (cortical -40%, medullary -53%) and medullary endothelial nitric oxide synthase (-48%) were decreased in old rats. Age-related intrarenal activation of the RAS preceded the development of severe renal fibrosis, suggesting that it contributes to the increased susceptibility to renal injury observed in the elderly. Copyright © 2010 S. Karger AG, Basel.

  13. Comparative effects of angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers on the risk of pneumonia and severe exacerbations in patients with COPD

    Directory of Open Access Journals (Sweden)

    Lai CC

    2018-03-01

    Full Text Available Chih-Cheng Lai,1 Ya-Hui Wang,2 Cheng-Yi Wang,3 Hao-Chien Wang,4 Chong-Jen Yu,4 Likwang Chen5 On behalf of the Taiwan Clinical Trial Consortium for Respiratory Diseases (TCORE 1Department of Intensive Care Medicine, Chi Mei Medical Center, Liouying, Taiwan; 2Medical Research Center, Cardinal Tien Hospital and School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan; 3Department of Internal Medicine, Cardinal Tien Hospital and School of Medicine, College of Medicine, Fu Jen Catholic University, New Taipei City, Taiwan; 4Department of Internal Medicine, National Taiwan University Hospital and College of Medicine, National Taiwan University, Taipei, Taiwan; 5Institute of Population Health Sciences, National Health Research Institutes, Zhunan, Taiwan Objectives: This study aimed to compare the effects of angiotensin-converting-enzyme inhibitors (ACEis and angiotensin receptor blockers (ARBs on the risk of pneumonia and severe exacerbations in patients with COPD.Patients and methods: All patients with COPD who used ACEis and ARBs for >90 days between 2000 and 2005 were recruited. Pairwise matching (1:1 of the ACEi and ARB groups resulted in two similar subgroups, with 6,226 patients in each. The primary outcomes were pneumonia and COPD exacerbations, and the secondary outcome was death.Results: During the follow-up period, the incidence of pneumonia was 7.20 per 100 person-years in the ACEi group and 5.89 per 100 person-years in the ARB group. The ACEi group had a higher risk of pneumonia (adjusted hazard ratio [aHR], 1.22; 95% CI, 1.15–1.29 than the ARB group. The incidence of severe exacerbations was 0.65 per person-year for the patients receiving ACEis and 0.52 per person-year for those receiving ARBs. The patients receiving ACEis had a higher risk of severe exacerbations (aHR, 1.19; 95% CI, 1.16–1.21 than those receiving ARBs. Similar trends were noted in terms of severe exacerbations requiring

  14. Role of Mas receptor in renal blood flow response to angiotensin (1-7) in male and female rats.

    Science.gov (United States)

    Nematbakhsh, Mehdi; Safari, Tahereh

    2014-01-01

    Epidemiologic and clinical studies have shown that progression of renal disease in male is faster than that in female. However, the exact mechanisms are not well recognized. Angiotensin (1-7) (Ang 1-7) receptor, called "Mas", is an element in the depressor arm of renin angiotensin system (RAS), and its expression is enhanced in females. We test the hypothesis that Mas receptor (MasR) blockade (A779) attenuates renal blood flow (RBF) in response to infusion of graded doses of Ang 1-7 in female rats. Male and female Wistar rats were anesthetized and catheterized. Then, the mean arterial pressure (MAP), RBF, and controlled renal perfusion pressure (RPP) responses to infusion of graded doses of Ang 1-7 (100-1000 ng/kg/min i.v.) with and without A779 were measured in the animals. Basal MAP, RPP, RBF, and renal vascular resistance (RVR) were not significantly different between the two groups. After Ang 1-7 administration, RPP was controlled at a constant level. However, RBF increased in a dose-related manner in response to Ang 1-7 infusion in both male and female rats (Pdoserenal diseases.

  15. A novel method to determine new potent angiotensin inhibitor ...

    African Journals Online (AJOL)

    that inhibit the action of angiotensin II by binding directly to the ..... Relatively high plasma protein binding of AZL (>99 .... Ghasemi JB, Zolfonoun E. Application of principal component analysis–multivariate adaptive regression splines for the ...

  16. Benzodiazepine receptor binding in vivo with (/sup 3/)-Ro 15-1788

    Energy Technology Data Exchange (ETDEWEB)

    Goeders, N.E.; Kuhar, M.J.

    1985-07-29

    In vivo benzodiazepine receptor binding has generally been studied by ex vivo techniques. In this investigation, the authors identify the conditions where (/sup 3/H)-Ro 15-1788 labels benzodiazepine receptors by true in vivo binding, i.e. where workable specific to nonspecific ratios are obtained in intact tissues without homogenization or washing. (/sup 3/H)-Flunitrazepam and (/sup 3/H)-clonazepam did not exhibit useful in vivo receptor binding. 39 references, 5 figures, 1 table.

  17. Renin-angiotensin system antagonists, glomerular filtration rate and blood pressure

    Directory of Open Access Journals (Sweden)

    D.D. Ivanov

    2018-02-01

    Full Text Available The article deals with the mModern data on the influence of renin-angiotensin system blockers on the glomerular filtration rate, the level of arterial pressure and the outcome of chronic kidney disease. The strategy of  rennin-angiotensine blockade is offered to be changed depending on the criteria va­lues of glomerular filtration rate: a combination of inhibitors of angiotensin-converting enzyme + angiotensin receptors blo­ckers, monotherapy and drug withdrawal in glomerular filtration rate under 15–30 ml/min/m2. The formula BRIMONEL for treatment of chronic kidney disease is given.

  18. Attenuation of myocardial fibrosis with curcumin is mediated by modulating expression of angiotensin II AT1/AT2 receptors and ACE2 in rats

    Directory of Open Access Journals (Sweden)

    Pang XF

    2015-11-01

    Full Text Available Xue-Fen Pang,1 Li-Hui Zhang,2 Feng Bai,1 Ning-Ping Wang,3 Ron E Garner,3 Robert J McKallip,4 Zhi-Qing Zhao1,3 1Department of Physiology, Shanxi Medical University, Taiyuan, Shanxi, People’s Republic of China; 2Department of Cardiology, Shanxi Academy of Medical Sciences and Shanxi Dayi Hospital, Shanxi Medical University, Taiyuan, Shanxi, People’s Republic of China; 3Department of Basic Biomedical Sciences, Mercer University School of Medicine, Savannah, GA, USA; 4Division of Basic Biomedical Sciences, Mercer University School of Medicine, Macon, GA, USA Abstract: Curcumin is known to improve cardiac function by balancing degradation and synthesis of collagens after myocardial infarction. This study tested the hypothesis that inhibition of myocardial fibrosis by curcumin is associated with modulating expression of angiotensin II (Ang II receptors and angiotensin-converting enzyme 2 (ACE2. Male Sprague Dawley rats were subjected to Ang II infusion (500 ng/kg/min using osmotic minipumps for 2 and 4 weeks, respectively, and curcumin (150 mg/kg/day was fed by gastric gavage during Ang II infusion. Compared to the animals with Ang II infusion, curcumin significantly decreased the mean arterial blood pressure during the course of the observation. The protein level of the Ang II type 1 (AT1 receptor was reduced, and the Ang II type 2 (AT2 receptor was up-regulated, evidenced by an increased ratio of the AT2 receptor over the AT1 receptor in the curcumin group (1.2±0.02% vs in the Ang II group (0.7±0.03%, P<0.05. These changes were coincident with less locally expressed AT1 receptor and enhanced AT2 receptor in the intracardiac vessels and intermyocardium. Along with these modulations, curcumin significantly decreased the populations of macrophages and alpha smooth muscle actin-expressing myofibroblasts, which were accompanied by reduced expression of transforming growth factor beta 1 and phosphorylated-Smad2/3. Collagen I synthesis was

  19. Association of angiotensin receptor 2 gene polymorphisms with pregnancy induced hypertension risk.

    Science.gov (United States)

    Li, Chenyang; Peng, Weijun; Zhang, Heng; Yan, Weirong

    2018-05-01

    To investigate the association of polymorphisms and haplotypes of angiotensin receptor 2 (AT2R) gene with pregnancy induced hypertension (PIH) in Chinese Han women. A case-control study was designed with 446 cases (gestational hypertension, GH: 124; pre-eclampsia, PE + eclampsia, E: 322) and 650 controls. rs5193, rs1403543 and rs12710567 of AT2R gene were genotyped. A logistic regression approach was applied to estimate the relationship between the polymorphisms and haplotypes of AT2Rgene with PIH risk. No relationship between AT2R gene polymorphisms and PIH was detected. The haplotype analysis also showed a negative result. rs5193, rs1403543 and rs12710567 of AT2R gene might have no effect on PIH risk among Chinese Han women.

  20. Angiotensin Receptor Blockers: Cardiovascular Protection in the Metabolic Syndrome

    Directory of Open Access Journals (Sweden)

    Prakash C Deedwania

    2006-03-01

    Full Text Available It is well recognised that the metabolic syndrome, a constellation of risk factors including obesity, hypertension, insulin resistance and dyslipidaemia, is associated with an increased risk of cardiovascular complications and the development of Type 2 diabetes. Consequently, timely identification and management of all components of the metabolic syndrome is warranted. In particular, guidelines have emphasised the importance of targeting elevated blood pressure (BP and dyslipidaemia as a method of reducing global cardiovascular risk.Findings from the Valsartan Antihypertensive Long-term Use Evaluation (VALUE trial show that the angiotensin receptor blocker, valsartan, reduces cardiovascular events and the development of Type 2 diabetes in high-risk individuals. This profile is being further explored in the ongoing Nateglinide And Valsartan in Impaired Glucose Tolerance Outcomes Research (NAVIGATOR trial.Given the potential advantages to patients and physicians of tackling more than one of the components of the metabolic syndrome, antihypertensive agents such as valsartan would appear to be an important addition to the management of vulnerable patients at high risk of cardiovascular events.

  1. Insight into the structural requirement of substituted quinazolinone biphenyl acylsulfonamides derivatives as Angiotensin II AT1 receptor antagonist: 2D and 3D QSAR approach

    Directory of Open Access Journals (Sweden)

    Mukesh C. Sharma

    2014-01-01

    Full Text Available A series of 19 molecules substituted quinazolinone biphenyl acylsulfonamides derivatives displaying variable inhibition of Angiotensin II receptor AT1 activity were selected to develop models for establishing 2D and 3D QSAR. The compounds in the selected series were characterized by spatial, molecular and electro topological descriptors using QSAR module of Molecular Design Suite (VLife MDS™ 3.5. The best 2D QSAR model was selected, having correlation coefficient r2 (0.8056 and cross validated squared correlation coefficient q2 (0.6742 with external predictive ability of pred_r2 0.7583 coefficient of correlation of predicted data set (pred_r2se 0.2165. The results obtained from QSAR studies could be used in designing better Ang II activity among the congeners in future. The optimum QSAR model showed that the parameters SsssCHE index, SddCE-index, T_2_Cl_4, and SssNHE-index contributed in the model. 3D QSAR analysis by kNN-molecular field analysis approach developed based on principles of the k-nearest neighbor method combined with Genetic algorithms, stepwise forward variable selection approach; a leave-one-out cross-validated correlation coefficient (q2 of 0.6516 and a non-cross-validated correlation coefficient (r2 of 0.8316 and pred_r2 0.6954 were obtained. Contour maps using this approach showed that steric, electrostatic, and hydrophobic field effects dominantly determine binding affinities. The information rendered by 3D QSAR models may lead to a better understanding of structural requirements of Angiotensin II receptor and can help in the design of novel potent antihypertensive molecules.

  2. Organisation and functional role of the brain angiotensin system

    Directory of Open Access Journals (Sweden)

    Catherine Llorens-Cortes

    2002-03-01

    Full Text Available The discovery that all components of the renin-angiotensin system (RAS are present in the brain led investigators to postulate the existence of a local brain RAS. Supporting this, angiotensin immunoreactive neurones have been visualised in the brain. Two major pathways were described: a forebrain pathway which connects circumventricular organs to the median preoptic nucleus, paraventricular and supraoptic nuclei, and a second pathway connecting the hypothalamus to the medulla oblongata. Blood-brain-barrier deficient circumventricular organs are rich in angiotensin II (Ang II receptors. By activating these receptors, circulating Ang II may act on central cardiovascular centres via angiotensinergic neurones, providing a link between peripheral and central Ang II systems. Among the effector peptides of the brain RAS, Ang II and angiotensin III (Ang III have the same affinity for type 1 and type 2 Ang II receptors. When injected into the brain, both peptides increase blood pressure (BP, water intake and pituitary hormone release and may modify learning and memory. Since Ang II is converted in vivo to Ang III, the nature of the true effector is unknown. This review summarises new insights into the predominant role of brain Ang III in the control of BP and underlines the fact that brain aminopeptidase A, the enzyme forming central Ang III, could constitute a putative central therapeutic target for the treatment of hypertension.

  3. Angiotensin II type 2 receptors and cardiac hypertrophy in women with hypertrophic cardiomyopathy

    NARCIS (Netherlands)

    J. Deinum (Jacob); J.M. van Gool (Jeanette); M.J.M. Kofflard (Marcel); A.H.J. Danser (Jan); F.J. ten Cate (Folkert)

    2001-01-01

    textabstractThe development of left ventricular hypertrophy in subjects with hypertrophic cardiomyopathy (HCM) is variable, suggesting a role for modifying factors such as angiotensin II. Angiotensin II mediates both trophic and antitrophic effects, via angiotensin II type 1

  4. Increased angiotensin II type 1 receptor expression in temporal arteries from patients with giant cell arteritis

    DEFF Research Database (Denmark)

    Dimitrijevic, Ivan; Malmsjö, Malin; Andersson, Christina

    2009-01-01

    PURPOSE: Currently, giant cell arteritis (GCA) is primarily treated with corticosteroids or immunomodulating agents, but there is interest in identifying other noncorticosteroid alternatives. Similarities exist in the injury pathways between GCA and atherosclerosis. Angiotensin II is a vasoactive......, internal elastic lamina degeneration, and band-shaped infiltrates of inflammatory cells, including lymphocytes, histocytes, and multinucleated giant cells. AT(1) receptor staining was primarily observed in the medial layer of the temporal arteries and was higher in the patients with GCA than in the control...

  5. Allelic association of the D2 dopamine receptor gene with receptor-binding characteristics in alcoholism

    International Nuclear Information System (INIS)

    Noble, E.P.; Blum, K.; Ritchie, T.; Montgomery, A.; Sheridan, P.J.

    1991-01-01

    The allelic association of the human D2 dopamine receptor gene with the binding characteristics of the D2 dopamine receptor was determined in 66 brains of alcoholic and non-alcoholic subjects. In a blinded experiment, DNA from the cerebral cortex was treated with the restriction endonuclease Taql and probed with a 1.5-kilobase (kb) digest of a clone (lambda hD2G1) of the human D2 dopamine receptor gene. The binding characteristics (Kd [binding affinity] and Bmax [number of binding sites]) of the D2 dopamine receptor were determined in the caudate nuclei of these brains using tritiated spiperone as the ligand. The adjusted Kd was significantly lower in alcoholic than in nonalcoholic subjects. In subjects with the A1 allele, in whom a high association with alcoholism was found, the Bmax was significantly reduced compared with the Bmax of subjects with the A2 allele. Moreover, a progressively reduced Bmax was found in subjects with A2/A2, A1/A2, and A1/A1 alleles, with subjects with A2/A2 having the highest mean values, and subjects with A1/A1, the lowest. The polymorphic pattern of the D2 dopamine receptor gene and its differential expression of receptors suggests the involvement of the dopaminergic system in conferring susceptibility to at least one subtype of severe alcoholism

  6. Therapeutic targeting of angiotensin II receptor type 1 to regulate androgen receptor in prostate cancer.

    Science.gov (United States)

    Takahashi, Satoru; Uemura, Hiroji; Seeni, Azman; Tang, Mingxi; Komiya, Masami; Long, Ne; Ishiguro, Hitoshi; Kubota, Yoshinobu; Shirai, Tomoyuki

    2012-10-01

    With the limited strategies for curative treatment of castration-resistant prostate cancer (CRPC), public interest has focused on the potential prevention of prostate cancer. Recent studies have demonstrated that an angiotensin II receptor blocker (ARB) has the potential to decrease serum prostate-specific antigen (PSA) level and improve performance status in CRPC patients. These facts prompted us to investigate the direct effects of ARBs on prostate cancer growth and progression. Transgenic rat for adenocarcinoma of prostate (TRAP) model established in our laboratory was used. TRAP rats of 3 weeks of age received ARB (telmisartan or candesartan) at the concentration of 2 or 10 mg/kg/day in drinking water for 12 weeks. In vitro analyses for cell growth, ubiquitylation or reporter gene assay were performed using LNCaP cells. We found that both telmisartan and candesartan attenuated prostate carcinogenesis in TRAP rats by augmentation of apoptosis resulting from activation of caspases, inactivation of p38 MAPK and down-regulation of the androgen receptor (AR). Further, microarray analysis demonstrated up-regulation of estrogen receptor β (ERβ) by ARB treatment. In both parental and androgen-independent LNCaP cells, ARB inhibited both cell growth and AR-mediated transcriptional activity. ARB also exerted a mild additional effect on AR-mediated transcriptional activation by the ERβ up-regulation. An intervention study revealed that PSA progression was prolonged in prostate cancer patients given an ARB compared with placebo control. These data provide a new concept that ARBs are promising potential chemopreventive and chemotherapeutic agents for prostate cancer. Copyright © 2012 Wiley Periodicals, Inc.

  7. Five of Five VHHs Neutralizing Poliovirus Bind the Receptor-Binding Site.

    Science.gov (United States)

    Strauss, Mike; Schotte, Lise; Thys, Bert; Filman, David J; Hogle, James M

    2016-01-13

    Nanobodies, or VHHs, that recognize poliovirus type 1 have previously been selected and characterized as candidates for antiviral agents or reagents for standardization of vaccine quality control. In this study, we present high-resolution cryo-electron microscopy reconstructions of poliovirus with five neutralizing VHHs. All VHHs bind the capsid in the canyon at sites that extensively overlap the poliovirus receptor-binding site. In contrast, the interaction involves a unique (and surprisingly extensive) surface for each of the five VHHs. Five regions of the capsid were found to participate in binding with all five VHHs. Four of these five regions are known to alter during the expansion of the capsid associated with viral entry. Interestingly, binding of one of the VHHs, PVSS21E, resulted in significant changes of the capsid structure and thus seems to trap the virus in an early stage of expansion. We describe the cryo-electron microscopy structures of complexes of five neutralizing VHHs with the Mahoney strain of type 1 poliovirus at resolutions ranging from 3.8 to 6.3Å. All five VHHs bind deep in the virus canyon at similar sites that overlap extensively with the binding site for the receptor (CD155). The binding surfaces on the VHHs are surprisingly extensive, but despite the use of similar binding surfaces on the virus, the binding surface on the VHHs is unique for each VHH. In four of the five complexes, the virus remains essentially unchanged, but for the fifth there are significant changes reminiscent of but smaller in magnitude than the changes associated with cell entry, suggesting that this VHH traps the virus in a previously undescribed early intermediate state. The neutralizing mechanisms of the VHHs and their potential use as quality control agents for the end game of poliovirus eradication are discussed. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  8. Study of plasma binding of receptor-specific peptides

    OpenAIRE

    Gregor, David

    2008-01-01

    The binding ability of two receptor specific peptides namely 90Y-DOTA-TATE and 111In-DOTA-TATE was studied in therm of interspecies comparison by the method of equilibrium dialysis. This plasma protein binding was different for the chosen animal species (human, rat, rabbit, bovine eventually pork) whereas binding of 90Y-DOTA- TATE was higher than binding of 111In-DOTA-TATE. KEYWORDS: Protein binding, radiofarmaceuticals, equilibrium dialysis, 90Y-DOTA-TATE, 111In- DOTA-TATE

  9. [3H]Ethynylbicycloorthobenzoate ([3H]EBOB) binding in recombinant GABAA receptors.

    Science.gov (United States)

    Yagle, Monica A; Martin, Michael W; de Fiebre, Christopher M; de Fiebre, NancyEllen C; Drewe, John A; Dillon, Glenn H

    2003-12-01

    Ethynylbicycloorthobenzoate (EBOB) is a recently developed ligand that binds to the convulsant site of the GABAA receptor. While a few studies have examined the binding of [3H]EBOB in vertebrate brain tissue and insect preparations, none have examined [3H]EBOB binding in preparations that express known configurations of the GABAA receptor. We have thus examined [3H]EBOB binding in HEK293 cells stably expressing human alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and the effects of CNS convulsants on its binding. The ability of the CNS convulsants to displace the prototypical convulsant site ligand, [35S]TBPS, was also assessed. Saturation analysis revealed [3H]EBOB binding at a single site, with a K(d) of approximately 9 nM in alpha1beta2gamma2 and alpha2beta2gamma2 receptors. Binding of both [3H]EBOB and [35S]TBPS was inhibited by dieldrin, lindane, tert-butylbicycloorthobenzoate (TBOB), PTX, TBPS, and pentylenetetrazol (PTZ) at one site in a concentration-dependent fashion. Affinities were in the high nM to low microM range for all compounds except PTZ (low mM range), and the rank order of potency for these convulsants to displace [3H]EBOB and [35S]TBPS was the same. Low [GABA] stimulated [3H]EBOB binding, while higher [GABA] (greater than 10 microM) inhibited [3H]EBOB binding. Overall, our data demonstrate that [3H]EBOB binds to a single, high affinity site in alpha1beta2gamma2 and alpha2beta2gamma2 GABAA receptors, and modulation of its binding is similar to that seen with [35S]TBPS. [3H]EBOB has a number of desirable traits that may make it preferable to [35S]TBPS for analysis of the convulsant site of the GABAA receptor.

  10. Ascorbic acid enables reversible dopamine receptor 3H-agonist binding

    International Nuclear Information System (INIS)

    Leff, S.; Sibley, D.R.; Hamblin, M.; Creese, I.

    1981-01-01

    The effects of ascorbic acid on dopaminergic 3 H-agonist receptor binding were studied in membrane homogenates of bovine anterior pituitary and caudate, and rat striatum. In all tissues virtually no stereospecific binding (defined using 1uM (+)butaclamol) of the 3 H-agonists N-propylnorapomorphine (NPA), apomorphine, or dopamine could be demonstrated in the absence of ascorbic acid. Although levels of total 3 H-agonist binding were three to five times greater in the absence than in the presence of 0.1% ascorbic acid, the increased binding was entirely non-stereospecific. Greater amounts of dopamine-inhibitable 3 H-NPA binding could be demonstrated in the absence of 0.1% ascorbic acid, but this measure of ''specific binding'' was demonstrated not to represent dopamine receptor binding since several other catecholamines and catechol were equipotent with dopamine and more potent than the dopamine agonist (+/-)amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) in inhibiting this binding. High levels of dopamine-displaceable 3 H-agonist binding were detected in fresh and boiled homogenates of cerebellum, an area of brain which receives no dopaminergic innervation, further demonstrating the non-specific nature of 3 H-agonist binding in the absence of ascorbic acid. These studies emphasize that under typical assay conditions ascorbic acid is required in order to demonstrate reversible and specific 3 H-agonist binding to dopamine receptors

  11. Reduced post-synaptic serotonin type 1A receptor binding in bipolar depression

    Science.gov (United States)

    Nugent, Allison C.; Bain, Earle E.; Carlson, Paul J.; Neumeister, Alexander; Bonne, Omer; Carson, Richard E.; Eckelman, William; Herscovitch, Peter; Zarate, Carlos A.; Charney, Dennis S.; Drevets, Wayne C.

    2013-01-01

    Multiple lines of evidence suggest that serotonin type 1A (5-HT1A) receptor dysfunction is involved in the pathophysiology of mood disorders, and that alterations in 5-HT1A receptor function play a role in the mechanisms of antidepressant and mood stabilizer treatment. The literature is in disagreement, however, as to whether 5-HT1A receptor binding abnormalities exist in bipolar disorder (BD). We acquired PET images of 5-HT1A receptor binding in 26 unmedicated BD subjects and 37 healthy controls using [18F]FCWAY, a highly selective 5-HT1A receptor radio-ligand. The mean 5-HT1A receptor binding potential (BPP) was significantly lower in BD subjects compared to controls in cortical regions where 5-HT1A receptors are expressed post-synaptically, most prominently in the mesiotemporal cortex. Post-hoc assessments involving other receptor specific binding parameters suggested that this difference particularly affected the females with BD. The mean BPP did not differ between groups in the raphe nucleus, however, where 5-HT1A receptors are predominantly expressed pre-synaptically. Across subjects the BPP in the mesiotemporal cortex was inversely correlated with trough plasma cortisol levels, consistent with preclinical literature indicating that hippocampal 5-HT1A receptor expression is inhibited by glucocorticoid receptor stimulation. These findings suggest that 5-HT1A receptor binding is abnormally reduced in BD, and this abnormality may particularly involve the postsynaptic 5-HT1A receptor system of individuals with a tendency toward cortisol hypersecretion. PMID:23434290

  12. Fetal betamethasone exposure attenuates angiotensin-(1-7)-Mas receptor expression in the dorsal medulla of adult sheep.

    Science.gov (United States)

    Marshall, Allyson C; Shaltout, Hossam A; Nautiyal, Manisha; Rose, James C; Chappell, Mark C; Diz, Debra I

    2013-06-01

    Glucocorticoids including betamethasone (BM) are routinely administered to women entering into early preterm labor to facilitate fetal lung development and decrease infant mortality; however, fetal steroid exposure may lead to deleterious long term consequences. In a sheep model of fetal programming, BM-exposed (BMX) offspring exhibit elevated mean arterial pressure (MAP) and decreased baroreflex sensitivity (BRS) for control of heart rate by 0.5-years of age associated with changes in the circulating and renal renin-angiotensin systems (RAS). In the brain solitary tract nucleus, angiotensin (Ang) II actions through the AT1 receptor oppose the beneficial actions of Ang-(1-7) at the Mas receptor for BRS regulation. Therefore, we examined Ang peptides, angiotensinogen (Aogen), and receptor expression in this brain region of exposed and control offspring of 0.5- and 1.8-years of age. Mas protein expression was significantly lower (>40%) in the dorsal medulla of BMX animals at both ages; however, AT1 receptor expression was not changed. BMX offspring exhibited a higher ratio of Ang II to Ang-(1-7) (2.30±0.36 versus 0.99±0.28; p<0.01) and Ang II to Ang I at 0.5-years. Although total Aogen was unchanged, Ang I-intact Aogen was lower in 0.5-year BMX animals (0.78±0.06 vs. 1.94±0.41; p<0.05) suggesting a greater degree of enzymatic processing of the precursor protein in exposed animals. We conclude that in utero BM exposure promotes an imbalance in the central RAS pathways of Ang II and Ang-(1-7) that may contribute to the elevated MAP and lower BRS in this model. Copyright © 2013 Elsevier Inc. All rights reserved.

  13. Characterization of 125ITSH binding to its receptor in thyroid hyperplasies

    International Nuclear Information System (INIS)

    Bianco, A.C.; Nunes, M.T.

    1985-01-01

    An unpredictable and unbalanced response to a stimulus like TSH is indeed a striking characteristic of the follicles of the simple goiter. Since it is known that the first step for TSH action on its target cell is binding to specific TSH plasma membrane receptors, the binding of 125 ITSH to these receptors was studied in normal and ''cold'' hyperplastic thyroid fragments obtained at surgery. Through the Scatchard analysis it was verified that there are no differences with regard to the binding capacity of TSH receptors between normal and hyperplastic tissues. On the other hand, a significant decrease of the dissociation constant (Kd) was observed in hyperplastic tissue indicating higher affinity for TSH binding. It is known that intracellular iodine content can interfere with the TSH induced modifications on the thyroid folicular cells. It is supposed that this is mediated by interference on TSH binding to its receptor and/or activation of adenylate cyclase. Due to impaired organification capacity of ''cold'' tissue it is assumed that these cells present decreased intracellular iodine content. Therefore it is proposed that alterations of TSH binding to its receptors detected in the present investigation are consequent of the low iodine content of the hyperplastic folicular cell. (author) [pt

  14. Binding Mode of Insulin Receptor and Agonist Peptide

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Insulin is a protein hormone secreted by pancreatic β cells. One of its main functions is to keep the balance of glucose inside the body by regulating the absorption and metabolism of glucose in the periphery tissue, as well as the production and storage of hepatic glycogen. The insulin receptor is a transmembrane glycoprotein in which two α subunits with a molecular weight of 135 kD and twoβ subunits with a molecular weight of 95 kD are joined by a disulfide bond to form a β-α-α-β structure. The extracellular α subunit, especially, its three domains near the N-terminal are partially responsible for signal transduction or ligand-binding, as indicated by the experiments. The extracellular α subunits are involved in binding the ligands. The experimental results indicate that the three domains of the N-terminal of the α subunits are the main determinative parts of the insulin receptor to bind the insulin or mimetic peptide.We employed the extracellular domain (PDBID: 1IGR) of the insulin-like growth factor-1 receptor (IGF-1 R ) as the template to simulate and optimize the spatial structures of the three domains in the extracellular domain of the insulin receptor, which includes 468 residues. The work was accomplished by making use of the homology program in the Insight Ⅱ package on an Origin3800 server. The docking calculations of the insulin receptor obtained by homology with hexapeptides were carried out by means of the program Affinity. The analysis indicated that there were hydrogen bonding, and electrostatic and hydrophobic effects in the docking complex of the insulin receptor with hexapeptides.Moreover, we described the spatial orientation of a mimetic peptide with agonist activity in the docking complex. We obtained a rough model of binding of DLAPSQ or STIVYS with the insulin receptor, which provides the powerful theoretical support for designing the minimal insulin mimetic peptide with agonist activity, making it possible to develop oral small

  15. Competitive inhibition of [3H]dexamethasone binding to mammary glucocorticoid receptor by leupeptin

    International Nuclear Information System (INIS)

    Hsieh, L.C.C.; Su, C.; Markland, F.S. Jr.

    1987-01-01

    The inhibitory effect of leupeptin on [ 3 H]dexamethasone binding to the glucocorticoid receptor from lactating goat mammary cytosol has been studied. Leupeptin (10 mM) caused a significant (about 35%) inhibition of [ 3 H]dexamethasone binding to glucocorticoid receptor. Binding inhibition is further increased following filtration of unlabeled cytosolic receptor through a Bio-Gel A 0.5-m column. Binding inhibition was partially reversed by monothioglycerol at 10 mM concentration. A double reciprocal plot revealed that leupeptin appears to be a competitive inhibitor of [ 3 H]dexamethasone binding to the glucocorticoid receptor. Low salt sucrose density gradient centrifugation revealed that the leupeptin-treated sample formed a slightly larger (approximately 9 S) receptor complex (leupeptin-free complex sediments at 8 S)

  16. The angiotensin-(1-7/Mas axis counteracts angiotensin II-dependent and –independent pro-inflammatory signaling in human vascular smooth muscle cells

    Directory of Open Access Journals (Sweden)

    Laura A Villalobos

    2016-12-01

    Full Text Available Background and aims: Targeting inflammation is nowadays considered as a challenging pharmacological strategy to prevent or delay the development of vascular diseases. Angiotensin-(1-7 is a member of the renin-angiotensin system (RAS that binds Mas receptors and has gained growing attention in the last years as a regulator of vascular homeostasis. Here, we explored the capacity of Ang-(1-7 to counteract human aortic smooth muscle cell (HASMC inflammation triggered by RAS-dependent and –independent stimuli, such as Ang II or interleukin (IL-1.Methods and Results: In cultured HASMC, the expression of iNOS and the release of nitric oxide were stimulated by both Ang II and IL-1, as determined by Western blot and indirect immunofluorescence or the Griess method, respectively. iNOS induction was inhibited by Ang-(1-7 in a concentration-dependent manner. This effect was equally blocked by two different Mas receptor antagonists, A779 and D-Pro7-Ang-(1-7, suggesting the participation of a unique Mas receptor subtype. Using pharmacological inhibitors, the induction of iNOS was proven to rely on the consecutive upstream activation of NADPH oxidase and NF-B. Indeed, Ang-(1-7 markedly inhibited the activation of the NADPH oxidase and subsequently of NF-B, as determined by lucigenin-derived chemiluminiscence and electromobility shift assay, respectively.Conclusion: Ang-(1-7 can act as a counter-regulator of the inflammation of vascular smooth muscle cells triggered by Ang II, but also by other stimuli beyond the RAS. Activating or mimicking the Ang-(1-7/Mas axis may represent a pharmacological opportunity to attenuate the pro-inflammatory environment that promotes and sustains the development of vascular diseases.

  17. Purification and characterization of the V1 vasopressin receptor from rat liver

    International Nuclear Information System (INIS)

    Fishman, J.B.; Dickey, B.F.; Attisano, C.; Fine, R.E.

    1987-01-01

    The rat liver V1 vasopressin receptor was purified approximately 21,000-fold from rat liver microsomes. The receptor was solubilized from membranes using the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate). Since the V1 receptor loses its ability to bind ligand when solubilized, the authors devised a liposome reconstitution system to assay vasopressin binding activity during purification. The purified receptor exhibits a K/sub d/ of 6 nm, when, prior to solubilization, the membranes were exposed to 1 μm vasopressin. This resulted in the association of a pertussis-toxin insensitive guanine-nucleotide binding protein with the receptor during most of the purification procedure. The authors are further characterizing the V1-associated G-proteins. In the absence of this association, the receptor has a K/sub d/ of 30 nM. Crosslinking of 125 I-vasopressin to a partially purified preparation of receptor demonstrated that the receptor had a molecular weight of approximately 68,000 under reducing conditions, and 58,000 under non-reducing conditions. The purification procedure may prove useful in purifying a number of small peptide hormone receptors (e.g., bradykinin, angiotensin II) and perhaps their associated G-proteins as well

  18. Interaction of chemokines with their receptors--from initial chemokine binding to receptor activating steps

    DEFF Research Database (Denmark)

    Thiele, Stefanie; Rosenkilde, Mette Marie

    2014-01-01

    and surveillance. Chemokines are a group of 8-12 kDa large peptides with a secondary structure consisting of a flexible N-terminus and a core-domain usually stabilized by two conserved disulfide bridges. They mainly interact with the extracellular domains of their cognate 7TM receptors. Affinityand activity......-contributing interactions are attributed to different domains and known to occur in two steps. Here, knowledge on chemokine and receptor domains involved in the first binding-step and the second activation-step is reviewed. A mechanism comprising at least two steps seems consistent; however, several intermediate...... interactions possibly occur, resulting in a multi-step process, as recently proposed for other 7TM receptors. Overall, the N-terminus of chemokine receptors is pivotal for binding of all chemokines. During receptor activation, differences between the two major chemokine subgroups occur, as CC-chemokines mainly...

  19. Dopamine receptors in the guinea-pig heart. A binding study

    International Nuclear Information System (INIS)

    Sandrini, M.; Benelli, A.; Baraldi, M.

    1984-01-01

    The binding of dopaminergic agonists and antagonists to guinea-pig myocardial membrane preparations was studied using 3 H-dopamine and 3 H-spiperone as radioligand. 3 H-Dopamine bound specifically to heart membranes while 3 H-spiperone did not. A Scatchard analysis of 3 H-dopamine binding showed a curvilinear plot indicating the presence of two dopamine receptor populations that we have termed high- (K/sub d/ = 1.2 nM, B/sub mx/ = 52.9 fmol/mg prot.) and low- (K/sub d/ = 11.8 nM, B/sub mx/ = 267.3 fmol/gm prot.) affinity binding sites, respectively. The charactization of the high-affinity component of 3 H-dopamine binding indicated that the binding is rapid, saturable, stereospecific, pH- and temperature-dependent, and displaced by dopaminergic agonists and antagonists known to act similarly in vivo. The finding that pretreatment with dibenamine (which has been described as an α-adrenoceptor irreversible blocker) did not affect the binding of dopamine to cardiac membrane preparations suggests that α-adrenoceptors and dopamine receptors have separate recognition sites in the heart. It is concluded that 3 H-dopamine binds to specific dopamine receptors in the heart of guinea-pigs

  20. Angiotensin converting enzyme (ACE and ACE2 bind integrins and ACE2 regulates integrin signalling.

    Directory of Open Access Journals (Sweden)

    Nicola E Clarke

    Full Text Available The angiotensin converting enzymes (ACEs are the key catalytic components of the renin-angiotensin system, mediating precise regulation of blood pressure by counterbalancing the effects of each other. Inhibition of ACE has been shown to improve pathology in cardiovascular disease, whilst ACE2 is cardioprotective in the failing heart. However, the mechanisms by which ACE2 mediates its cardioprotective functions have yet to be fully elucidated. Here we demonstrate that both ACE and ACE2 bind integrin subunits, in an RGD-independent manner, and that they can act as cell adhesion substrates. We show that cellular expression of ACE2 enhanced cell adhesion. Furthermore, we present evidence that soluble ACE2 (sACE2 is capable of suppressing integrin signalling mediated by FAK. In addition, sACE2 increases the expression of Akt, thereby lowering the proportion of the signalling molecule phosphorylated Akt. These results suggest that ACE2 plays a role in cell-cell interactions, possibly acting to fine-tune integrin signalling. Hence the expression and cleavage of ACE2 at the plasma membrane may influence cell-extracellular matrix interactions and the signalling that mediates cell survival and proliferation. As such, ectodomain shedding of ACE2 may play a role in the process of pathological cardiac remodelling.

  1. Long-term use of angiotensin receptor blockers and the risk of cancer.

    Directory of Open Access Journals (Sweden)

    Laurent Azoulay

    Full Text Available The association between angiotensin receptor blockers (ARBs and cancer is controversial with meta-analyses of randomized controlled trials and observational studies reporting conflicting results. Thus, the objective of this study was to determine whether ARBs are associated with an overall increased risk of the four most common cancers, namely, lung, colorectal, breast and prostate cancers, and to explore these effects separately for each cancer type. We conducted a retrospective cohort study using a nested case-control analysis within the United Kingdom (UK General Practice Research Database. We assembled a cohort of patients prescribed antihypertensive agents between 1995, the year the first ARB (losartan entered the UK market, and 2008, with follow-up until December 31, 2010. Cases were patients newly-diagnosed with lung, colorectal, breast and prostate cancer during follow-up. We used conditional logistic regression to estimate adjusted rate ratios (RRs and 95% confidence intervals (CIs of cancer incidence, comparing ever use of ARBs with ever use of diuretics and/or beta-blockers. The cohort included 1,165,781 patients, during which 41,059 patients were diagnosed with one of the cancers under study (rate 554/100,000 person-years. When compared to diuretics and/or beta-blockers, ever use of ARBs was not associated with an increased rate of cancer overall (RR: 1.00; 95% CI: 0.96-1.03 or with each cancer site separately. The use of angiotensin-converting enzyme inhibitors and calcium channel blockers was associated with an increased rate of lung cancer (RR: 1.13; 95% CI: 1.06-1.20 and RR: 1.19; 95% CI: 1.12-1.27, respectively. This study provides additional evidence that the use of ARBs does not increase the risk of cancer overall or any of the four major cancer sites. Additional research is needed to further investigate a potentially increased risk of lung cancer with angiotensin-converting enzyme inhibitors and calcium channel blockers.

  2. Disposition and metabolism of [(14)C] Sacubitril/Valsartan (formerly LCZ696) an angiotensin receptor neprilysin inhibitor, in healthy subjects.

    Science.gov (United States)

    Flarakos, Jimmy; Du, Yancy; Bedman, Timothy; Al-Share, Qusai; Jordaan, Pierre; Chandra, Priya; Albrecht, Diego; Wang, Lai; Gu, Helen; Einolf, Heidi J; Huskey, Su-Er; Mangold, James B

    2016-11-01

    1. Sacubitril/valsartan (LCZ696) is an angiotensin receptor neprilysin inhibitor (ARNI) providing simultaneous inhibition of neprilysin (neutral endopeptidase 24.11; NEP) and blockade of the angiotensin II type-1 (AT1) receptor. 2. Following oral administration, [(14)C]LCZ696 delivers systemic exposure to valsartan and AHU377 (sacubitril), which is rapidly metabolized to LBQ657 (M1), the biologically active neprilysin inhibitor. Peak sacubitril plasma concentrations were reached within 0.5-1 h. The mean terminal half-lives of sacubitril, LBQ657 and valsartan were ∼1.3, ∼12 and ∼21 h, respectively. 3. Renal excretion was the dominant route of elimination of radioactivity in human. Urine accounted for 51.7-67.8% and feces for 36.9 to 48.3 % of the total radioactivity. The majority of the drug was excreted as the active metabolite LBQ657 in urine and feces, total accounting for ∼85.5% of the total dose. 4. Based upon in vitro studies, the potential for LCZ696 to inhibit or induce cytochrome P450 (CYP) enzymes and cause CYP-mediated drug interactions clinically was found to be low.

  3. In vitro binding and receptor-mediated activity of terlipressin at vasopressin receptors V1 and V2.

    Science.gov (United States)

    Jamil, Khurram; Pappas, Stephen Chris; Devarakonda, Krishna R

    2018-01-01

    Terlipressin, a synthetic, systemic vasoconstrictor with selective activity at vasopressin-1 (V 1 ) receptors, is a pro-drug for the endogenous/natural porcine hormone [Lys 8 ]-vasopressin (LVP). We investigated binding and receptor-mediated cellular activities of terlipressin, LVP, and endogenous human hormone [Arg 8 ]-vasopressin (AVP) at V 1 and vasopressin-2 (V 2 ) receptors. Cell membrane homogenates of Chinese hamster ovary cells expressing human V 1 and V 2 receptors were used in competitive binding assays to measure receptor-binding activity. These cells were used in functional assays to measure receptor-mediated cellular activity of terlipressin, LVP, and AVP. Binding was measured by [ 3 H]AVP counts, and the activity was measured by fluorometric detection of intracellular calcium mobilization (V 1 ) and cyclic adenosine monophosphate (V 2 ). Binding potency at V 1 and V 2 was AVP>LVP>terlipressin. LVP and terlipressin had approximately sixfold higher affinity for V 1 than for V 2 . Cellular activity potency was also AVP>LVP>terlipressin. Terlipressin was a partial agonist at V 1 and a full agonist at V 2 ; LVP was a full agonist at both V 1 and V 2 . The in vivo response to terlipressin is likely due to the partial V 1 agonist activity of terlipressin and full V 1 agonist activity of its metabolite, LVP. These results provide supportive evidence for previous findings and further establish terlipressin pharmacology for vasopressin receptors.

  4. Guanine nucleotide-binding protein regulation of melatonin receptors in lizard brain

    International Nuclear Information System (INIS)

    Rivkees, S.A.; Carlson, L.L.; Reppert, S.M.

    1989-01-01

    Melatonin receptors were identified and characterized in crude membrane preparations from lizard brain by using 125 I-labeled melatonin ( 125 I-Mel), a potent melatonin agonist. 125 I-Mel binding sites were saturable; Scatchard analysis revealed high-affinity and lower affinity binding sites, with apparent K d of 2.3 ± 1.0 x 10 -11 M and 2.06 ± 0.43 x 10 -10 M, respectively. Binding was reversible and inhibited by melatonin and closely related analogs but not by serotonin or norepinephrine. Treatment of crude membranes with the nonhydrolyzable GTP analog guanosine 5'-[γ-thio]triphosphate (GTP[γS]), significantly reduced the number of high-affinity receptors and increased the dissociation rate of 125 I-Mel from its receptor. Furthermore, GTP[γS] treatment of ligand-receptor complexes solubilized by Triton X-100 also led to a rapid dissociation of 125 I-Mel from solubilized ligand-receptor complexes. Gel filtration chromatography of solubilized ligand-receptor complexes revealed two major peaks of radioactivity corresponding to M r > 400,000 and M r ca. 110,000. This elution profile was markedly altered by pretreatment with GTP[γS] before solubilization; only the M r 110,000 peak was present in GTP[γS]-pretreated membranes. The results strongly suggest that 125 I-mel binding sites in lizard brain are melatonin receptors, with agonist-promoted guanine nucleotide-binding protein (G protein) coupling and that the apparent molecular size of receptors uncoupled from G proteins is about 110,000

  5. Molecular characterization of the receptor binding structure-activity relationships of influenza B virus hemagglutinin.

    Science.gov (United States)

    Carbone, V; Kim, H; Huang, J X; Baker, M A; Ong, C; Cooper, M A; Li, J; Rockman, S; Velkov, T

    2013-01-01

    Selectivity of α2,6-linked human-like receptors by B hemagglutinin (HA) is yet to be fully understood. This study integrates binding data with structure-recognition models to examine the impact of regional-specific sequence variations within the receptor-binding pocket on selectivity and structure activity relationships (SAR). The receptor-binding selectivity of influenza B HAs corresponding to either B/Victoria/2/1987 or the B/Yamagata/16/88 lineages was examined using surface plasmon resonance, solid-phase ELISA and gel-capture assays. Our SAR data showed that the presence of asialyl sugar units is the main determinant of receptor preference of α2,6 versus α2,3 receptor binding. Changes to the type of sialyl-glycan linkage present on receptors exhibit only a minor effect upon binding affinity. Homology-based structural models revealed that structural properties within the HA pocket, such as a glyco-conjugate at Asn194 on the 190-helix, sterically interfere with binding to avian receptor analogs by blocking the exit path of the asialyl sugars. Similarly, naturally occurring substitutions in the C-terminal region of the 190-helix and near the N-terminal end of the 140-loop narrows the horizontal borders of the binding pocket, which restricts access of the avian receptor analog LSTa. This study helps bridge the gap between ligand structure and receptor recognition for influenza B HA; and provides a consensus SAR model for the binding of human and avian receptor analogs to influenza B HA.

  6. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

    International Nuclear Information System (INIS)

    Wang, Xianwei; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L.

    2012-01-01

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22 phox , p47 phox , p67 phox , NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H 2 O 2 . Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast growth and collagen

  7. Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

    2012-03-15

    Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

  8. Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy.

    Science.gov (United States)

    Kadam, Parnika; McAllister, Ryan; Urbach, Jeffrey S; Sandberg, Kathryn; Mueller, Susette C

    2017-03-27

    Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

  9. Identification of steroid-binding and phosphorylated sites within the glucocorticoid receptor

    International Nuclear Information System (INIS)

    Smith, L.I.

    1989-01-01

    The primary goal of these studies was to localize the steroid-binding and phosphorylated sites of the glucocorticoid receptor. The synthetic steroid, dexamethasone 21-mesylate (DM) forms a covalent thioether bond via the sulfhydryl group of a cysteine residue in the receptor. To determine the covalent site of attachment of this ligand, receptors in WEHI-7 mouse thymoma cells were labeled with [ 3 H]DM and purified with a monoclonal antibody. The receptor was completely digested with trypsin and a single peptide covalently labeled with steroid identified by reversed-phase HPLC. This peptide was analyzed by automated Edman degradation to determine the location of the steroid-labeled residue. A similar analysis was performed on an overlapping peptide produced by Staphylococcus aureus protease digestion. Analysis of tryptic peptides from receptors labeled with both [ 3 H]DM and L-[ 35 S]methionine indicated that this peptide contained methionine. These analyses, coupled with the published amino acid sequence of the receptor, identified Cysteine-644 in the steroid-binding domain of the mouse glucocorticoid receptor as the residue involved in covalent steroid-binding. A synthetic peptide representing amino acids 640-650 of the mouse receptor was prepared and analyzed to confirm the identification. These biochemical studies represent a direct demonstration of an amino acid important in receptor function. It has been proposed that the receptor functions through a phosphorylation-dephosphorylation cycle to explain the dependence of hormone binding capacity upon cellular ATP. The glucocorticoid receptor has been shown to be a phosphoprotein. As an initial step to identifying a role of phosphorylation in receptor action, phosphorylated sites within the functional domains of the protein were identified

  10. Human corpus luteum: presence of epidermal growth factor receptors and binding characteristics

    International Nuclear Information System (INIS)

    Ayyagari, R.R.; Khan-Dawood, F.S.

    1987-01-01

    Epidermal growth factor receptors are present in many reproductive tissues but have not been demonstrated in the human corpus luteum. To determine the presence of epidermal growth factor receptors and its binding characteristics, we carried out studies on the plasma cell membrane fraction of seven human corpora lutea (days 16 to 25) of the menstrual cycle. Specific epidermal growth factor receptors were present in human corpus luteum. Insulin, nerve growth factor, and human chorionic gonadotropin did not competitively displace epidermal growth factor binding. The optimal conditions for corpus luteum-epidermal growth factor receptor binding were found to be incubation for 2 hours at 4 degrees C with 500 micrograms plasma membrane protein and 140 femtomol 125 I-epidermal growth factor per incubate. The number (mean +/- SEM) of epidermal growth factor binding sites was 12.34 +/- 2.99 X 10(-19) mol/micrograms protein; the dissociation constant was 2.26 +/- 0.56 X 10(-9) mol/L; the association constant was 0.59 +/- 0.12 X 10(9) L/mol. In two regressing corpora lutea obtained on days 2 and 3 of the menstrual cycle, there was no detectable specific epidermal growth factor receptor binding activity. Similarly no epidermal growth factor receptor binding activity could be detected in ovarian stromal tissue. Our findings demonstrate that specific receptors for epidermal growth factor are present in the human corpus luteum. The physiologic significance of epidermal growth factor receptors in human corpus luteum is unknown, but epidermal growth factor may be involved in intragonadal regulation of luteal function

  11. Glycosylation of immunoglobulin A influences its receptor binding.

    Science.gov (United States)

    Basset, C; Devauchelle, V; Durand, V; Jamin, C; Pennec, Y L; Youinou, P; Dueymes, M

    1999-12-01

    Immunoglobulin A (IgA), which is heavily glycosylated, interacts with a variety of receptors, e.g. the asialoglycoprotein receptor (ASGP-R), which binds terminal galactose residues, and the Fcalpha receptor (FcalphaRI). It has thus been proposed that elevated serum levels of IgA in primary Sjögren's syndrome (pSS) are caused by its defective clearance. To test this hypothesis, we developed a method (based on sialyl transferases eluted from a hepatoma cell line) to increase the amount of sialic acid (SA) on IgA, and used a battery of IgA1- and IgA2-specific glycosidases to reduce this amount. Binding of IgA1 and IgA2 to ASGP-R and FcalphaRI was found to be sugar dependent because oversialylated IgA bound less than native or desialylated IgA. However, individual sugars did not play a direct role in this binding. Given that IgA are oversialylated in pSS, defective clearance of IgA may indeed be ascribed to an excess of SA in IgA1 and IgA2.

  12. The effect of angiotensin 1-7 on tyrosine kinases activity in rat anterior pituitary

    International Nuclear Information System (INIS)

    Rebas, Elzbieta; Zabczynska, Joanna; Lachowicz, Agnieszka

    2006-01-01

    Angiotensin 1-7 (Ang 1-7) is a peptide originated from Ang II. It is known that in vessels Ang 1-7 shows opposite effects to Ang II. Ang 1-7 can modify processes of proliferation. However, Ang 1-7 action in pituitary gland cells was never studied. Moreover, the specific binding sites for Ang 1-7 are still unknown. The aim of this study was to examine the effects of Ang 1-7 on tyrosine kinases (PTKs) activity in the anterior pituitary. The reaction of phosphorylation was carrying out in presence of different concentration of Ang 1-7 and losartan (antagonist of AT1 receptor) and PD123319 (antagonist of AT2). Our results show that Ang 1-7 inhibited activity of PTK to 60% of basic activity. Losartan did not change the Ang 1-7-induced changes in PTKs activity. The presence of PD123319 together with Ang 1-7 caused stronger inhibition PTKs activity than Ang 1-7 alone. These observations suggest that Ang 1-7 binds to the novel, unknown, specific for this peptide receptor

  13. Preadmission use of renin-angiotensin blockers and rupture of abdominal aortic aneurysm

    DEFF Research Database (Denmark)

    Wemmelund, Holger; Høgh, Annette; Hundborg, Heidi H.

    2016-01-01

    PURPOSE: Rupture of abdominal aortic aneurysms (rAAA) is associated with high mortality. Use of angiotensin converting enzyme inhibitors (ACE-inhibitors) and angiotensin receptor blockers (ARBs) has been suggested to reduce the risk of rAAA. This nationwide, combined case-control and follow-up st...

  14. Induction of human adiponectin gene transcription by telmisartan, angiotensin receptor blocker, independently on PPAR-γ activation

    International Nuclear Information System (INIS)

    Moriuchi, Akie; Yamasaki, Hironori; Shimamura, Mika; Kita, Atsushi; Kuwahara, Hironaga; Fujishima, Keiichiro; Satoh, Tsuyoshi; Fukushima, Keiko; Fukushima, Tetsuya; Hayakawa, Takao; Mizuguchi, Hiroyuki; Nagayama, Yuji; Abiru, Norio; Kawasaki, Eiji; Eguchi, Katsumi

    2007-01-01

    Adiponectin, an adipose tissue-specific plasma protein, has been shown to ameliorate insulin resistance and inhibit the process of atherosclerosis. Recently, several reports have stated that angiotensin type 1 receptor blockers (ARBs), increase adiponectin plasma level, and ameliorate insulin resistance. Telmisartan, a subclass of ARBs, has been shown to be a partial agonist of the peroxisome proliferator-activated receptor (PPAR)-γ, and to increase the plasma adiponectin level. However, the transcriptional regulation of the human adiponectin gene by telmisartan has not been determined yet. To elucidate the effect of telmisartan on adiponectin, the stimulatory regulation of human adiponectin gene by telmisartan was investigated in 3T3-L1 adipocytes, utilizing adenovirus-mediated luciferase reporter gene-transferring technique. This study indicates that telmisartan may stimulate adiponectin transcription independent of PPAR-γ

  15. Low 5-HT1B receptor binding in the migraine brain

    DEFF Research Database (Denmark)

    Deen, Marie; Hansen, Hanne D; Hougaard, Anders

    2018-01-01

    Background The pathophysiology of migraine may involve dysfunction of serotonergic signaling. In particular, the 5-HT1B receptor is considered a key player due to the efficacy of 5-HT1B receptor agonists for treatment of migraine attacks. Aim To examine the cerebral 5-HT1B receptor binding....... Patients who reported migraine brain regions involved in pain modulation as regions of interest and applied a latent variable model (LVM) to assess the group effect on binding across these regions. Results Our data...... support a model wherein group status predicts the latent variable ( p = 0.038), with migraine patients having lower 5-HT1B receptor binding across regions compared to controls. Further, in a whole-brain voxel-based analysis, time since last migraine attack correlated positively with 5-HT1B receptor...

  16. Pitting type of pretibial edema in a patient with silent thyroiditis successfully treated by angiotensin ii receptor blockade.

    Science.gov (United States)

    Kazama, Itsuro; Mori, Yoko; Baba, Asuka; Nakajima, Toshiyuki

    2014-01-01

    Female, 56 FINAL DIAGNOSIS: Thyroiditis - silent Symptoms: Palpitations • pretibial pitting edema • short of breath • sweating - Clinical Procedure: - Specialty: Endocrinology and Metabolic. Unknown etiology. Hyper- or hypothyroidism sometimes causes pretibial myxedema characterized by non-pitting infiltration of a proteinaceous ground substance. However, in those patients, the "pitting" type of pretibial edema as a result of increased sodium and fluid retention or vascular hyper-permeability rarely occurs, except in cases complicated by heart failures due to severe cardiomyopathy or pulmonary hypertension. A 56-year-old woman developed bilateral pretibial pitting edema, followed by occasional sweating, palpitations, and shortness of breath, which persisted for more than 2 months. The diagnosis of hyperthyroidism due to silent thyroiditis was supported by elevated levels of free thyroxine (T4) and triiodothyronine (T3), with a marked decrease in thyroid-stimulating hormone (TSH), and the negative results for TSH receptor antibodies with typical findings of destructive thyrotoxicosis. Despite her "pitting" type of pretibial edema, a chest radio-graph demonstrated the absence of cardiomyopathy or congestive heart failure. Oral administration of angiotensin II receptor blocker (ARB) was initiated for her systolic hypertension, with a relatively higher elevation of plasma renin activity compared to that of the aldosterone level. Although the symptoms characteristic to hyperthyroidism, such as increased sweating, palpitations and shortness of breath, slowly improved with a spontaneous resolution of the disease, ARB quickly resolved the pretibial pitting edema shortly after the administration.. In this case, increased activity of the renin-angiotensin-aldosterone system stimulated by thyroid hormone was likely responsible for the patient's pitting type of edema. The pharmacological blockade of the renin-angiotensin-aldosterone system was thought to be effective for

  17. Effect of the AT1-receptor antagonists losartan, irbesartan, and telmisartan on angiotensin II-induced facilitation of sympathetic neurotransmission in the rat mesenteric artery

    NARCIS (Netherlands)

    Balt, J. C.; Mathy, M. J.; Nap, A.; Pfaffendorf, M.; van Zwieten, P. A.

    2001-01-01

    SUMMARY: The effect of the AT1-receptor antagonists losartan, irbesartan, and telmisartan on angiotensin II (Ang II)-induced facilitation of noradrenergic neurotransmission was investigated in the isolated rat mesenteric artery under isometric conditions. Electrical field stimulation (2, 4, and 8

  18. Association of Angiotensin-Converting Enzyme Intron 16 Insertion/Deletion and Angiotensin II Type 1 Receptor A1166C Gene Polymorphisms with Preeclampsia in South East of Iran

    Directory of Open Access Journals (Sweden)

    Saeedeh Salimi

    2011-01-01

    Full Text Available Some evidence suggests that a variety of genetic factors contributed in pathogenesis of the preeclampsia. The aim of this study was to assess the association between the angiotensin-converting enzyme (ACE I/D and angiotensin II type1 receptor A1166C polymorphisms with preeclampsia. This study was performed in 125 preeclamptic pregnant women and 132 controls. The I/D Polymorphism of the ACE gene was assessed by polymerase chain reaction and the A1166C Polymorphism of the AT1R gene was determined by restriction fragment length polymorphism. The genotype and allele frequencies of I/D polymorphism differed between two groups. The risk of preeclampsia was 3.2-fold in pregnant women with D allele (OR, 3.2 [95% CI, 1.1 to 3.8]; P=0.01. The distribution of the AT1R gene A1166C polymorphism was similar in affected and control groups. Our results supported that presence of the I/D polymorphism of ACE gene is a marker for the increased risk of preeclampsia.

  19. RTA, a candidate G protein-coupled receptor: Cloning, sequencing, and tissue distribution

    International Nuclear Information System (INIS)

    Ross, P.C.; Figler, R.A.; Corjay, M.H.; Barber, C.M.; Adam, N.; Harcus, D.R.; Lynch, K.R.

    1990-01-01

    Genomic and cDNA clones, encoding a protein that is a member of the guanine nucleotide-binding regulatory protein (G protein)-coupled receptor superfamily, were isolated by screening rat genomic and thoracic aorta cDNA libraries with an oligonucleotide encoding a highly conserved region of the M 1 muscarinic acetylcholine receptor. Sequence analyses of these clones showed that they encode a 343-amino acid protein (named RTA). The RTA gene is single copy, as demonstrated by restriction mapping and Southern blotting of genomic clones and rat genomic DNA. RTA RNA sequences are relatively abundant throughout the gut, vas deferens, uterus, and aorta but are only barely detectable (on Northern blots) in liver, kidney, lung, and salivary gland. In the rat brain, RTA sequences are markedly abundant in the cerebellum. TRA is most closely related to the mas oncogene (34% identity), which has been suggested to be a forebrain angiotensin receptor. They conclude that RTA is not an angiotensin receptor; to date, they have been unable to identify its ligand

  20. Synthesis and receptor binding studies of (+/-)1-iodo-MK-801

    International Nuclear Information System (INIS)

    Yang, D.J.; Ciliax, B.J.; Van Dort, M.E.; Gildersleeve, D.; Pirat, J.L.; Young, A.B.; Wieland, D.M.

    1989-01-01

    The glutamate analogue N-methyl-D-aspartate (NMDA) binds to a subset of glutamate receptors that are coupled to a voltage-sensitive cation channel. This NMDA-linked channel is the likely binding locus of the potent anticonvulsant MK-801. To develop single-photon emission computed tomography (SPECT) probes of this brain channel, we synthesized (+/)1-iodo-MK-801 and (+/-)1-[ 125 I]iodo-MK-801. The effect of (+/-)1-iodo-MK-801 on ligand binding to the NMDA-linked glutamate receptor site was assessed using a rat brain homogenate assay. (+/-)1-Iodo-MK-801 displaced the dissociative anesthetic ligand [ 3 H]N-[1-(2-thienyl)cyclohexyl]piperidine ([ 3 H]TCP) binding with an IC50 of 1 microM, which is a 10-fold lower binding affinity than that of (+/-)MK-801. In in vivo autoradiographic studies, (+/-)MK-801 failed to block selective uptake of (+/-)1-iodo-MK-801 in rat brain. These results suggest that (+/-)1-iodo-MK-801 may not be a suitable ligand for mapping NMDA-linked glutamate receptor channels

  1. A novel mechanism of angiotensin II-regulated placental vascular tone in the development of hypertension in preeclampsia.

    Science.gov (United States)

    Gao, Qinqin; Tang, Jiaqi; Li, Na; Zhou, Xiuwen; Li, Yongmei; Liu, Yanping; Wu, Jue; Yang, Yuxian; Shi, Ruixiu; He, Axin; Li, Xiang; Zhang, Yingying; Chen, Jie; Zhang, Lubo; Sun, Miao; Xu, Zhice

    2017-05-09

    The present study tested the hypothesis that angiotensin II plays a role in the regulation of placental vascular tone, which contributes to hypertension in preeclampsia. Functional and molecular assays were performed in large and micro placental and non-placental vessels from humans and animals. In human placental vessels, angiotensin II induced vasoconstrictions in 78.7% vessels in 155 tests, as referenced to KCl-induced contractions. In contrast, phenylephrine only produced contractions in 3.0% of 133 tests. In non-placental vessels, phenylephrine induced contractions in 76.0% of 67 tests, whereas angiotensin II failed to produce contractions in 75 tests. Similar results were obtained in animal placental and non-placental vessels. Compared with non-placental vessels, angiotensin II receptors and β-adrenoceptors were significantly increased in placental vessels. Compared to the vessels from normal pregnancy, angiotensin II-induced vasoconstrictions were significantly reduced in preeclamptic placentas, which was associated with a decrease in angiotensin II receptors. In addition, angiotensin II and angiotensin converting enzyme in the maternal-placenta circulation in preeclampsia were increased, whereas angiotensin I and angiotensin1-7 concentrations were unchanged. The study demonstrates a selective effect of angiotensin II in maintaining placental vessel tension, which may play an important role in development of hypertension in preeclampsia.

  2. Association between exposure to angiotensin-converting enzyme inhibitors and angiotensin receptor blockers prior to septic shock and acute kidney injury.

    Science.gov (United States)

    Suberviola, B; Rodrigo, E; González-Castro, A; Serrano, M; Heras, M; Castellanos-Ortega, Á

    To evaluate the association between angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) use prior to a septic shock episode and the development, prognosis and long-term recovery from acute kidney injury (AKI). A single-centre, prospective observational study was carried out between September 2005 and August 2010. Patients admitted to the ICU of a third level hospital. A total of 386 septic shock patients were studied. None. Use of ACEIs/ARBs, AKI development, recovery of previous creatinine levels and time to recovery. A total of 386 patients were included, of which 312 (80.8%) developed AKI during ICU stay and 23% were receiving ACEIs/ARBs. The percentage of patients on ACEIs/ARBs increased significantly in relation to more severe stages of AKI irrespective of the kind of AKI score. After adjusting for confounders, the development of AKI was independently associated to the use of ACEIs/ARBs (OR 2.19; 95%CI 1.21-3.84; p=.04). With respect to the recovery of kidney function, the group of patients on ACEIs/ARBs had significantly higher creatinine levels at ICU discharge and needed hemodialysis more frequently thereafter. However, use of ACEIs/ARBs affected neither recovery of previous creatinine levels nor significantly delayed recovery. The use of ACEIs/ARBs before septic shock episodes was correlated to AKI development and severity, but did not affect the recovery of kidney function after sepsis resolution. Copyright © 2016 Elsevier España, S.L.U. y SEMICYUC. All rights reserved.

  3. Angiotensin-converting enzyme inhibitors and angiotensin II receptor blockers reduced dementia risk in patients with diabetes mellitus and hypertension.

    Science.gov (United States)

    Kuan, Yi-Chun; Huang, Kuang-Wei; Yen, Der-Jen; Hu, Chaur-Jong; Lin, Cheng-Li; Kao, Chia-Hung

    2016-10-01

    The effects of angiotensin-converting enzyme inhibitors (ACEI) and angiotensin II receptor blockers (ARB) on dementia risk in patients with type 2 diabetes mellitus (DM) and hypertension remain unknown. We investigated the effects of ACEIs and ARBs on dementia risk in patients with type 2 DM and hypertension. We conducted a cohort study by using the Taiwan National Health Insurance Research Database. We included 2377 patients receiving ACEIs and 1780 patients receiving ARBs in the ACEI and ARB cohorts, respectively. We included a comparable number of patients not receiving ACEIs and ARBs as controls in the non-ACEI and non-ARB cohorts through propensity score matching. The effect of ACEIs and ARBs on dementia risk was estimated through multivariate Cox proportional hazard regression after adjustment for several confounding factors. During the 12-year follow-up period, compared with the non-ACEI cohort, all-cause dementia risk decreased by 26% in the ACEI cohort [hazard ratio (HR)=0.74, 95% confidence interval (CI)=0.56-0.96]. The all-cause dementia risk was nearly 40% lower in the ARB cohort than in the non-ARB cohort (HR=0.60, 95% CI=0.37-0.97). These drugs prevented the occurrence of vascular dementia (VD), however, this effect was nonsignificant for Alzheimer's dementia (AD). Treatment duration- and dosage-related protection effects on dementia occurrence were observed. ACEIs and ARBs may effectively prevent all-cause dementia, particularly VD, in patients with type 2 DM and hypertension. Moreover, compared with ACEIs, ARBs appear to be more advantageous in dementia prevention. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  4. Differential effects of Rho-kinase inhibitor and angiotensin II type-1 receptor antagonist on the vascular function in hypertensive rats induced by chronic l-NAME treatment

    Directory of Open Access Journals (Sweden)

    Bainian Chen

    2012-10-01

    Full Text Available Little attention has been paid to the effect of Rho-kinase inhibitor on the vascular dysfunction of nitric oxide-deficient hypertension. We aimed to investigate whether the Rho-kinase inhibitor fasudil showed beneficial effect on the vascular dysfunction of the NG-nitro-l-arginine methyl ester (l-NAME treated rat, as well as to compare the differential effects of fasudil and angiotensin II receptor antagonist valsartan on vascular function. In the present study, both valsartan and fasudil exerted antihypertensive action on the l-NAME-treated rats, while only valsartan attenuated the cardiac hypertrophy. Treatment with valsartan showed improvement on vascular reactivity to norepinephrine, KCl and CaCl2, whereas fasudil therapy showed little effect on vasoconstriction. Endothelium-dependent vasodilation to acetylcholine was reduced in the NO-deficient group but was normalized by the fasudil therapy. The increased expression of RhoA and Rho-kinase (ROCK in the vasculature was corrected well to normal level by either valsartan or fasudil administration, which seemed to be at least partially responsible for the beneficial effect of the drug infusion. These findings suggest that the angiotensin II receptor antagonist interferes more with the contractile response than Rho-kinase inhibitor, whereas inhibition of Rho-kinase activity exhibits a better improvement on vasorelaxation than blockade of angiotensin II receptor.

  5. The effect of hyperthyroidism on opiate receptor binding and pain sensitivity

    International Nuclear Information System (INIS)

    Edmondson, E.A.; Bonnet, K.A.; Friedhoff, A.J.

    1990-01-01

    This study was conducted to determine the effect of thyroid hormone on opiate receptor ligand-binding and pain sensitivity. Specific opiate receptor-binding was performed on brain homogenates of Swiss-Webster mice. There was a significant increase in 3 H-naloxone-binding in thyroxine-fed subjects (hyperthyroid). Scatchard analysis revealed that the number of opiate receptors was increased in hyperthyroid mice (Bmax = 0.238 nM for hyperthyroid samples vs. 0.174 nM for controls). Binding affinity was unaffected (Kd = 1.54 nM for hyperthyroid and 1.58 nM for control samples). When mice were subjected to hotplate stimulation, the hyperthyroid mice were noted to be more sensitive as judged by pain aversion response latencies which were half that of control animals. After morphine administration, the hyperthyroid animals demonstrated a shorter duration of analgesia. These findings demonstrate that thyroxine increases opiate receptor number and native pain sensitivity but decreases the duration of analgesia from morphine

  6. The effect of hyperthyroidism on opiate receptor binding and pain sensitivity

    Energy Technology Data Exchange (ETDEWEB)

    Edmondson, E.A. (Baylor College of Medicine, Houston, TX (USA)); Bonnet, K.A.; Friedhoff, A.J. (New York Univ. School of Medicine, NY (USA))

    1990-01-01

    This study was conducted to determine the effect of thyroid hormone on opiate receptor ligand-binding and pain sensitivity. Specific opiate receptor-binding was performed on brain homogenates of Swiss-Webster mice. There was a significant increase in {sup 3}H-naloxone-binding in thyroxine-fed subjects (hyperthyroid). Scatchard analysis revealed that the number of opiate receptors was increased in hyperthyroid mice (Bmax = 0.238 nM for hyperthyroid samples vs. 0.174 nM for controls). Binding affinity was unaffected (Kd = 1.54 nM for hyperthyroid and 1.58 nM for control samples). When mice were subjected to hotplate stimulation, the hyperthyroid mice were noted to be more sensitive as judged by pain aversion response latencies which were half that of control animals. After morphine administration, the hyperthyroid animals demonstrated a shorter duration of analgesia. These findings demonstrate that thyroxine increases opiate receptor number and native pain sensitivity but decreases the duration of analgesia from morphine.

  7. "Assessment of human AT1 Binding Affinity of Some Novel 2-alkylthio-1-[4-(N-α-ethoxycarbonyl-nzylaminobenzyl-5-hydroxymethylimidazoles "

    Directory of Open Access Journals (Sweden)

    Setareh Badakhshannoory

    2004-06-01

    Full Text Available Antagonists of various components of the renin-angiotensin system have been the subject of many studies for the control of blood pressure. Compounds with a phenoxyphenylacetic acid moiety that mimic the structure of losartan which is a powerful competitive antagonist of angiotensin receptor, have shown to be effective. In this study, the affinity of some 2-alkylthio-1-[4-(N-α-ethoxycarbonylbenzylaminobenzyl]-5-hydroxymethyl imidazoles for the human AT1 receptor was assessed in a radioligand binding assay. It was found that an alkyl chain of appropriate length would be most suitable if situated on the imidazole ring. Furthermore, variations of the lower phenyl rings demonstrated that introduction of a methyl group in this position will account for the most desired effect.

  8. Rationale for combination therapy in hypertension management: focus on angiotensin receptor blockers and thiazide diuretics.

    Science.gov (United States)

    Nash, David T

    2007-04-01

    Despite recognition that hypertension is a major risk factor for cardiovascular events and mortality, blood pressure control rates remain low in the US population. Reflecting clinical trial results, hypertension management guidelines assert the clinical benefit of achieving current blood pressure goals and indicate that most patients will require 2 or more drugs to reach goal. Well-designed drug combinations counter hypertension via complementary mechanisms that increase antihypertensive efficacy, potentially with lower rates of adverse events than higher dose monotherapy regimens. Lower adverse event rates, in turn, may contribute to greater adherence with treatment. The combination of a low-dose diuretic with agents that block the effects of the renin-angiotensin system (RAS), such as angiotensin receptor blockers, has been found in numerous clinical trials to be highly effective for lowering blood pressure in patients with uncomplicated as well as high-risk hypertension, with a comparable favorable side effect profile compared with monotherapy. Moreover, agents that block the RAS are associated with a lower risk of new-onset diabetes mellitus than other antihypertensive classes. Complementary combinations of antihypertensive agents provide an efficient and effective approach to hypertension management.

  9. Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

    Science.gov (United States)

    Ha, Sookhee N; Hey, Pat J; Ransom, Rick W; Harrell, C Meacham; Murphy, Kathryn L; Chang, Ray; Chen, Tsing-Bau; Su, Dai-Shi; Markowitz, M Kristine; Bock, Mark G; Freidinger, Roger M; Hess, Fred J

    2005-05-27

    We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

  10. Unsaturated free fatty acids increase benzodiazepine receptor agonist binding depending on the subunit composition of the GABAA receptor complex.

    Science.gov (United States)

    Witt, M R; Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nielsen, M

    1996-11-01

    It has been shown previously that unsaturated free fatty acids (FFAs) strongly enhance the binding of agonist benzodiazepine receptor ligands and GABAA receptor ligands in the CNS in vitro. To investigate the selectivity of this effect, recombinant human GABAA/benzodiazepine receptor complexes formed by different subunit compositions (alpha x beta y gamma 2, x = 1, 2, 3, and 5; y = 1, 2, and 3) were expressed using the baculovirus-transfected Sf9 insect cell system. At 10(-4) M, unsaturated FFAs, particularly arachidonic (20:4) and docosahexaenoic (22:6) acids, strongly stimulated (> 200% of control values) the binding of [3H]flunitrazepam ([3H]FNM) to the alpha 3 beta 2 gamma 2 receptor combination in whole cell preparations. No effect or small increases in levels of unsaturated FFAs on [3H]FNM binding to alpha 1 beta x gamma 2 and alpha 2 beta x gamma 2 receptor combinations were observed, and weak effects (130% of control values) were detected using the alpha 5 beta 2 gamma 2 receptor combination. The saturated FFAs, stearic and palmitic acids, were without effect on [3H]FNM binding to any combination of receptor complexes. The hydroxylated unsaturated FFAs, ricinoleic and ricinelaidic acids, were shown to decrease the binding of [3H]FNM only if an alpha 1 beta 2 gamma 2 receptor combination was used. Given the heterogeneity of the GABAA/ benzodiazepine receptor subunit distribution in the CNS, the effects of FFAs on the benzodiazepine receptor can be assumed to vary at both cellular and regional levels.

  11. G-CSF receptor-binding cyclic peptides designed with artificial amino-acid linkers

    International Nuclear Information System (INIS)

    Shibata, Kenji; Maruyama-Takahashi, Kumiko; Yamasaki, Motoo; Hirayama, Noriaki

    2006-01-01

    Designing small molecules that mimic the receptor-binding local surface structure of large proteins such as cytokines or growth factors is fascinating and challenging. In this study, we designed cyclic peptides that reproduce the receptor-binding loop structures of G-CSF. We found it is important to select a suitable linker to join two or more discontinuous sequences and both termini of the peptide corresponding to the receptor-binding loop. Structural simulations based on the crystallographic structure of KW-2228, a stable and potent analog of human G-CSF, led us to choose 4-aminobenzoic acid (Abz) as a part of the linker. A combination of 4-Abz with β-alanine or glycine, and disulfide bridges between cysteins or homocysteins, gave a structure suitable for receptor binding. In this structure, the side-chains of several amino acids important for the interactions with the receptor are protruding from one side of the peptide ring. This artificial peptide showed G-CSF antagonistic activity in a cell proliferation assay

  12. Between-patient differences in the renal response to renin-angiotensin system intervention : clue to optimising renoprotective therapy?

    NARCIS (Netherlands)

    Laverman, GD; de Zeeuw, D; Navis, G

    2002-01-01

    Renin-angiotensin-aldosterone system (RAAS) blockade with angiotensin-converting enzyme inhibitors (ACE-I) or angiotensin II (Ang II), AT(1)-receptor blockers (ARB) is the cornerstone of renoprotective therapy. Still, the number of patients with end-stage renal disease is increasing worldwide,

  13. Bovine ovarian cells have (pro)renin receptors and prorenin induces resumption of meiosis in vitro.

    Science.gov (United States)

    Dau, Andressa Minussi Pereira; da Silva, Eduardo Pradebon; da Rosa, Paulo Roberto Antunes; Bastiani, Felipe Tusi; Gutierrez, Karina; Ilha, Gustavo Freitas; Comim, Fabio Vasconcellos; Gonçalves, Paulo Bayard Dias

    2016-07-01

    The discovery of a receptor that binds prorenin and renin in human endothelial and mesangial cells highlights the possible effect of renin-independent prorenin in the resumption of meiosis in oocytes that was postulated in the 1980s.This study aimed to identify the (pro)renin receptor in the ovary and to assess the effect of prorenin on meiotic resumption. The (pro)renin receptor protein was detected in bovine cumulus-oocyte complexes, theca cells, granulosa cells, and in the corpus luteum. Abundant (pro)renin receptor messenger ribonucleic acid (mRNA) was detected in the oocytes and cumulus cells, while prorenin mRNA was identified in the cumulus cells only. Prorenin at concentrations of 10(-10), 10(-9), and 10(-8)M incubated with oocytes co-cultured with follicular hemisections for 15h caused the resumption of oocyte meiosis. Aliskiren, which inhibits free renin and receptor-bound renin/prorenin, at concentrations of 10(-7), 10(-5), and 10(-3)M blocked this effect (Pmeiosis resumption, cumulus-oocyte complexes and follicular hemisections were treated with prorenin and with angiotensin II or saralasin (angiotensin II antagonist). Prorenin induced the resumption of meiosis independently of angiotensin II. Furthermore, cumulus-oocyte complexes cultured with forskolin (200μM) and treated with prorenin and aliskiren did not exhibit a prorenin-induced resumption of meiosis (Pmeiosis in cattle. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

    International Nuclear Information System (INIS)

    Waser, Beatrice; Reubi, Jean Claude

    2014-01-01

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the 125 iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer 125 I-GLP-1(7-36)amide. Receptor autoradiography studies with 125 I-GLP-1(7-36)amide agonist or 125 I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist 125 I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer 125 I-GLP-1(7-36)amide. For comparison, 125 I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with 125 I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

  15. Modelling the interdependence between the stoichiometry of receptor oligomerization and ligand binding for a coexisting dimer/tetramer receptor system.

    Science.gov (United States)

    Rovira, X; Vivó, M; Serra, J; Roche, D; Strange, P G; Giraldo, J

    2009-01-01

    Many G protein-coupled receptors have been shown to exist as oligomers, but the oligomerization state and the effects of this on receptor function are unclear. For some G protein-coupled receptors, in ligand binding assays, different radioligands provide different maximal binding capacities. Here we have developed mathematical models for co-expressed dimeric and tetrameric species of receptors. We have considered models where the dimers and tetramers are in equilibrium and where they do not interconvert and we have also considered the potential influence of the ligands on the degree of oligomerization. By analogy with agonist efficacy, we have considered ligands that promote, inhibit or have no effect on oligomerization. Cell surface receptor expression and the intrinsic capacity of receptors to oligomerize are quantitative parameters of the equations. The models can account for differences in the maximal binding capacities of radioligands in different preparations of receptors and provide a conceptual framework for simulation and data fitting in complex oligomeric receptor situations.

  16. Angiotensin II promotes development of the renal microcirculation through AT1 receptors

    DEFF Research Database (Denmark)

    Madsen, Kirsten; Marcussen, Niels; Pedersen, Michael

    2010-01-01

    Pharmacologic or genetic deletion of components of the renin-angiotensin system leads to postnatal kidney injury, but the roles of these components in kidney development are unknown. To test the hypothesis that angiotensin II supports angiogenesis during postnatal kidney development, we quantifie...

  17. In vivo (/sup 3/H)flunitrazepam binding: imaging of receptor regulation

    Energy Technology Data Exchange (ETDEWEB)

    Ciliax, B.J.; Penney, J.B. Jr.; Young, A.B.

    1986-08-01

    The use of (/sup 3/H)flunitrazepam as a ligand to measure alterations in benzodiazepine receptors in vivo in rats was investigated. Animals were injected with (/sup 3/H)flunitrazepam i.v., arterial samples of (/sup 3/H)flunitrazepam were obtained and, later, the animals were sacrificed to assay brain binding. (/sup 3/H)flunitrazepam enters the brain rapidly and binds to benzodiazepine receptors. About two-thirds of this binding is blocked by predosing the animals with 5 mg/kg of clonazepam. The amount of remaining (nonspecific) binding correlates very well (r = 0.88) with the amount of radioactivity found in plasma at the time of death. A series of rats were lesioned unilaterally with kainic acid in the caudate-putamen several months before the infusion of (/sup 3/H)flunitrazepam. In vivo autoradiography in lesioned rats showed that benzodiazepine binding in globus pallidus and substantia nigra on the side of the lesion was increased significantly as compared to the intact side. The observed changes in benzodiazepine binding were similar to those observed previously in lesioned rats using in vitro techniques. Thus, benzodiazepine receptor regulation can be imaged quantitatively using in vivo binding techniques.

  18. Chronic blockade of angiotensin II action prevents glomerulosclerosis, but induces graft vasculopathy in experimental kidney transplantation

    NARCIS (Netherlands)

    Smit-van Oosten, A; Navis, G; Stegeman, CA; Joles, JA; Klok, PA; Kuipers, F; Tiebosch, ATMG; van Goor, H

    Long-term renin-angiotensin system blockade is beneficial in a variety of renal diseases, This study examines the long-term (34 weeks) effects of the angiotensin-converting enzyme inhibitor lisinopril and the angiotensin II receptor type I blocker L158,809 in the Fisher to Lewis rat model of chronic

  19. Expression of the Components of the Renin-Angiotensin System in Venous Malformation

    Directory of Open Access Journals (Sweden)

    Sam eSiljee

    2016-05-01

    Full Text Available Background Venous malformation (VM is the most common form of vascular malformation, consisting of a network of thin-walled ectatic venous channels with deficient or absent media. This study investigated the expression of the components of the renin-angiotensin system (RAS, namely (prorenin receptor (PRR, angiotensin converting enzyme (ACE, angiotensin II receptor 1 (ATIIR1 and angiotensin II receptor 2 (AIITR2 in subcutaneous (SC and intramuscular (IM VM. Materials and Methods SC (n=7 and IM (n=7 VM were analyzed for the expression of PRR, ACE, ATIIR1, and ATIIR2 using 3,3-diaminobenzidine (DAB and immunofluorescent (IF immunohistochemical (IHC staining and NanoString gene expression analysis. Results IHC staining showed expression of PRR, ACE, ATIIR1 and faint expression of ATIIR2 in the endothelium of SC and IM VM. Furthermore, ATIIR2 was expressed by cells away from the endothelium in both SC and IM VM lesions examined. NanoString analysis demonstrated the presence of PRR, ACE and ATIIR1 but not ATIIR2.Conclusions The presence of PRR, ACE, ATIIR1 and potentially ATIIR2, in both SC and IM VM suggests a role for the RAS in the biology of VM. This novel finding may lead to a mechanism-based therapy for VM.

  20. Renin-angiotensin system: an old player with novel functions in skeletal muscle.

    Science.gov (United States)

    Cabello-Verrugio, Claudio; Morales, María Gabriela; Rivera, Juan Carlos; Cabrera, Daniel; Simon, Felipe

    2015-05-01

    Skeletal muscle is a tissue that shows the most plasticity in the body; it can change in response to physiological and pathological stimuli. Among the diseases that affect skeletal muscle are myopathy-associated fibrosis, insulin resistance, and muscle atrophy. A common factor in these pathologies is the participation of the renin-angiotensin system (RAS). This system can be functionally separated into the classical and nonclassical RAS axis. The main components of the classical RAS pathway are angiotensin-converting enzyme (ACE), angiotensin II (Ang-II), and Ang-II receptors (AT receptors), whereas the nonclassical axis is composed of ACE2, angiotensin 1-7 [Ang (1-7)], and the Mas receptor. Hyperactivity of the classical axis in skeletal muscle has been associated with insulin resistance, atrophy, and fibrosis. In contrast, current evidence supports the action of the nonclassical RAS as a counter-regulator axis of the classical RAS pathway in skeletal muscle. In this review, we describe the mechanisms involved in the pathological effects of the classical RAS, advances in the use of pharmacological molecules to inhibit this axis, and the beneficial effects of stimulation of the nonclassical RAS pathway on insulin resistance, atrophy, and fibrosis in skeletal muscle. © 2015 Wiley Periodicals, Inc.

  1. Angiotensin II induces kidney inflammatory injury and fibrosis through binding to myeloid differentiation protein-2 (MD2).

    Science.gov (United States)

    Xu, Zheng; Li, Weixin; Han, Jibo; Zou, Chunpeng; Huang, Weijian; Yu, Weihui; Shan, Xiaoou; Lum, Hazel; Li, Xiaokun; Liang, Guang

    2017-03-21

    Growing evidence indicates that angiotensin II (Ang II), a potent biologically active product of RAS, is a key regulator of renal inflammation and fibrosis. In this study, we tested the hypothesis that Ang II induces renal inflammatory injury and fibrosis through interaction with myeloid differentiation protein-2 (MD2), the accessory protein of toll-like receptor 4 (TLR4) of the immune system. Results indicated that in MD2 -/- mice, the Ang II-induced renal fibrosis, inflammation and kidney dysfunction were significantly reduced compared to control Ang II-infused wild-type mice. Similarly, in the presence of small molecule MD2 specific inhibitor L6H21 or siRNA-MD2, the Ang II-induced increases of pro-fibrotic and pro-inflammatory molecules were prevented in tubular NRK-52E cells. MD2 blockade also inhibited activation of NF-κB and ERK. Moreover, MD2 blockade prevented the Ang II-stimulated formation of the MD2/TLR4/MyD88 signaling complex, as well as the increased surface binding of Ang II in NRK-52E cells. In addition, Ang II directly bound recombinant MD2 protein, rather than TLR4 protein. We conclude that MD2 is a significant contributor in the Ang II-induced kidney inflammatory injury in chronic renal diseases. Furthermore, MD2 inhibition could be a new and important therapeutic strategy for preventing progression of chronic renal diseases.

  2. Receptor-ligand binding sites and virtual screening.

    Science.gov (United States)

    Hattotuwagama, Channa K; Davies, Matthew N; Flower, Darren R

    2006-01-01

    Within the pharmaceutical industry, the ultimate source of continuing profitability is the unremitting process of drug discovery. To be profitable, drugs must be marketable: legally novel, safe and relatively free of side effects, efficacious, and ideally inexpensive to produce. While drug discovery was once typified by a haphazard and empirical process, it is now increasingly driven by both knowledge of the receptor-mediated basis of disease and how drug molecules interact with receptors and the wider physiome. Medicinal chemistry postulates that to understand a congeneric ligand series, or set thereof, is to understand the nature and requirements of a ligand binding site. Likewise, structural molecular biology posits that to understand a binding site is to understand the nature of ligands bound therein. Reality sits somewhere between these extremes, yet subsumes them both. Complementary to rules of ligand design, arising through decades of medicinal chemistry, structural biology and computational chemistry are able to elucidate the nature of binding site-ligand interactions, facilitating, at both pragmatic and conceptual levels, the drug discovery process.

  3. Allosteric regulation by oleamide of the binding properties of 5-hydroxytryptamine7 receptors.

    Science.gov (United States)

    Hedlund, P B; Carson, M J; Sutcliffe, J G; Thomas, E A

    1999-12-01

    Oleamide belongs to a family of amidated lipids with diverse biological activities, including sleep induction and signaling modulation of several 5-hydroxytryptamine (5-HT) receptor subtypes, including 5-HT1A, 5-HT2A/2C, and 5-HT7. The 5-HT7 receptor, predominantly localized in the hypothalamus, hippocampus, and frontal cortex, stimulates cyclic AMP formation and is thought to be involved in the regulation of sleep-wake cycles. Recently, it was proposed that oleamide acts at an allosteric site on the 5-HT7 receptor to regulate cyclic AMP formation. We have further investigated the interaction between oleamide and 5-HT7 receptors by performing radioligand binding assays with HeLa cells transfected with the 5-HT7 receptor. Methiothepin, clozapine, and 5-HT all displaced specific [3H]5-HT (100 nM) binding, with pK(D) values of 7.55, 7.85, and 8.39, respectively. Oleamide also displaced [3H]5-HT binding, but the maximum inhibition was only 40% of the binding. Taking allosteric (see below) cooperativity into account, a K(D) of 2.69 nM was calculated for oleamide. In saturation binding experiments, oleamide caused a 3-fold decrease in the affinity of [3H]5-HT for the 5-HT7 receptor, without affecting the number of binding sites. A Schild analysis showed that the induced shift in affinity of [3H]5-HT reached a plateau, unlike that of a competitive inhibitor, illustrating the allosteric nature of the interaction between oleamide and the 5-HT7 receptor. Oleic acid, the product of oleamide hydrolysis, had a similar effect on [3H]5-HT binding, whereas structural analogs of oleamide, trans-9,10-octadecenamide, cis-8,9-octadecenamide, and erucamide, did not alter [3H]5-HT binding significantly. The findings support the hypothesis that oleamide acts via an allosteric site on the 5-HT7 receptor regulating receptor affinity.

  4. Mu receptor binding of some commonly used opioids and their metabolites

    International Nuclear Information System (INIS)

    Chen, Zhaorong; Irvine, R.J.; Somogyi, A.A.; Bochner, F.

    1991-01-01

    The binding affinity to the μ receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with 3 H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding. Decreasing the length of the alkyl group at position 3 decreased the K i values (morphine < codeine < ethylmorphine < pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 had relatively weak receptor binding, while their O-demethylated metabolites had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites

  5. Mu receptor binding of some commonly used opioids and their metabolites

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Zhaorong; Irvine, R.J. (Univ. of Adelaide (Australia)); Somogyi, A.A.; Bochner, F. (Univ. of Adelaide (Australia) Royal Adelaide Hospital (Australia))

    1991-01-01

    The binding affinity to the {mu} receptor of some opioids chemically related to morphine and some of their metabolites was examined in rat brain homogenates with {sup 3}H-DAMGO. The chemical group at position 6 of the molecule had little effect on binding. Decreasing the length of the alkyl group at position 3 decreased the K{sub i} values (morphine < codeine < ethylmorphine < pholcodine). Analgesics with high clinical potency containing a methoxyl group at position 3 had relatively weak receptor binding, while their O-demethylated metabolites had much stronger binding. Many opioids may exert their pharmacological actions predominantly through metabolites.

  6. Delineation of the peptide binding site of the human galanin receptor.

    Science.gov (United States)

    Kask, K; Berthold, M; Kahl, U; Nordvall, G; Bartfai, T

    1996-01-01

    Galanin, a neuroendocrine peptide of 29 amino acids, binds to Gi/Go-coupled receptors to trigger cellular responses. To determine which amino acids of the recently cloned seven-transmembrane domain-type human galanin receptor are involved in the high-affinity binding of the endogenous peptide ligand, we performed a mutagenesis study. Mutation of the His264 or His267 of transmembrane domain VI to alanine, or of Phe282 of transmembrane domain VII to glycine, results in an apparent loss of galanin binding. The substitution of Glu271 to serine in the extracellular loop III of the receptor causes a 12-fold loss in affinity for galanin. We combined the mutagenesis results with data on the pharmacophores (Trp2, Tyr9) of galanin and with molecular modelling of the receptor using bacteriorhodopsin as a model. Based on these studies, we propose a binding site model for the endogenous peptide ligand in the galanin receptor where the N-terminus of galanin hydrogen bonds with Glu271 of the receptor, Trp2 of galanin interacts with the Zn2+ sensitive pair of His264 and His267 of transmembrane domain VI, and Tyr9 of galanin interacts with Phe282 of transmembrane domain VII, while the C-terminus of galanin is pointing towards the N-terminus of th Images PMID:8617199

  7. Intracerebroventricular infusion of the (Pro)renin receptor antagonist PRO20 attenuates deoxycorticosterone acetate-salt-induced hypertension.

    Science.gov (United States)

    Li, Wencheng; Sullivan, Michelle N; Zhang, Sheng; Worker, Caleb J; Xiong, Zhenggang; Speth, Robert C; Feng, Yumei

    2015-02-01

    We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT(1) receptor-dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension. © 2014 American Heart Association, Inc.

  8. Altered [125I]epidermal growth factor binding and receptor distribution in psoriasis

    International Nuclear Information System (INIS)

    Nanney, L.B.; Stoscheck, C.M.; Magid, M.; King, L.E. Jr.

    1986-01-01

    Stimulation of growth and differentiation of human epidermis by epidermal growth factor (EGF) is mediated by its binding to specific receptors. Whether EGF receptors primarily mediate cell division or differentiation in hyperproliferative disease such as psoriasis vulgaris is unclear. To study the pathogenesis of psoriasis, 4-mm2 punch biopsy specimens of normal, uninvolved, and involved psoriatic skin were assayed for EGF receptors by autoradiographic, immunohistochemical, and biochemical methods. Using autoradiographic and immunohistochemical methods, basal keratinocytes were found to contain the greatest number of EGF binding sites and immunoreactive receptors as compared to the upper layers of the epidermis in both normal epidermis and psoriatic skin. No EGF receptor differences between normal and psoriatic epidermis were observed in this layer. In the upper layers of the epidermis, a 2-fold increase in EGF binding capacity was observed in psoriatic skin as compared with normal thin or thick skin. Biochemical methods indicated that [ 125 I]EGF binding was increased in psoriatic epidermis as compared with similar thickness normal epidermis when measured on a protein basis. Epidermal growth factor was shown to increase phosphorylation of the EGF receptor in skin. EGF receptors retained in the nonmitotic stratum spinosum and parakeratotic stratum corneum may reflect the incomplete, abnormal differentiation that occurs in active psoriatic lesions. Alternatively, retained EGF receptors may play a direct role in inhibiting cellular differentiation in the suprabasal layers

  9. Interaction of cadmium with atrial natriuretic factor receptors: Ligand binding and cellular processing

    International Nuclear Information System (INIS)

    Giridhar, J.; Rathinavelu, A.; Isom, G.E.

    1990-01-01

    ANF is a peptide hormone secreted by the heart and produces potent diuresis and vascular smooth muscle relaxation. It is well known that Cd produces cardiovascular toxicity and is implicated in the pathogenesis of hypertension. Hence the effects of Cd on ANF receptor dynamics and ligand binding were studied in PC12 cells. Receptor internalization using 125 I-ANF as the ligand at 37 degree C displayed a decrease in endocytic rate constants (ERC) when either preincubated with Cd (500 μM for 30 min, ERC = 0.183/min) or coincubated with Cd (500 μM, ERC = 0.196) when compared to control value (ERC = 0.259/min). Ligand binding ( 125 I-ANF) was changed by Cd as reflected by a decrease in the number of binding sites/cell in both Cd preincubated (Kd = 3.81 x 10 -10 M, B max = 1 x 10 -10 M, binding sites/cell = 9333) and coincubated cells (Kd = 1.76 x 10 -10 M, B max = 3.92 x 10 -11 M, binding sites/cell = 5960) from control (Kd = 3.87 x 10 -10 M, B max = 9.58 x 10 -11 M, binding sites/cell = 12141). Photoaffinity labelling with 125 I-ANF as the ligand was used to measure receptor subtype binding. Coincubation of cells with Cd (500 μM) and ligand decreased both high and low mol. wt. receptor binding, whereas preincubation with Cd (500μM) for 60 min produced a slight decrease in binding of both receptor subtypes. These results indicate that the cardiovascular toxicity of Cd may be partially mediated by altered ANF receptor function

  10. Receptor macroautoradiography of 3H-spiroperidol binding in rat brain

    International Nuclear Information System (INIS)

    Mori, Hirofumi; Shiba, Kazuhiro; Tsuji, Shiro; Matsuda, Hiroshi; Hisada, Kinichi; Kojima, Kazuhiko

    1985-01-01

    The kinetic and pharmacological characteristics of 3 H-spiroperidol binding sites were studied in slide mounted sections of rat forebrain, and optical binding conditions were defined. Using the receptor macroautoradiographic techniques with tritium-sensitive LKB sheet film, the distribution of dopamine (D 2 ) receptor was determined in slices including striatum of rat brain. The autoradiograms were analyzed using Video Digitizer System combined with video camera and minicomputer, and the subtraction images were obtained. These studies suggest that this quantitative receptor macroautoradiography might be useful in the explanation of etiology in the field of neuro-psychiatric diseases and the fundamental studies of positron emission computed tomography, since this method has several advantages over in vivo autoradiography and in vitro receptor assay. (author)

  11. Angiotensin II reduces cardiac AdipoR1 expression through AT1 receptor/ROS/ERK1/2/c-Myc pathway.

    Directory of Open Access Journals (Sweden)

    Li Li

    Full Text Available Adiponectin, an abundant adipose tissue-derived protein, exerts protective effect against cardiovascular disease. Adiponectin receptors (AdipoR1 and AdipoR2 mediate the beneficial effects of adiponectin on the cardiovascular system. However, the alteration of AdipoRs in cardiac remodeling is not fully elucidated. Here, we investigated the effect of angiotensin II (AngII on cardiac AdipoRs expression and explored the possible molecular mechanism. AngII infusion into rats induced cardiac hypertrophy, reduced AdipoR1 but not AdipoR2 expression, and attenuated the phosphorylations of adenosine monophosphate-activated protein kinase and acetyl coenzyme A carboxylase, and those effects were all reversed by losartan, an AngII type 1 (AT1 receptor blocker. AngII reduced expression of AdipoR1 mRNA and protein in cultured neonatal rat cardiomyocytes, which was abolished by losartan, but not by PD123319, an AT2 receptor antagonist. The antioxidants including reactive oxygen species (ROS scavenger NAC, NADPH oxidase inhibitor apocynin, Nox2 inhibitor peptide gp91 ds-tat, and mitochondrial electron transport chain complex I inhibitor rotenone attenuated AngII-induced production of ROS and phosphorylation of extracellular signal-regulated kinase (ERK 1/2. AngII-reduced AdipoR1 expression was reversed by pretreatment with NAC, apocynin, gp91 ds-tat, rotenone, and an ERK1/2 inhibitor PD98059. Chromatin immunoprecipitation assay demonstrated that AngII provoked the recruitment of c-Myc onto the promoter region of AdipoR1, which was attenuated by PD98059. Moreover, AngII-induced DNA binding activity of c-Myc was inhibited by losartan, NAC, apocynin, gp91 ds-tat, rotenone, and PD98059. c-Myc small interfering RNA abolished the inhibitory effect of AngII on AdipoR1 expression. Our results suggest that AngII inhibits cardiac AdipoR1 expression in vivo and in vitro and AT1 receptor/ROS/ERK1/2/c-Myc pathway is required for the downregulation of AdipoR1 induced by AngII.

  12. Angiotensin II Regulates Th1 T Cell Differentiation Through Angiotensin II Type 1 Receptor-PKA-Mediated Activation of Proteasome.

    Science.gov (United States)

    Qin, Xian-Yun; Zhang, Yun-Long; Chi, Ya-Fei; Yan, Bo; Zeng, Xiang-Jun; Li, Hui-Hua; Liu, Ying

    2018-01-01

    Naive CD4+ T cells differentiate into T helper cells (Th1 and Th2) that play an essential role in the cardiovascular diseases. However, the molecular mechanism by which angiotensin II (Ang II) promotes Th1 differentiation remains unclear. The aim of this study was to determine whether the Ang II-induced Th1 differentiation regulated by ubiquitin-proteasome system (UPS). Jurkat cells were treated with Ang II (100 nM) in the presence or absence of different inhibitors. The gene mRNA levels were detected by real-time quantitative PCR analysis. The protein levels were measured by ELISA assay or Western blot analysis, respectively. Ang II treatment significantly induced a shift from Th0 to Th1 cell differentiation, which was markedly blocked by angiotensin II type 1 receptor (AT1R) inhibitor Losartan (LST). Moreover, Ang II significantly increased the activities and the expression of proteasome catalytic subunits (β1, β1i, β2i and β5i) in a dose- and time-dependent manner. However, Ang II-induced proteasome activities were remarkably abrogated by LST and PKA inhibitor H-89. Mechanistically, Ang II-induced Th1 differentiation was at least in part through proteasome-mediated degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB. This study for the first time demonstrates that Ang II activates AT1R-PKA-proteasome pathway, which promotes degradation of IκBα and MKP-1 and activation of STAT1 and NF-κB thereby leading to Th1 differentiation. Thus, inhibition of proteasome activation might be a potential therapeutic target for Th1-mediated diseases. © 2018 The Author(s). Published by S. Karger AG, Basel.

  13. Neurotensin receptor binding levels in basal ganglia are not altered in Huntington's chorea or schizophrenia

    International Nuclear Information System (INIS)

    Palacios, J.M.; Chinaglia, G.; Rigo, M.; Ulrich, J.; Probst, A.

    1991-01-01

    Autoradiographic techniques were used to examine the distribution and levels of neurotensin receptor binding sites in the basal ganglia and related regions of the human brain. Monoiodo ( 125 I-Tyr3)neurotensin was used as a ligand. High amounts of neurotensin receptor binding sites were found in the substantia nigra pars compacta. Lower but significant quantities of neurotensin receptor binding sites characterized the caudate, putamen, and nucleus accumbens, while very low quantities were seen in both medial and lateral segments of the globus pallidus. In Huntington's chorea, the levels of neurotensin receptor binding sites were found to be comparable to those of control cases. Only slight but not statistically significant decreases in amounts of receptor binding sites were detected in the dorsal part of the head and in the body of caudate nucleus. No alterations in the levels of neurotensin receptor binding sites were observed in the substantia nigra pars compacta and reticulata. These results suggest that a large proportion of neurotensin receptor binding sites in the basal ganglia are located on intrinsic neurons and on extrinsic afferent fibers that do not degenerate in Huntington's disease

  14. Partial separation of platelet and placental adenosine receptors from adenosine A2-like binding protein

    International Nuclear Information System (INIS)

    Zolnierowicz, S.; Work, C.; Hutchison, K.; Fox, I.H.

    1990-01-01

    The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-[ 3 H]ethylcarboxamidoadenosine [( 3 H]NECA). To solve this problem, we developed a rapid and simple method to separate adenosine receptors from the adenosine A2-like binding protein. Human platelet and placental membranes were solubilized with 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. The soluble platelet extract was precipitated with polyethylene glycol and the fraction enriched in adenosine receptors was isolated from the precipitate by differential centrifugation. The adenosine A2-like binding protein was removed from the soluble placental extract with hydroxylapatite and adenosine receptors were precipitated with polyethylene glycol. The specificity of the [ 3 H]NECA binding is typical of an adenosine A2 receptor for platelets and an adenosine A1 receptor for placenta. This method leads to enrichment of adenosine A2 receptors for platelets and adenosine A1 receptors for placenta. This provides a useful preparation technique for pharmacologic studies of adenosine receptors

  15. Radiolabelled GLP-1 receptor antagonist binds to GLP-1 receptor-expressing human tissues

    Energy Technology Data Exchange (ETDEWEB)

    Waser, Beatrice; Reubi, Jean Claude [University of Berne, Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, PO Box 62, Berne (Switzerland)

    2014-06-15

    Radiolabelled glucagon-like peptide 1 (GLP-1) receptor agonists have recently been shown to successfully image benign insulinomas in patients. For the somatostatin receptor targeting of tumours, however, it was recently reported that antagonist tracers were superior to agonist tracers. The present study therefore evaluated various forms of the {sup 125}iodinated-Bolton-Hunter (BH)-exendin(9-39) antagonist tracer for the in vitro visualization of GLP-1 receptor-expressing tissues in rats and humans and compared it with the agonist tracer {sup 125}I-GLP-1(7-36)amide. Receptor autoradiography studies with {sup 125}I-GLP-1(7-36)amide agonist or {sup 125}I-BH-exendin(9-39) antagonist radioligands were performed in human and rat tissues. The antagonist {sup 125}I-BH-exendin(9-39) labelled at lysine 19 identifies all human and rat GLP-1 target tissues and GLP-1 receptor-expressing tumours. Binding is of high affinity and is comparable in all tested tissues in its binding properties with the agonist tracer {sup 125}I-GLP-1(7-36)amide. For comparison, {sup 125}I-BH-exendin(9-39) with the BH labelled at lysine 4 did identify the GLP-1 receptor in rat tissues but not in human tissues. The GLP-1 receptor antagonist exendin(9-39) labelled with {sup 125}I-BH at lysine 19 is an excellent GLP-1 radioligand that identifies human and rat GLP-1 receptors in normal and tumoural tissues. It may therefore be the molecular basis to develop suitable GLP-1 receptor antagonist radioligands for in vivo imaging of GLP-1 receptor-expressing tissues in patients. (orig.)

  16. The Roles of Hemagglutinin Phe-95 in Receptor Binding and Pathogenicity of Influenza B Virus

    Science.gov (United States)

    Ni, Fengyun; Mbawuike, Innocent Nnadi; Kondrashkina, Elena; Wang, Qinghua

    2014-01-01

    Diverged ~4,000 years ago, influenza B virus has several important differences from influenza A virus, including lower receptor-binding affinity and highly restricted host range. Based on our prior structural studies, we hypothesized that a single-residue difference in the receptor-binding site of hemagglutinin (HA), Phe-95 in influenza B virus versus Tyr-98 in influenza A/H1~H15, is possibly a key determinant for the low receptor-binding affinity. Here we demonstrate that the mutation Phe95→Tyr in influenza B virus HA restores all three hydrogen bonds made by Tyr-98 in influenza A/H3 HA and has the potential to enhance receptor binding. However, the full realization of this potential is influenced by the local environment into which the mutation is introduced. The binding and replication of the recombinant viruses correlate well with the receptor-binding capabilities of HA. These results are discussed in relation to the roles of Phe-95 in receptor binding and pathogenicity of influenza B virus. PMID:24503069

  17. Targeting the renin-angiotensin system as novel therapeutic strategy for pulmonary diseases.

    Science.gov (United States)

    Tan, Wan Shun Daniel; Liao, Wupeng; Zhou, Shuo; Mei, Dan; Wong, Wai-Shiu Fred

    2017-12-27

    The renin-angiotensin system (RAS) plays a major role in regulating electrolyte balance and blood pressure. RAS has also been implicated in the regulation of inflammation, proliferation and fibrosis in pulmonary diseases such as asthma, acute lung injury (ALI), chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and pulmonary arterial hypertension (PAH). Current therapeutics suffer from some drawbacks like steroid resistance, limited efficacies and side effects. Novel intervention is definitely needed to offer optimal therapeutic strategy and clinical outcome. This review compiles and analyses recent investigations targeting RAS for the treatment of inflammatory lung diseases. Inhibition of the upstream angiotensin (Ang) I/Ang II/angiotensin receptor type 1 (AT 1 R) pathway and activation of the downstream angiotensin-converting enzyme 2 (ACE2)/Ang (1-7)/Mas receptor pathway are two feasible strategies demonstrating efficacies in various pulmonary disease models. More recent studies favor the development of targeting the downstream ACE2/Ang (1-7)/Mas receptor pathway, in which diminazene aceturate, an ACE2 activator, GSK2586881, a recombinant ACE2, and AV0991, a Mas receptor agonist, showed much potential for further development. As the pathogenesis of pulmonary diseases is so complex that RAS modulation may be used alone or in combination with existing drugs like corticosteroids, pirfenidone/nintedanib or endothelin receptor antagonists for different pulmonary diseases. Personalized medicine through genetic screening and phenotyping for angiotensinogen or ACE would aid treatment especially for non-responsive patients. This review serves to provide an update on the latest development in the field of RAS targeting for pulmonary diseases, and offer some insights into future direction. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. The Role of Vasodilator Receptors of Renin-angiotensin System on Nitric Oxide Formation and Kidney Circulation after Angiotensin II Infusion in Renal Ischemia/Reperfusion Rats.

    Science.gov (United States)

    Maleki, Maryam; Hasanshahi, Jalal; Moslemi, Fatemeh

    2018-01-01

    Nitric oxide (NO) as a vasodilator factor has renoprotective effect against renal ischemia. The balance between angiotensin II (Ang II) and NO can affect kidney homeostasis. The aim of this study was to determine NO alteration in response to renin-Ang system vasodilator receptors antagonists (PD123319; Ang II type 2 receptor antagonist and A779; Mas receptor antagonist) in renal ischemia/reperfusion injury (IRI) in rats. Sixty-three Wistar male and female rats were used. Animals from each gender were divided into four groups received saline, Ang II, PD123319 + Ang II, and A779 + Ang II after renal IRI. Renal IRI induced with an adjustable hook. Blood pressure and renal blood flow (RBF) measured continuously. The nitrite levels were measured in serum, kidney, and urine samples. In female rats, the serum and kidney nitrite levels increased significantly by Ang II ( P < 0.05) and decreased significantly ( P < 0.05) when PD123319 was accompanied with Ang II. Such observation was not seen in male. Ang II decreased RBF significantly in all groups ( P < 0.05), while PD + Ang II group showed significant decrease in RBF in comparison with the other groups in female rats ( P < 0.05). Males show more sensibility to Ang II infusion; in fact, it is suggested that there is gender dimorphism in the Ang II and NO production associated with vasodilator receptors.

  19. Neuronal low-density lipoprotein receptor-related protein 1 binds and endocytoses prion fibrils via receptor cluster 4

    DEFF Research Database (Denmark)

    Jen, Angela; Parkyn, Celia J; Mootoosamy, Roy C

    2010-01-01

    For infectious prion protein (designated PrP(Sc)) to act as a template to convert normal cellular protein (PrP(C)) to its distinctive pathogenic conformation, the two forms of prion protein (PrP) must interact closely. The neuronal receptor that rapidly endocytoses PrP(C) is the low......-density lipoprotein receptor-related protein 1 (LRP1). We show here that on sensory neurons LRP1 is also the receptor that binds and rapidly endocytoses smaller oligomeric forms of infectious prion fibrils, and recombinant PrP fibrils. Although LRP1 binds two molecules of most ligands independently to its receptor...... both prion and LRP1 biology....

  20. Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Yogi, Alvaro; Callera, Glaucia E. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Mecawi, André S. [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Batalhão, Marcelo E.; Carnio, Evelin C. [Department of General and Specialized Nursing, College of Nursing of Ribeirão Preto, USP, São Paulo (Brazil); Antunes-Rodrigues, José [Department of Physiology, Faculty of Medicine of Ribeirão Preto, University of São Paulo (USP), Ribeirão Preto, SP (Brazil); Queiroz, Regina H. [Department of Clinical, Toxicological and Food Science Analysis, Faculty of Pharmaceutical Sciences, USP, São Paulo (Brazil); Touyz, Rhian M. [Kidney Research Centre, Ottawa Hospital Research Institute, University of Ottawa, Ontario (Canada); Tirapelli, Carlos R., E-mail: crtirapelli@eerp.usp.br [Department of Psychiatric Nursing and Human Sciences, Laboratory of Pharmacology, College of Nursing of Ribeirão Preto, USP, Ribeirão Preto, SP (Brazil)

    2012-11-01

    Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1 g/kg; p.o. gavage) effects were assessed within 30 min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10 mg/kg; p.o. gavage), a selective AT{sub 1} receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT{sub 1}-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage. -- Highlights: ► Acute ethanol intake stimulates RAS activity and vascular oxidative stress. ► RAS plays a role in acute ethanol-induced oxidative damage via AT{sub 1} receptor activation.

  1. Nuclear thyroid hormone receptors in rabbit heart: reduced triiodothyronine binding in atrium compared with ventricle

    International Nuclear Information System (INIS)

    Banerjee, S.K.; Ulrich, J.M.; Kaldor, G.J.

    1988-01-01

    Radiolabeled triiodothyronine (T3) binding to isolated nuclei was measured to compare the binding characteristics of the nuclear receptors in rabbit ventricular and atrial muscle cells. Scatchard analysis of the binding data yielded a maximum binding capacity of 170 +/- 20 fmol per mg DNA and apparent dissociation constant of 525 +/- 100 pM for ventricular nuclei. The binding capacity and the dissociation constant for the atrial muscle cell nuclei were 55 +/- 10 fmol per mg DNA and 500 +/- 75 pM, respectively. The results suggest that the binding capacity for T3 receptor in the atrium is considerably lower than that found in the ventricle. The reduced binding capacity of the T3 receptor in the atrium might reflect differences in the nuclear T3 receptors between ventricle and atrium

  2. Role of the renin-angiotensin system in hepatic fibrosis and portal hypertension.

    Science.gov (United States)

    Shim, Kwang Yong; Eom, Young Woo; Kim, Moon Young; Kang, Seong Hee; Baik, Soon Koo

    2018-05-01

    The renin-angiotensin system (RAS) is an important regulator of cirrhosis and portal hypertension. As hepatic fibrosis progresses, levels of the RAS components angiotensin (Ang) II, Ang-(1-7), angiotensin-converting enzyme (ACE), and Ang II type 1 receptor (AT1R) are increased. The primary effector Ang II regulates vasoconstriction, sodium homoeostasis, fibrosis, cell proliferation, and inflammation in various diseases, including liver cirrhosis, through the ACE/Ang II/AT1R axis in the classical RAS. The ACE2/Ang-(1-7)/Mas receptor and ACE2/Ang-(1-9)/AT2R axes make up the alternative RAS and promote vasodilation, antigrowth, proapoptotic, and anti-inflammatory effects; thus, countering the effects of the classical RAS axis to reduce hepatic fibrogenesis and portal hypertension. Patients with portal hypertension have been treated with RAS antagonists such as ACE inhibitors, Ang receptor blockers, and aldosterone antagonists, with very promising hemodynamic results. In this review, we examine the RAS, its roles in hepatic fibrosis and portal hypertension, and current therapeutic approaches based on the use of RAS antagonists in patients with portal hypertension.

  3. Monoclonal antibody to the rat glucocorticoid receptor. Relationship between the immunoreactive and DNA-binding domain

    International Nuclear Information System (INIS)

    Eisen, L.P.; Reichman, M.E.; Thompson, E.B.; Gametchu, B.; Harrison, R.W.; Eisen, H.J.

    1985-01-01

    The region of the glucocorticoid receptor that reacted with a monoclonal antibody (BUGR-1) was identified. In order to identify the immunoreactive region, the rat liver glucocorticoid receptor was subjected to limited proteolysis; immunoreactive fragments were identified by Western blotting. The monoclonal antibody reacted with both the undigested Mr approximately 97,000 receptor subunit and a Mr approximately 45,000 fragment containing the steroid-binding and DNA-binding domains. Digestion by trypsin also produced two steroid-binding fragments of Mr approximately 27,000 and 31,000 which did not react with the antibody and an immunoreactive Mr approximately 16,000 fragment. This Mr approximately 16,000 fragment was shown to bind to DNA-cellulose, indicating that it contained a DNA-binding domain of the receptor. The undigested receptor must have steroid associated with it to undergo activation to a DNA-binding form. However, the Mr approximately 16,000 immunoreactive fragment binds to DNA-cellulose even if it is obtained by digestion of the steroid-free holoreceptor which does not itself bind to DNA

  4. Diminished hepatic growth hormone receptor binding in sex-linked dwarf broiler and leghorn chickens.

    Science.gov (United States)

    Leung, F C; Styles, W J; Rosenblum, C I; Lilburn, M S; Marsh, J A

    1987-02-01

    Hepatic growth hormone (GH) receptor binding was compared in normal and sex-linked dwarfs (SLD) from both Hubbard and Cornell strain chickens. At 6, 8, and 20 weeks of age, hepatic GH receptor binding in the Hubbard SLD chickens was significantly lower than that of normal fast-growing birds. At 20 weeks of age, only 2 of 22 SLD chickens in the Hubbard broiler strain showed positive binding at a high enough level to allow for Scatchard analysis. The affinity constants and binding capacities of these two SLD chickens were numerically (but not significantly) lower than those of the normal fast-growing birds. We further examined hepatic GH receptor binding in two closely related White Leghorn strains of chickens that have been maintained as closed breeding populations for many years. We observed no detectable hepatic GH binding in the Cornell SLD chickens (N = 20), as compared to the normal-growing control strain (K strain). In both SLD strains, pretreatment with 4 M MgCl2 did not enhance GH binding, suggesting that there was no endogenous GH binding to the receptor. Based on these data, we suggest that the lack, or greatly reduced number, of GH receptors may be a major contributing factor to the dwarfism observed in these strains.

  5. Analysis of the hormone-binding domain of steroid receptors using chimeras generated by homologous recombination

    International Nuclear Information System (INIS)

    Martinez, Elisabeth D.; Pattabiraman, Nagarajan; Danielsen, Mark

    2005-01-01

    The glucocorticoid receptor and the mineralocorticoid receptor are members of the steroid receptor family that exhibit ligand cross-reactivity. Specificity of steroid receptor action is investigated in the present work by the construction and characterization of chimeras between the glucocorticoid receptor and the mineralocorticoid receptor. We used an innovative approach to make novel steroid receptor proteins in vivo that in general, contrary to our expectations, show increased ligand specificity compared to the parental receptors. We describe a receptor that is specific for the potent synthetic glucocorticoid triamcinolone acetonide and does not bind aldosterone. A further set of chimeras has an increased ability to discriminate between ligands, responding potently to mineralocorticoids and only very weakly to synthetic glucocorticoids. A chimera with the fusion site in the hinge highlights the importance of the region between the DNA-binding and the hormone-binding domains since, unlike both the glucocorticoid and mineralocorticoid receptors, it only responds to mineralocorticoids. One chimera has reduced specificity in that it acts as a general corticoid receptor, responding to glucocorticoids and mineralocorticoids with similar potency and efficacy. Our data suggest that regions of the glucocorticoid and mineralocorticoid receptor hormone-binding domains are functionally non-reciprocal. We present transcriptional, hormone-binding, and structure-modeling evidence that suggests that receptor-specific interactions within and across domains mediate aspects of specificity in transcriptional responses to steroids

  6. Relationship Between Aldosterone and Parathyroid Hormone, and the Effect of Angiotensin and Aldosterone Inhibition on Bone Health

    DEFF Research Database (Denmark)

    L.S., Bislev; T., Sikjaer; L., Rolighed

    2015-01-01

    Emerging evidence suggests a stimulating effect of parathyroid hormone (PTH) on the reninnullangiotensinnullaldosterone system (RAAS). In primary hyperparathyroidism, chronic-elevated PTH levels seem to stimulate the RAAS which may explain the increased risk of cardiovascular disease (CVD......). In addition to increased PTH levels, low vitamin D levels may also directly increase risk of CVD, as vitamin D, itself, has been shown to inhibit the RAAS. Angiotensin II, aldosterone and cortisol all negatively impact bone health. Hyperaldosteronism is associated with a reversible secondary...... hyperparathyroidism due to increased renal calcium excretion. Moreover, the angiotensin II receptor is expressed by human parathyroid tissue, and angiotensin may therefore directly stimulates PTH secretion. An increased bone loss is found in patients with hyperaldosteronism. The angiotensin II receptor seems main...

  7. Characterisation of the human NMDA receptor subunit NR3A glycine binding site

    DEFF Research Database (Denmark)

    Nilsson, A; Duan, J; Mo-Boquist, L-L

    2007-01-01

    In this study, we characterise the binding site of the human N-methyl-d-aspartate (NMDA) receptor subunit NR3A. Saturation radioligand binding of the NMDA receptor agonists [(3)H]-glycine and [(3)H]-glutamate showed that only glycine binds to human NR3A (hNR3A) with high affinity (K(d)=535nM (277...

  8. Exploration of N-arylpiperazine Binding Sites of D2 Dopaminergic Receptor.

    Science.gov (United States)

    Soskic, Vukic; Sukalovic, Vladimir; Kostic-Rajacic, Sladjana

    2015-01-01

    The crystal structures of the D3 dopamine receptor and several other G-protein coupled receptors (GPCRs) were published in recent times. Those 3D structures are used by us and other scientists as a template for the homology modeling and ligand docking analysis of related GPCRs. Our main scientific interest lies in the field of pharmacologically active N-arylpiperazines that exhibit antipsychotic and/or antidepressant properties, and as such are dopaminergic and serotonergic receptor ligands. In this short review article we are presenting synthesis and biological data on the new N-arylpipereazine as well our results on molecular modeling of the interactions of those N-arylpiperazines with the model of D2 dopamine receptors. To obtain that model the crystal structure of the D3 dopamine receptor was used. Our results show that the N-arylpiperazines binding site consists of two pockets: one is the orthosteric binding site where the N-arylpiperazine part of the ligand is docked and the second is a non-canonical accessory binding site for N-arylpipereazine that is formed by a second extracellular loop (ecl2) of the receptor. Until now, the structure of this receptor region was unresolved in crystal structure analyses of the D3 dopamine receptor. To get a more complete picture of the ligand - receptor interaction, DFT quantum mechanical calculations on N-arylpiperazine were performed and the obtained models were used to examine those interactions.

  9. Changes in angiotensin AT1 receptor mRNA levels in the rat brain after immobilization stress and inhibition of central nitric oxide synthase.

    Science.gov (United States)

    Kiss, A; Jurkovicova, D; Jezova, D; Krizanova, O

    2001-06-01

    To study functional interactions between angiotensin II AT1 receptors and nitric oxide (NO) activity in different brain areas in rats exposed to immobilization stress. Central inhibition of nitric oxide synthase (NOS) was provided by intracerebroventricular (i.c.v.) administration of (N-omega-nitro-L-arginine-methylester) L-NAME and analysis of AT1 receptor mRNA was performed using semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The immobilization in prone position lasted 2 hrs and the rats were sacrificed 24 hr later. The hypothalamus, hippocampus, thalamus, and cortex were isolated from fresh brains. In the cortex, gene expression of AT1 receptors was unaffected either by L-NAME treatment, or by a single exposure to immobilization stress for 2 hours followed by 24 hours of rest. In the hippocampus, the repeated treatment with L-NAME increased mRNA levels of AT1 receptors approximately 9-times compared to those in the control (untreated) group. Immobilization also increased AT1 receptor mRNA levels in the hippocampus which was similar to that induced by the L-NAME. The increase of AT1 receptor mRNA levels in the hippocampus of immobilized rats was not further altered when the animals were pretreated with L-NAME. In control rats, exposure to immobilization resulted in a significant rise in mRNA levels coding for AT1 receptors in the hypothalamus, but not in the thalamus. L-NAME treatment showed a tendency of increase in AT1 receptor mRNA levels in the hypothalamus. Moreover, when animals treated with L-NAME were subjected to immobilization, a further increase in AT1 receptor mRNA levels was observed in the hypothalamus in comparison with corresponding controls. The present data indicate that a single immobilization stress results in increased gene expression of AT1 receptors in the hypothalamus and hippocampus. The rise in AT1 mRNA levels in the same brain structures after repeated treatment with L-NAME allow to suggest an

  10. Meta-analysis of genome-wide association studies on the intolerance of angiotensin-converting enzyme inhibitors

    NARCIS (Netherlands)

    Mahmoudpour, Seyed H.; Veluchamy, Abirami; Siddiqui, Moneeza K.; Asselbergs, Folkert W.; Souverein, Patrick C.; De Keyser, Catherine E.; Hofman, Albert; Lang, Chim C.; Doney, Alexander S.F.; Stricker, Bruno H.; De Boer, Anthonius; Maitland-Van Der Zee, Anke H.; Palmer, Colin N.A.

    2017-01-01

    Objectives To identify single nucleotide polymorphisms (SNPs) associated with switching from an angiotensin-converting enzyme (ACE)-inhibitor to an angiotensin receptor blocker. Methods Two cohorts of patients starting ACE-inhibitors were identified within the Rotterdam Study in the Netherlands and

  11. G-protein mediates voltage regulation of agonist binding to muscarinic receptors: effects on receptor-Na+ channel interaction

    International Nuclear Information System (INIS)

    Cohen-Armon, M.; Garty, H.; Sokolovsky, M.

    1988-01-01

    The authors previous experiments in membranes prepared from rat heart and brain led them to suggest that the binding of agonist to the muscarinic receptors and to the Na + channels is a coupled event mediated by guanine nucleotide binding protein(s) [G-protein(s)]. These in vitro findings prompted us to employ synaptoneurosomes from brain stem tissue to examine (i) the binding properties of [ 3 H] acetylcholine at resting potential and under depolarization conditions in the absence and presence of pertussis toxin; (ii) the binding of [ 3 H]batrachotoxin to Na + channel(s) in the presence of the muscarinic agonists; and (iii) muscarinically induced 22 Na + uptake in the presence and absence of tetrodotoxin, which blocks Na + channels. The findings indicate that agonist binding to muscarinic receptors is voltage dependent, that this process is mediated by G-protein(s), and that muscarinic agonists induce opening of Na + channels. The latter process persists even after pertussis toxin treatment, indicating that it is not likely to be mediated by pertussis toxin sensitive G-protein(s). The system with its three interacting components-receptor, G-protein, and Na + channel-is such that at resting potential the muscarinic receptor induces opening of Na + channels; this property may provide a possible physiological mechanism for the depolarization stimulus necessary for autoexcitation or repetitive firing in heart or brain tissues

  12. Syntheses of 7-Substituted α-Cyperone Derivatives for Selective Sigma-1 Receptor over Cannabinoid-1 Receptor Binding Affinities

    Energy Technology Data Exchange (ETDEWEB)

    Park, Juyoung; Shin, Younggyun; Yoon, Sunghwa [Ajou Univ., Suwon (Korea, Republic of); Kim, Keewon; Kwon, Youngbae [ChonBuk National Univ., Jeonju (Korea, Republic of)

    2013-11-15

    We have successfully synthesized seven α-cyperone derivatives and found that the presence of a hydrogen bond donor/acceptor groups at the C7 position of α-cyperone significantly affects specificity and potency of CB{sub 1} receptor binding affinity over sigma-1 receptor binding affinity. In particular, the presence of the amino moiety at the C7 position of α-cyperone is beneficial for binding to sigmia-1 receptor. The molecular mechanism of compound 8 involved in the high binding affinity to sigma-1 receptor is under investigation. We first synthesized α-cyperone 1 by following the previously reported synthetic routes.15-19 In brief, azeotropic imination of (+)-dihydrocarvone and (R)-(+)-1-phenylethylamine followed by alkylation with a slight excess of ethyl vinyl ketone (EVK) in THF at 40 .deg. C produced the Micheal adduct. The resulting adduct was hydrolyzed and then treated with sodium methoxide at room temperature to give an easily separable mixture of α-cyperone 1 and its side product. Flash chromatography resulted in pure α-cyperone 1 in a 30% yield from (+)-dihydrocarvone.

  13. A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor

    International Nuclear Information System (INIS)

    Quitterer, Ursula; Pohl, Armin; Langer, Andreas; Koller, Samuel; AbdAlla, Said

    2011-01-01

    Highlights: → A new FRET-based method detects AT1/B2 receptor heterodimerization. → First time application of AT1-Cerulean as a FRET donor. → Method relies on signal peptide-enhanced cell surface delivery of AT1-Cerulean. → A high FRET efficiency revealed efficient heterodimerization of AT1/B2R proteins. → AT1/B2R heterodimers were functionally coupled to desensitization mechanisms. -- Abstract: Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1-EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor's amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R heterodimerization, confocal FRET imaging of

  14. A cleavable signal peptide enhances cell surface delivery and heterodimerization of Cerulean-tagged angiotensin II AT1 and bradykinin B2 receptor

    Energy Technology Data Exchange (ETDEWEB)

    Quitterer, Ursula, E-mail: ursula.quitterer@pharma.ethz.ch [Molecular Pharmacology Unit, Swiss Federal Institute of Technology and University of Zurich, Zurich (Switzerland); Pohl, Armin; Langer, Andreas; Koller, Samuel; AbdAlla, Said [Molecular Pharmacology Unit, Swiss Federal Institute of Technology and University of Zurich, Zurich (Switzerland)

    2011-06-10

    Highlights: {yields} A new FRET-based method detects AT1/B2 receptor heterodimerization. {yields} First time application of AT1-Cerulean as a FRET donor. {yields} Method relies on signal peptide-enhanced cell surface delivery of AT1-Cerulean. {yields} A high FRET efficiency revealed efficient heterodimerization of AT1/B2R proteins. {yields} AT1/B2R heterodimers were functionally coupled to desensitization mechanisms. -- Abstract: Heterodimerization of the angiotensin II AT1 receptor with the receptor for the vasodepressor bradykinin, B2R, is known to sensitize the AT1-stimulated response of hypertensive individuals in vivo. To analyze features of that prototypic receptor heterodimer in vitro, we established a new method that uses fluorescence resonance energy transfer (FRET) and applies for the first time AT1-Cerulean as a FRET donor. The Cerulean variant of the green fluorescent protein as donor fluorophore was fused to the C-terminus of AT1, and the enhanced yellow fluorescent protein (EYFP) as acceptor fluorophore was fused to B2R. In contrast to AT1-EGFP, the AT1-Cerulean fusion protein was retained intracellularly. To facilitate cell surface delivery of AT1-Cerulean, a cleavable signal sequence was fused to the receptor's amino terminus. The plasma membrane-localized AT1-Cerulean resembled the native AT1 receptor regarding ligand binding and receptor activation. A high FRET efficiency of 24.7% between membrane-localized AT1-Cerulean and B2R-EYFP was observed with intact, non-stimulated cells. Confocal FRET microscopy further revealed that the AT1/B2 receptor heterodimer was functionally coupled to receptor desensitization mechanisms because activation of the AT1-Cerulean/B2R-EYFP heterodimer with a single agonist triggered the co-internalization of AT1/B2R. Receptor co-internalization was sensitive to inhibition of G protein-coupled receptor kinases, GRKs, as evidenced by a GRK-specific peptide inhibitor. In agreement with efficient AT1/B2R

  15. Identification of the A2 adenosine receptor binding subunit by photoaffinity crosslinking

    International Nuclear Information System (INIS)

    Barrington, W.W.; Jacobson, K.A.; Hutchison, A.J.; Williams, M.; Stiles, G.L.

    1989-01-01

    A high-affinity iodinated agonist radioligand for the A2 adenosine receptor has been synthesized to facilitate studies of the A2 adenosine receptor binding subunit. The radioligand 125I-labeled PAPA-APEC (125I-labeled 2-[4-(2-[2-[(4- aminophenyl)methylcarbonylamino]ethylaminocarbonyl]- ethyl)phenyl]ethylamino-5'-N-ethylcarboxamidoadenosine) was synthesized and found to bind to the A2 adenosine receptor in bovine striatal membranes with high affinity (Kd = 1.5 nM) and A2 receptor selectivity. Competitive binding studies reveal the appropriate A2 receptor pharmacologic potency order with 5'-N-ethylcarboxamidoadenosine (NECA) greater than (-)-N6-[(R)-1-methyl- 2-phenylethyl]adenosine (R-PIA) greater than (+)-N6-[(S)-1-methyl-2- phenylethyl]adenosine (S-PIA). Adenylate cyclase assays, in human platelet membranes, demonstrate a dose-dependent stimulation of cAMP production. PAPA-APEC (1 microM) produces a 43% increase in cAMP production, which is essentially the same degree of increase produced by 5'-N- ethylcarboxamidoadenosine (the prototypic A2 receptor agonist). These findings combined with the observed guanine nucleotide-mediated decrease in binding suggest that PAPA-APEC is a full A2 agonist. The A2 receptor binding subunit was identified by photoaffinity-crosslinking studies using 125I-labeled PAPA-APEC and the heterobifunctional crosslinking agent N-succinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate (SANPAH). After covalent incorporation, a single specifically radiolabeled protein with an apparent molecular mass of 45 kDa was observed on NaDodSO4/PAGE/autoradiography. Incorporation of 125I-labeled PAPA-APEC into this polypeptide is blocked by agonists and antagonists with the expected potency for A2 receptors and is decreased in the presence of 10(-4) M guanosine 5'-[beta, gamma-imido]triphosphate

  16. Combined sodium ion sensitivity in agonist binding and internalization of vasopressin V1b receptors.

    Science.gov (United States)

    Koshimizu, Taka-Aki; Kashiwazaki, Aki; Taniguchi, Junichi

    2016-05-03

    Reducing Na(+) in the extracellular environment may lead to two beneficial effects for increasing agonist binding to cell surface G-protein coupled receptors (GPCRs): reduction of Na(+)-mediated binding block and reduce of receptor internalization. However, such combined effects have not been explored. We used Chinese Hamster Ovary cells expressing vasopressin V1b receptors as a model to explore Na(+) sensitivity in agonist binding and receptor internalization. Under basal conditions, a large fraction of V1b receptors is located intracellularly, and a small fraction is in the plasma membrane. Decreases in external Na(+) increased cell surface [(3)H]AVP binding and decreased receptor internalization. Substitution of Na(+) by Cs(+) or NH4(+) inhibited agonist binding. To suppress receptor internalization, the concentration of NaCl, but not of CsCl, had to be less than 50 mM, due to the high sensitivity of the internalization machinery to Na(+) over Cs(+). Iso-osmotic supplementation of glucose or NH4Cl maintained internalization of the V1b receptor, even in a low-NaCl environment. Moreover, iodide ions, which acted as a counter anion, inhibited V1b agonist binding. In summary, we found external ionic conditions that could increase the presence of high-affinity state receptors at the cell surface with minimum internalization during agonist stimulations.

  17. Brain Renin-Angiotensin System and Microglial Polarization: Implications for Aging and Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Jose L. Labandeira-Garcia

    2017-05-01

    Full Text Available Microglia can transform into proinflammatory/classically activated (M1 or anti-inflammatory/alternatively activated (M2 phenotypes following environmental signals related to physiological conditions or brain lesions. An adequate transition from the M1 (proinflammatory to M2 (immunoregulatory phenotype is necessary to counteract brain damage. Several factors involved in microglial polarization have already been identified. However, the effects of the brain renin-angiotensin system (RAS on microglial polarization are less known. It is well known that there is a “classical” circulating RAS; however, a second RAS (local or tissue RAS has been observed in many tissues, including brain. The locally formed angiotensin is involved in local pathological changes of these tissues and modulates immune cells, which are equipped with all the components of the RAS. There are also recent data showing that brain RAS plays a major role in microglial polarization. Level of microglial NADPH-oxidase (Nox activation is a major regulator of the shift between M1/proinflammatory and M2/immunoregulatory microglial phenotypes so that Nox activation promotes the proinflammatory and inhibits the immunoregulatory phenotype. Angiotensin II (Ang II, via its type 1 receptor (AT1, is a major activator of the NADPH-oxidase complex, leading to pro-oxidative and pro-inflammatory effects. However, these effects are counteracted by a RAS opposite arm constituted by Angiotensin II/AT2 receptor signaling and Angiotensin 1–7/Mas receptor (MasR signaling. In addition, activation of prorenin-renin receptors may contribute to activation of the proinflammatory phenotype. Aged brains showed upregulation of AT1 and downregulation of AT2 receptor expression, which may contribute to a pro-oxidative pro-inflammatory state and the increase in neuron vulnerability. Several recent studies have shown interactions between the brain RAS and different factors involved in microglial polarization

  18. Differences in both glycosylation and binding properties between rat and mouse liver prolactin receptors.

    Science.gov (United States)

    Lascols, O; Cherqui, G; Munier, A; Picard, J; Capeau, J

    1994-05-01

    To investigate whether glycanic chains of prolactin receptors (PRL-R) play a role in hormone binding activity, comparison was made of rat and mouse liver solubilized receptors with respect to both their affinity for the hormone and their glycosylation properties. As compared with rat receptors, mouse receptors exhibited a 2-fold higher affinity for human growth hormone (hGH), the hormone being bound by both tissues with a lactogenic specificity. Along with this increased affinity, mouse receptors had a 2 lower M(r) relative to rat receptors (62 kDa versus 64 kDa as measured on hGH cross-linked receptors). These differences could be ascribed to different glycosylation properties of the receptors from the two species, as supported by the followings. 1) After treatment with endoglycosidase F (endo F), rat and mouse PRL-R no longer exhibited any difference in their M(r) (54 kDa for both cross-linked receptors). 2) Neuraminidase treatment increased by 37% the binding of hGH to mouse receptors, but was ineffective on the hormone-binding to rat receptors. Conversely, wheat germ agglutinin (WGA), another sialic acid specific probe, decreased hGH binding to rat receptors by 25%, but had no effect on this process for mouse ones. 3) Marked differences were observed in the recoveries of rat and mouse hormone-receptor (HR) complexes from ricin-1- (RCA1-), concanavalin A- (ConA-) and WGA-immobilized lectins. These differences were reduced (RCA1 and ConA) or abolished (WGA) after rat and mouse receptor desialylation by neuraminidase, a treatment which decreased the M(r) of both receptors by 2 kDa. Taken together, these results strongly suggest that the PRL-R from rat and mouse liver contain biantennary N-linked oligosaccharidic chains with distinct type of sialylation, which may account for their differential hormone-binding affinities.

  19. A Synthetic Thiourea-Based Tripodal Receptor that Impairs the Function of Human First Trimester Cytotrophoblast Cells

    Directory of Open Access Journals (Sweden)

    Darijana Horvat

    2014-07-01

    Full Text Available A synthetic tripodal-based thiourea receptor (PNTTU was used to explore the receptor/ligand binding affinity using CTB cells. The human extravillous CTB cells (Sw.71 used in this study were derived from first trimester chorionic villus tissue. The cell proliferation, migration and angiogenic factors were evaluated in PNTTU-treated CTB cells. The PNTTU inhibited the CTBs proliferation and migration. The soluble fms-like tyrosine kinase-1 (sFlt-1 secretion was increased while vascular endothelial growth factor (VEGF was decreased in the culture media of CTB cells treated with ≥1 nM PNTTU. The angiotensin II receptor type 2 (AT2 expression was significantly upregulated in ≥1 nM PNTTU-treated CTB cells in compared to basal; however, the angiotensin II receptor, type 1 (AT1 and vascular endothelial growth factor receptor 1 (VEGFR-1 expression was downregulated. The anti-proliferative and anti-angiogenic effect of this compound on CTB cells are similar to the effect of CTSs. The receptor/ligand affinity of PNTTU on CTBs provides us the clue to design a potent inhibitor to prevent the CTS-induced impairment of CTB cells.

  20. Characterization of glucagon-like peptide-1 receptor-binding determinants.

    Science.gov (United States)

    Xiao, Q; Jeng, W; Wheeler, M B

    2000-12-01

    Glucagon-like peptide 1 (GLP-1) is a potent insulinotropic hormone currently under study as a therapeutic agent for type 2 diabetes. Since an understanding of the molecular mechanisms leading to high-affinity receptor (R) binding and activation may facilitate the development of more potent GLP-1R agonists, we have localized specific regions of GLP-1R required for binding. The purified N-terminal fragment (hereafter referred to as NT) of the GLP-1R produced in either insect (Sf9) or mammalian (COS-7) cells was shown to bind GLP-1. The physical interaction of NT with GLP-1 was first demonstrated by cross-linking ((125)I-GLP-1/NT complex band at approximately 28 kDa) and secondly by attachment to Ni(2+)-NTA beads. The GLP-1R NT protein attached to beads bound GLP-1, but with lower affinity (inhibitory concentration (IC(50)): 4.5 x 10(-7) M) than wild-type (WT) GLP-1R (IC(50): 5.2 x 10(-9)M). The low affinity of GLP-1R NT suggested that other receptor domains may contribute to GLP-1 binding. This was supported by studies using chimeric glucose-dependent insulinotropic polypeptide (GIP)/GLP-1 receptors. GIP(1-151)/GLP-1R, but not GIP(1-222)/GLP-1R, exhibited specific GLP-1 binding and GLP-1-induced cAMP production, suggesting that the region encompassing transmembrane (TM) domain 1 through to TM3 was required for binding. Since it was hypothesized that certain charged or polar amino acids in this region might be involved in binding, these residues (TM2-TM3) were analyzed by substitution mutagenesis. Five mutants (K197A, D198A, K202A, D215A, R227A) displayed remarkably reduced binding affinity. These studies indicate that the NT domain of the GLP-1R is able to bind GLP-1, but charged residues concentrated at the distal TM2/extracellular loop-1 (EC1) interface (K197, D198, K202) and in EC1 (D215 and R227) probably contribute to the binding determinants of the GLP-1R.

  1. Angiotensin Type-2 Receptors Influence the Activity of Vasopressin Neurons in the Paraventricular Nucleus of the Hypothalamus in Male Mice.

    Science.gov (United States)

    de Kloet, Annette D; Pitra, Soledad; Wang, Lei; Hiller, Helmut; Pioquinto, David J; Smith, Justin A; Sumners, Colin; Stern, Javier E; Krause, Eric G

    2016-08-01

    It is known that angiotensin-II acts at its type-1 receptor to stimulate vasopressin (AVP) secretion, which may contribute to angiotensin-II-induced hypertension. Less well known is the impact of angiotensin type-2 receptor (AT2R) activation on these processes. Studies conducted in a transgenic AT2R enhanced green fluorescent protein reporter mouse revealed that although AT2R are not themselves localized to AVP neurons within the paraventricular nucleus of the hypothalamus (PVN), they are localized to neurons that extend processes into the PVN. In the present set of studies, we set out to characterize the origin, phenotype, and function of nerve terminals within the PVN that arise from AT2R-enhanced green fluorescent protein-positive neurons and synapse onto AVP neurons. Initial experiments combined genetic and neuroanatomical techniques to determine that γ-aminobutyric acid (GABA)ergic neurons derived from the peri-PVN area containing AT2R make appositions onto AVP neurons within the PVN, thereby positioning AT2R to negatively regulate neuroendocrine secretion. Subsequent patch-clamp electrophysiological experiments revealed that selective activation of AT2R in the peri-PVN area using compound 21 facilitates inhibitory (ie, GABAergic) neurotransmission and leads to reduced activity of AVP neurons within the PVN. Final experiments determined the functional impact of AT2R activation by testing the effects of compound 21 on plasma AVP levels. Collectively, these experiments revealed that AT2R expressing neurons make GABAergic synapses onto AVP neurons that inhibit AVP neuronal activity and suppress baseline systemic AVP levels. These findings have direct implications in the targeting of AT2R for disorders of AVP secretion and also for the alleviation of high blood pressure.

  2. Immunohistochemical Localization of AT1a, AT1b, and AT2 Angiotensin II Receptor Subtypes in the Rat Adrenal, Pituitary, and Brain with a Perspective Commentary

    Directory of Open Access Journals (Sweden)

    Courtney Premer

    2013-01-01

    Full Text Available Angiotensin II increases blood pressure and stimulates thirst and sodium appetite in the brain. It also stimulates secretion of aldosterone from the adrenal zona glomerulosa and epinephrine from the adrenal medulla. The rat has 3 subtypes of angiotensin II receptors: AT1a, AT1b, and AT2. mRNAs for all three subtypes occur in the adrenal and brain. To immunohistochemically differentiate these receptor subtypes, rabbits were immunized with C-terminal fragments of these subtypes to generate receptor subtype-specific antibodies. Immunofluorescence revealed AT1a and AT2 receptors in adrenal zona glomerulosa and medulla. AT1b immunofluorescence was present in the zona glomerulosa, but not the medulla. Ultrastructural immunogold labeling for the AT1a receptor in glomerulosa and medullary cells localized it to plasma membrane, endocytic vesicles, multivesicular bodies, and the nucleus. AT1b and AT2, but not AT1a, immunofluorescence was observed in the anterior pituitary. Stellate cells were AT1b positive while ovoid cells were AT2 positive. In the brain, neurons were AT1a, AT1b, and AT2 positive, but glia was only AT1b positive. Highest levels of AT1a, AT1b, and AT2 receptor immunofluorescence were in the subfornical organ, median eminence, area postrema, paraventricular nucleus, and solitary tract nucleus. These studies complement those employing different techniques to characterize Ang II receptors.

  3. Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor.

    Science.gov (United States)

    Miao, Yinglong; McCammon, J Andrew

    2018-03-20

    Protein-protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein-protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. These simulations provided important insights into the mechanism of GPCR-nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein-protein interactions.

  4. Pharmacological properties of angiotensin II antagonists: Examining all the therapeutic implications

    Directory of Open Access Journals (Sweden)

    Thomas Unger

    2001-06-01

    Full Text Available Angiotensin II (Ang II, the effector peptide of the renin-angiotensin system (RAS, exerts a variety of actions in physiological blood pressure and body fluid regulation, and is implicated as a major pathogenic factor in the development of cardiovascular disease. Inhibition of the RAS, via treatment with the angiotensin-converting enzyme inhibitors (ACE-I, or more recently the Ang II AT1-receptor blockers (ARBs, has been used as a therapeutic approach to the treatment of hypertension and other cardiovascular dysfunction. Evidence from animal and clinical studies shows that the antihypertensive and overall organ-protective actions of the ARBs are similar to those of ACE-I. However, as the ARBs selectively block the AT1-receptor, which is responsible for the known cardiovascular actions of Ang II, leave the AT2-receptor unopposed and do not interfere with the breakdown of bradykinin, there is the potential for beneficial effects in hypertensive patients with cardiovascular diseases such as left ventricular hypertrophy. Furthermore, there may be additional benefits when the ARBs are combined with ACE-I in such patients. Animal studies contribute to the elucidation and understanding of the role of AT1- and AT2-receptors in the cardiovascular system, and may help in the design of clinical studies aimed at investigating the effects of ACE-I, ARBs, and their combination, on cardiovascular outcomes in hypertensive patients.

  5. Pharmacological properties of angiotensin II antagonists: examining all the therapeutic implications

    Directory of Open Access Journals (Sweden)

    Thomas Unger

    2001-06-01

    Full Text Available Angiotensin II (Ang II, the effector peptide of the renin-angiotensin system (RAS, exerts a variety of actions in physiological blood pressure and body fluid regulation, and is implicated as a major pathogenic factor in the development of cardiovascular disease. Inhibition of the RAS, via treatment with the angiotensin-converting enzyme inhibitors (ACE-I, or more recently the Ang II AT1-receptor blockers (ARBs, has been used as a therapeutic approach to the treatment of hypertension and other cardiovascular dysfunction. Evidence from animal and clinical studies shows that the antihypertensive and overall organ-protective actions of the ARBs are similar to those of ACE-I. However, as the ARBs selectively block the AT1-receptor, which is responsible for the known cardiovascular actions of Ang II, leave the AT2-receptor unopposed and do not interfere with the breakdown of bradykinin, there is the potential for beneficial effects in hypertensive patients with cardiovascular diseases such as left ventricular hypertrophy. Furthermore, there may be additional benefits when the ARBs are combined with ACE-I in such patients. Animal studies contribute to the elucidation and understanding of the role of AT1- and AT2-receptors in the cardiovascular system, and may help in the design of clinical studies aimed at investigating the effects of ACE-I, ARBs, and their combination, on cardiovascular outcomes in hypertensive patients.

  6. Randomised trial on episodic cluster headache with an angiotensin II receptor blocker

    DEFF Research Database (Denmark)

    Tronvik, Erling; Wienecke, Troels; Monstad, Inge

    2013-01-01

    OBJECTIVES: The aim of this study was to evaluate the angiotensin II receptor antagonist candesartan as prophylactic medication in patients with episodic cluster headache. METHODS: This study comprised a prospective, placebo-controlled, double-blind, parallel-designed trial performed in seven cen...... the candesartan and placebo group was not significant with the pre-planned non-parametric ranking test, but a post-hoc exact Poisson test, which takes into account the temporal properties of the data, revealed a significant result ( P  ...... (primary efficacy variable) during the three-week treatment period was reduced from 14.3 ± 9.2 attacks in week 1 to 5.6 ± 7.0 attacks in week 3 (-61%) in the candesartan group and from 16.8 ± 14.1 attacks in week 1 to 10.5 ± 11.3 attacks in week 3 (-38%) in the placebo group. The difference between...

  7. Muscarinic cholinergic receptor binding sites differentiated by their affinity for pirenzepine do not interconvert

    International Nuclear Information System (INIS)

    Gil, D.W.; Wolfe, B.B.

    1986-01-01

    Although it has been suggested by many investigators that subtypes of muscarinic cholinergic receptors exist, physical studies of solubilized receptors have indicated that only a single molecular species may exist. To test the hypothesis that the putative muscarinic receptor subtypes in rat forebrain are interconvertible states of the same receptor, the selective antagonist pirenzepine (PZ) was used to protect muscarinic receptors from blockade by the irreversible muscarinic receptor antagonist propylbenzilylcholine mustard (PBCM). If interconversion of high (M1) and low (M2) affinity binding sites for PZ occurs, incubation of cerebral cortical membranes with PBCM in the presence of PZ should not alter the proportions of M1 and M2 binding sites that are unalkylated (i.e., protected). If, on the other hand, the binding sites are not interconvertible, PZ should be able to selectively protect M1 sites and alter the proportions of unalkylated M1 and M2 binding sites. In the absence of PZ, treatment of cerebral cortical membranes with 20 nM PBCM at 4 degrees C for 50 min resulted in a 69% reduction in the density of M1 binding sites and a 55% reduction in the density of M2 binding sites with no change in the equilibrium dissociation constants of the radioligands [ 3 H]quinuclidinyl benzilate or [ 3 H]PZ. The reasons for this somewhat selective effect of PBCM are not apparent. In radioligand binding experiments using cerebral cortical membranes, PZ inhibited the binding of [ 3 H]quinuclidinyl benzilate in a biphasic manner

  8. C5a binding to human polymorphonuclear leukocyte plasma membrane (PMNLM) receptors

    International Nuclear Information System (INIS)

    Conway, R.G.; Mollison, K.W.; Carter, G.W.; Lane, B.

    1986-01-01

    Previous investigations of the C5a receptor have been performed using intact human PMNL. To circumvent some of the potential problems with such whole cell assays (e.g. internalization or metabolism of radioligand) the authors have developed a PMNLM binding assay. Human PMNLM were prepared by nitrogen cavitation and Percoll gradient centrifugation. Specific binding of [ 125 I]C5a to PMNLM was: high affinity, K/sub D/ = 0.6 nM; saturable, B/sub max/ = 8.7 pmol/mg protein; and reversible. Kinetic measurements agree with the K/sub D/ value obtained by Scatchard analysis. Furthermore, the binding activity of C5a correlates with biological activity as measured by myeloperoxidase release from human PMNL. Human serum C5a and recombinant C5a bind with similar affinities when measured by competition or direct binding and label the same number of sites in human PMNLM. The nonhydrolyzable GTP analog, GppNHp, induces a low affinity state of the C5a receptor (4-6 fold shift in K/sub D/) with little effect on B/sub max/. In summary, the criteria have been satisfied for identification of a biologically relevant C5a binding site in human PMNLM. Regulation of the C5a receptor and its membrane transduction mechanism(s) appears to involve guanyl nucleotides, as has been found for other chemoattractant receptors

  9. Increased expression of vascular endothelin type B and angiotensin type 1 receptors in patients with ischemic heart disease

    DEFF Research Database (Denmark)

    Dimitrijevic, Ivan; Edvinsson, Lars; Chen, Qingwen

    2009-01-01

    expression in subcutaneous arteries from patients with different degrees of ischemic heart disease. METHODS: Subcutaneous arteries were obtained, by biopsy from the abdomen, from patients undergoing coronary artery bypass graft (CABG) surgery because of ischemic heart disease (n = 15), patients with angina...... pectoris without established myocardial infarction (n = 15) and matched cardiovascular healthy controls (n = 15). Endothelin type A (ETA) and type B (ETB), and angiotensin type 1 (AT1) and type 2 (AT2) receptors expression and function were examined using immunohistochemistry, Western blot and in vitro...

  10. Reversible Fetal Renal Impairment following Angiotensin Receptor Blocking Treatment during Third Trimester of Pregnancy: Case Report and Review of the Literature

    Directory of Open Access Journals (Sweden)

    Tal Saar

    2016-01-01

    Full Text Available Background. Late pregnancy usage of angiotensin converting enzyme inhibitors (ACE-I and angiotensin II receptor blockers (ARB may cause severe oligohydramnios due to fetal renal impairment. Affected neonates will often suffer from fatal, renal, and respiratory failure. Case. A 39-year-old multigravida admitted due to anhydramnios secondary to valsartan (ARB exposure at 30 weeks’ gestation. Following secession of treatment amniotic fluid volume returned to normal. Delivery was induced at 34 weeks’ gestation following premature rupture of membranes and maternal fever. During the two-year follow-up, no signs of renal insufficiency were noted. Conclusions. This description of reversible fetal renal damage due to ARB intake during pregnancy is the first to show no adverse renal function in a two-year follow-up period. This case may help clinicians counsel patients with pregnancies complicated by exposure to these drugs.

  11. Aging-induced changes in brain regional serotonin receptor binding: Effect of Carnosine.

    Science.gov (United States)

    Banerjee, S; Poddar, M K

    2016-04-05

    Monoamine neurotransmitter, serotonin (5-HT) has its own specific receptors in both pre- and post-synapse. In the present study the role of carnosine on aging-induced changes of [(3)H]-5-HT receptor binding in different brain regions in a rat model was studied. The results showed that during aging (18 and 24 months) the [(3)H]-5-HT receptor binding was reduced in hippocampus, hypothalamus and pons-medulla with a decrease in their both Bmax and KD but in cerebral cortex the [(3)H]-5-HT binding was increased with the increase of its only Bmax. The aging-induced changes in [(3)H]-5-HT receptor binding with carnosine (2.0 μg/kg/day, intrathecally, for 21 consecutive days) attenuated in (a) 24-month-aged rats irrespective of the brain regions with the attenuation of its Bmax except hypothalamus where both Bmax and KD were significantly attenuated, (b) hippocampus and hypothalamus of 18-month-aged rats with the attenuation of its Bmax, and restored toward the [(3)H]-5-HT receptor binding that observed in 4-month-young rats. The decrease in pons-medullary [(3)H]-5-HT binding including its Bmax of 18-month-aged rats was promoted with carnosine without any significant change in its cerebral cortex. The [(3)H]-5-HT receptor binding with the same dosages of carnosine in 4-month-young rats (a) increased in the cerebral cortex and hippocampus with the increase in their only Bmax whereas (b) decreased in hypothalamus and pons-medulla with a decrease in their both Bmax and KD. These results suggest that carnosine treatment may (a) play a preventive role in aging-induced brain region-specific changes in serotonergic activity (b) not be worthy in 4-month-young rats in relation to the brain regional serotonergic activity. Copyright © 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

  12. Oxygen dependency of epidermal growth factor receptor binding and DNA synthesis of rat hepatocytes

    International Nuclear Information System (INIS)

    Hirose, Tetsuro; Terajima, Hiroaki; Yamauchi, Akira

    1997-01-01

    Background/Aims: Changes in oxygen availability modulate replicative responses in several cell types, but the effects on hepatocyte replication remain unclear. We have studied the effects of transient nonlethal hypoxia on epidermal growth factor receptor binding and epidermal growth factor-induced DNA synthesis of rat hepatocytes. Methods: Lactate dehydrogenase activity in culture supernatant, intracellular adenosine triphosphate content, 125 I-epidermal growth factor specific binding, epidermal growth factor receptor protein expression, and 3 H-thymidine incorporation were compared between hepatocytes cultured in hypoxia and normoxia. Results: Hypoxia up to 3 h caused no significant increase in lactate dehydrogenase activity in the culture supernatant, while intracellular adenosine triphosphate content decreased time-dependently and was restored to normoxic levels by reoxygenation (nonlethal hypoxia). Concomitantly, 125 I-epidermal growth factor specific binding to hepatocytes decreased time-dependently (to 54.1% of normoxia) and was restored to control levels by reoxygenation, although 125 I-insulin specific binding was not affected. The decrease in 125 I-epidermal growth factor specific binding was explained by the decrease in the number or available epidermal growth factor receptors (21.37±3.08 to 12.16±1.42 fmol/10 5 cells), while the dissociation constant of the receptor was not affected. The change in the number of available receptors was not considered to be due to receptor degradation-resynthesis, since immuno-detection of the epidermal growth factor receptor revealed that the receptor protein expression did not change during hypoxia and reoxygenation, and since neither actinomycin D nor cycloheximide affected the recovery of 125 I-epidermal growth factor binding by reoxygenation. Inhibition of epidermal growth factor-induced DNA synthesis after hypoxia (to 75.4% of normoxia by 3 h hypoxia) paralleled the decrease in 125 I-epidermal growth factor binding

  13. A local renal renin-angiotensin system activation via renal uptake of prorenin and angiotensinogen in diabetic rats.

    Science.gov (United States)

    Tojo, Akihiro; Kinugasa, Satoshi; Fujita, Toshiro; Wilcox, Christopher S

    2016-01-01

    The mechanism of activation of local renal renin-angiotensin system (RAS) has not been clarified in diabetes mellitus (DM). We hypothesized that the local renal RAS will be activated via increased glomerular filtration and tubular uptake of prorenin and angiotensinogen in diabetic kidney with microalbuminuria. Streptozotocin (STZ)-induced DM and control rats were injected with human prorenin and subsequently with human angiotensinogen. Human prorenin uptake was increased in podocytes, proximal tubules, macula densa, and cortical collecting ducts of DM rats where prorenin receptor (PRR) was expressed. Co-immunoprecipitation of kidney homogenates in DM rats revealed binding of human prorenin to the PRR and to megalin. The renal uptake of human angiotensinogen was increased in DM rats at the same nephron sites as prorenin. Angiotensin-converting enzyme was increased in podocytes, but decreased in the proximal tubules in DM rats, which may have contributed to unchanged renal levels of angiotensin despite increased angiotensinogen. The systolic blood pressure increased more after the injection of 20 μg of angiotensinogen in DM rats than in controls, accompanied by an increased uptake of human angiotensinogen in the vascular endothelium. In conclusion, endocytic uptake of prorenin and angiotensinogen in the kidney and vasculature in DM rats was contributed to increased tissue RAS and their pressor response to angiotensinogen.

  14. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways.

    Science.gov (United States)

    Fantl, W J; Escobedo, J A; Martin, G A; Turck, C W; del Rosario, M; McCormick, F; Williams, L T

    1992-05-01

    The receptor for platelet-derived growth factor (PDGF) binds two proteins containing SH2 domains, GTPase activating protein (GAP) and phosphatidylinositol 3-kinase (PI3-kinase). The sites on the receptor that mediate this interaction were identified by using phosphotyrosine-containing peptides representing receptor sequences to block specifically binding of either PI3-kinase or GAP. These results suggested that PI3-kinase binds two phosphotyrosine residues, each located in a 5 aa motif with an essential methionine at the fourth position C-terminal to the tyrosine. Point mutations at these sites caused a selective elimination of PI3-kinase binding and loss of PDGF-stimulated DNA synthesis. Mutation of the binding site for GAP prevented the receptor from associating with or phosphorylating GAP, but had no effect on PI3-kinase binding and little effect on DNA synthesis. Therefore, GAP and PI3-kinase interact with the receptor by binding to different phosphotyrosine-containing sequence motifs.

  15. Cross talk between AT1 receptors and Toll-like receptor 4 in microglia contributes to angiotensin II-derived ROS production in the hypothalamic paraventricular nucleus.

    Science.gov (United States)

    Biancardi, Vinicia Campana; Stranahan, Alexis M; Krause, Eric G; de Kloet, Annette D; Stern, Javier E

    2016-02-01

    ANG II is thought to increase sympathetic outflow by increasing oxidative stress and promoting local inflammation in the paraventricular nucleus (PVN) of the hypothalamus. However, the relative contributions of inflammation and oxidative stress to sympathetic drive remain poorly understood, and the underlying cellular and molecular targets have yet to be examined. ANG II has been shown to enhance Toll-like receptor (TLR)4-mediated signaling on microglia. Thus, in the present study, we aimed to determine whether ANG II-mediated activation of microglial TLR4 signaling is a key molecular target initiating local oxidative stress in the PVN. We found TLR4 and ANG II type 1 (AT1) receptor mRNA expression in hypothalamic microglia, providing molecular evidence for the potential interaction between these two receptors. In hypothalamic slices, ANG II induced microglial activation within the PVN (∼65% increase, P receptors and TLR4 in mediating ANG II-dependent microglial activation and oxidative stress within the PVN. More broadly, our results support a functional interaction between the central renin-angiotensin system and innate immunity in the regulation of neurohumoral outflows from the PVN. Copyright © 2016 the American Physiological Society.

  16. Angiotensin II Protects Primary Rat Hepatocytes against Bile Salt-Induced Apoptosis

    NARCIS (Netherlands)

    Karimian, Golnar; Buist-Homan, Manon; Mikus, Bojana; Henning, Robert H.; Faber, Klaas Nico; Moshage, Han

    2012-01-01

    Angiotensin II (AT-II) is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R) and thereby activates hepatic stellate cells (HSCs). AT-II receptor blockers (ARBs) are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that

  17. DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells

    International Nuclear Information System (INIS)

    Cuthill, S.; Poellinger, L.

    1988-01-01

    The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, the authors have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [ 3 H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin

  18. Characterization of the receptor-binding domain of Ebola glycoprotein in viral entry.

    Science.gov (United States)

    Wang, Jizhen; Manicassamy, Balaji; Caffrey, Michael; Rong, Lijun

    2011-06-01

    Ebola virus infection causes severe hemorrhagic fever in human and non-human primates with high mortality. Viral entry/infection is initiated by binding of glycoprotein GP protein on Ebola virion to host cells, followed by fusion of virus-cell membrane also mediated by GP. Using an human immunodeficiency virus (HIV)-based pseudotyping system, the roles of 41 Ebola GP1 residues in the receptor-binding domain in viral entry were studied by alanine scanning substitutions. We identified that four residues appear to be involved in protein folding/structure and four residues are important for viral entry. An improved entry interference assay was developed and used to study the role of these residues that are important for viral entry. It was found that R64 and K95 are involved in receptor binding. In contrast, some residues such as I170 are important for viral entry, but do not play a major role in receptor binding as indicated by entry interference assay and/or protein binding data, suggesting that these residues are involved in post-binding steps of viral entry. Furthermore, our results also suggested that Ebola and Marburg viruses share a common cellular molecule for entry.

  19. Sulfhydryl group content of chicken progesterone receptor: effect of oxidation on DNA binding activity

    International Nuclear Information System (INIS)

    Peleg, S.; Schrader, W.T.; O'Malley, B.W.

    1988-01-01

    DNA binding activity of chicken progesterone receptor B form (PRB) and A form (PRA) has been examined. This activity is strongly dependent upon the presence of thiols in the buffer. Stability studies showed that PRB was more sensitive to oxidation that was PRA. Receptor preparations were fractionated by DNA-cellulose chromatography to DNA-positive and DNA-negative subpopulations, and sulfhydryl groups were quantified on immunopurified receptor by labeling with [ 3 H]-N-ethylmaleimide. Labeling of DNA-negative receptors with [ 3 H]-N-ethylmaleimide showed 21-23 sulfhydryl groups on either PRA or PRB form when the proteins were reduced and denatured. A similar number was seen without reduction if denatured DNA-positive receptor species were tested. In contrast, the DNA-negative PRB had only 10-12 sulfhydryl groups detectable without reduction. A similar number (12-13 sulfhydryl groups) was found for PRA species that lost DNA binding activity after exposure to a nonreducing environment in vitro. The authors conclude that the naturally occurring receptor forms unable to bind to DNA, as well as receptor forms that have lost DNA binding activity due to exposure to nonreducing environment in vitro, contain 10-12 oxidized cysteine residues, likely present as disulfide bonds. Since they were unable to reduce the disulfide bonds when the native DNA-negative receptor proteins were treated with dithiothreitol (DTT), they speculate that irreversible loss of DNA binding activity of receptor in vitro is due to oxidation of cysteine residues that are not accessible to DTT in the native state

  20. Potential ligand-binding residues in rat olfactory receptors identified by correlated mutation analysis

    Science.gov (United States)

    Singer, M. S.; Oliveira, L.; Vriend, G.; Shepherd, G. M.

    1995-01-01

    A family of G-protein-coupled receptors is believed to mediate the recognition of odor molecules. In order to identify potential ligand-binding residues, we have applied correlated mutation analysis to receptor sequences from the rat. This method identifies pairs of sequence positions where residues remain conserved or mutate in tandem, thereby suggesting structural or functional importance. The analysis supported molecular modeling studies in suggesting several residues in positions that were consistent with ligand-binding function. Two of these positions, dominated by histidine residues, may play important roles in ligand binding and could confer broad specificity to mammalian odor receptors. The presence of positive (overdominant) selection at some of the identified positions provides additional evidence for roles in ligand binding. Higher-order groups of correlated residues were also observed. Each group may interact with an individual ligand determinant, and combinations of these groups may provide a multi-dimensional mechanism for receptor diversity.

  1. Emerging drugs which target the renin-angiotensin-aldosterone system.

    Science.gov (United States)

    Steckelings, Ulrike Muscha; Paulis, Ludovit; Unger, Thomas; Bader, Michael

    2011-12-01

    The renin-angiotensin-aldosterone system (RAAS) is already the most important target for drugs in the cardiovascular system. However, still new developments are underway to interfere with the system on different levels. The novel strategies to interfere with RAAS aim to reduce the synthesis of the two major RAAS effector hormones, angiotensin (Ang) II and aldosterone, or interfere with their receptors, AT1 and mineralocorticoid receptor, respectively. Moreover, novel targets have been identified in RAAS, such as the (pro)renin receptor, and molecules, which counteract the classical actions of Ang II and are therefore beneficial in cardiovascular diseases. These include the AT2 receptor and the ACE2/Ang-(1-7)/Mas axis. The search for drugs activating these tissue-protective arms of RAAS is therefore the most innovative field in RAAS pharmacology. Most of the novel pharmacological strategies to inhibit the classical RAAS need to prove their superiority above the existing treatment in clinical trials and then have to compete against these now quite cheap drugs in a competitive market. The newly discovered targets have functions beyond the cardiovascular system opening up novel therapeutic areas for drugs interfering with RAAS components.

  2. Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Lianying [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); College of Life Science, Dezhou University, Dezhou 253023 (China); Ren, Xiao-Min; Wan, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China); Guo, Liang-Hong, E-mail: LHGuo@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, P.O. Box 2871, 18 Shuangqing Road, Beijing 100085 (China)

    2014-09-15

    Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity. - Highlights: • Binding affinity between PFCs and PPARγ was evaluated for the first time. • The binding strength was dependent on fluorinated carbon chain and functional group. • PFC binding induced distinctive structural change of the receptor. • PFCs could act as hPPARγ agonists in Hep G2 cells.

  3. Angiotensin II type 1 receptor (A1166C gene polymorphism and essential hypertension in Egyptian population

    Directory of Open Access Journals (Sweden)

    Marium M. Shamaa

    2016-09-01

    Full Text Available The pathogenesis of essential hypertension (EH is affected by genetic and environmental factors. Mutations in hypertension-related genes can affect blood pressure (BP via alteration of salt and water reabsorption by the nephron. The genes of the renin-angiotensin system (RAS have been extensively studied because of the well documented role of this system in the control of BP. It has been previously shown that Angiotensin II type 1 receptor (ATR1 gene polymorphism could be associated with increased risk of EH. So, in the current study, we evaluated the frequency of ATR1 (A1166C polymorphism in relation to EH in a group of Egyptian population. The study population included 83 hypertensive patients and 60 age and sex matched healthy control subjects. Restriction fragment length polymorphism – Polymerase chain reaction (RFLP – PCR was used for the analysis of A1166C polymorphism of ATR1 genes in peripheral blood samples of all patients and controls. The results revealed that there was a positive risk of developing EH when having the T allele whether in homozygous or heterozygous state. From this work, it was concluded that there was an association between ATR1 (A1166C gene polymorphism and the risk of developing EH.

  4. Combined blockade of angiotensin II and prorenin receptors ameliorates podocytic apoptosis induced by IgA-activated mesangial cells.

    Science.gov (United States)

    Leung, Joseph C K; Chan, Loretta Y Y; Saleem, M A; Mathieson, P W; Tang, Sydney C W; Lai, Kar Neng

    2015-07-01

    Glomerulo-podocytic communication plays an important role in the podocytic injury in IgA nephropathy (IgAN). In this study, we examine the role of podocytic angiotensin II receptor subtype 1 (AT1R) and prorenin receptor (PRR) in podocytic apoptosis in IgAN. Polymeric IgA (pIgA) was isolated from patients with IgAN and healthy controls. Conditioned media were prepared from growth arrested human mesangial cells (HMC) incubated with pIgA from patients with IgAN (IgA-HMC media) or healthy controls (Ctl-HMC media). A human podocyte cell line was used as a model to examine the regulation of the expression of AT1R, PRR, TNF-α and CTGF by IgA-HMC media. Podocytic nephrin expression, annexin V binding and caspase 3 activity were used as the functional readout of podocytic apoptosis. IgA-HMC media had no effect on AngII release by podocytes. IgA-HMC media significantly up-regulated the expression of AT1R and PRR, down-regulated nephrin expression and induced apoptosis in podocytes. Mono-blockade of AT1R, PRR, TNF-α or CTGF partially reduced podocytic apoptosis. IgA-HMC media activated NFκB, notch1 and HEY1 expression by podocytes and dual blockade of AT1R with PRR, or anti-TNF-α with anti-CTGF, effectively rescued the podocytic apoptosis induced by IgA-HMC media. Our data suggests that pIgA-activated HMC up-regulates the expression of AT1R and PRR expression by podocytes and the associated activation of NFκB and notch signalling pathways play an essential role in the podocytic apoptosis induced by glomerulo-podocytic communication in IgAN. Simultaneously targeting the AT1R and PRR could be a potential therapeutic option to reduce the podocytic injury in IgAN.

  5. Two high-affinity ligand binding states of uterine estrogen receptor distinguished by modulation of hydrophobic environment

    International Nuclear Information System (INIS)

    Hutchens, T.W.; Li, C.M.; Zamah, N.M.; Besch, P.K.

    1987-01-01

    The steroid binding function of soluble (cytosolic) estrogen receptors from calf uteri was evaluated under conditions known to modify the extent of hydrophobic interaction with receptor-associated proteins. Receptor preparations were equilibrated into 6 M urea buffers and control buffers by chromatography through small columns of Sephadex G-25 or by dialysis at 0.6 0 C. Equilibrium dissociation constants (K/sub d/) and binding capacities (n) of experimental and control receptor preparations were determined by 13-point Scatchard analyses using concentrations of 17β-[ 3 H]estradiol from 0.05 to 10 nM. Nonspecific binding was determined at each concentration by parallel incubations with a 200-fold molar excess of the receptor-specific competitor diethylstilbestrol. The control receptor population was consistently found to be a single class of binding sites with a high affinity for estradiol which was unaffected by G-25 chromatography, by dialysis, by dilution, or by the presence of 0.4 M KCl. However, equilibration into 6 M urea induced a discrete (10-fold) reduction in receptor affinity to reveal a second, thermodynamically stable, high-affinity binding state. The presence of 0.4 M KCl did not significantly influence the discrete change in receptor affinity induced by urea. The effects of urea on both receptor affinity and binding capacity were reversible, suggesting a lack of covalent modification. These results demonstrate nonenzymatic means by which not only the binding capacity but also the affinity of receptor for estradiol can be reversibly controlled, suggesting that high concentrations of urea might be more effectively utilized during the physicochemical characterization and purification of steroid receptor proteins

  6. Impact of cell type and epitope tagging on heterologous expression of G protein-coupled receptor: a systematic study on angiotensin type II receptor.

    Directory of Open Access Journals (Sweden)

    Lili Jiang

    Full Text Available Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2 receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression.

  7. Familial Risk for Major Depression is Associated with Lower Striatal 5-HT4 Receptor Binding

    DEFF Research Database (Denmark)

    Madsen, Karine; Torstensen, Eva; Holst, Klaus K

    2014-01-01

    was to determine whether familial risk for MDD is associated with cerebral 5-HT4 receptor binding as measured with [(11)C]SB207145 brain PET imaging. Familial risk is the most potent risk factor of MDD. METHODS: We studied 57 healthy individuals (mean age 36 yrs, range 20-86; 21 women), 26 of which had first......-degree relatives treated for MDD. RESULTS: We found that having a family history of MDD was associated with lower striatal 5-HT4 receptor binding (p = 0.038; in individuals below 40 years, p = 0.013). Further, we found evidence for a "risk-dose effect" on 5-HT4 receptor binding, since the number of first......-degree relatives with a history of MDD binding correlated negatively with 5-HT4 receptor binding in both the striatum (p = 0.001) and limbic regions (p = 0.012). CONCLUSIONS: Our data suggest that the 5-HT4 receptor is involved in the neurobiological mechanism underlying familial risk for depression...

  8. Excess of Aminopeptidase A in the Brain Elevates Blood Pressure via the Angiotensin II Type 1 and Bradykinin B2 Receptors without Dipsogenic Effect

    Directory of Open Access Journals (Sweden)

    Takuto Nakamura

    2017-01-01

    Full Text Available Aminopeptidase A (APA cleaves angiotensin (Ang II, kallidin, and other related peptides. In the brain, it activates the renin angiotensin system and causes hypertension. Limited data are available on the dipsogenic effect of APA and pressor effect of degraded peptides of APA such as bradykinin. Wistar-Kyoto rats received intracerebroventricular (icv APA in a conscious, unrestrained state after pretreatment with (i vehicle, (ii 80 μg of telmisartan, an Ang II type-1 (AT1 receptor blocker, (iii 800 nmol of amastatin, an aminopeptidase inhibitor, and (iv 1 nmol of HOE-140, a bradykinin B2 receptor blocker. Icv administration of 400 and 800 ng of APA increased blood pressure by 12.6 ± 3.0 and 19.0 ± 3.1 mmHg, respectively. APA did not evoke drinking behavior. Pressor response to APA was attenuated on pretreatment with telmisartan (vehicle: 22.1 ± 2.2 mmHg versus telmisartan: 10.4 ± 3.2 mmHg. Pressor response to APA was also attenuated with amastatin and HOE-140 (vehicle: 26.5 ± 1.1 mmHg, amastatin: 14.4 ± 4.2 mmHg, HOE-140: 16.4 ± 2.2 mmHg. In conclusion, APA increase in the brain evokes a pressor response via enzymatic activity without dipsogenic effect. AT1 receptors and B2 receptors in the brain may contribute to the APA-induced pressor response.

  9. Inhibition of angiotensin II-induced facilitation of sympathetic neurotransmission in the pithed rat: a comparison between losartan, irbesartan, telmisartan, and captopril

    NARCIS (Netherlands)

    Balt, J. C.; Mathy, M. J.; Pfaffendorf, M.; van Zwieten, P. A.

    2001-01-01

    Numerous studies have shown that angiotensin II enhances sympathetic nervous transmission. The objective of the present study was to quantify the inhibitory effect of the angiotensin II type 1 (AT1) receptor blockers losartan, irbesartan and telmisartan and the angiotensin converting enzyme (ACE)

  10. Cholinergic, opioid and glycine receptor binding sites localized in human spinal cord by in vitro autoradiography

    International Nuclear Information System (INIS)

    Gillberg, P.-G.; Aquilonius, S.-M.

    1985-01-01

    Binding sites for the receptor ligands 3 H-quinuclidinylbenzilate, 3 H-alpha-bungarotoxin ( 3 H-alpha-Btx), 3 H-etorphine and 3 H-strychnine were localized autoradiographically at cervical, thoracic and lumbar levels of spinal cords from post-mortem human control subjects and subjects with amyotrophic lateral sclerosis (ALS). The highest densities of muscarinic binding sites were found in the motor neuron areas and in the substantia gelatinosa, while the grey matter binding was very low within Clarke's column. Both 3 H-alpha-Btx and opioid receptor binding sites were numerous within the substantia gelatinosa, while glycine receptor binding sites were more uniformly distribute within the spinal grey matter. In ALS cases, muscarinic receptor binding sites were markedly reduced in motor neuron areas and slightly reduced in the dorsal horn, while the other binding sites studied were relatively unchanged. (author)

  11. Recruitment of macrophages from the spleen contributes to myocardial fibrosis and hypertension induced by angiotensin II

    Directory of Open Access Journals (Sweden)

    Ning-Ping Wang

    2017-05-01

    Full Text Available Introduction: The purpose of this study was to determine whether macrophages migrated from the spleen are associated with angiotensin II-induced cardiac fibrosis and hypertension. Methods: Sprague-Dawley rats were subjected to angiotensin II infusion in vehicle (500 ng/kg/min for up to four weeks. In splenectomy, the spleen was removed before angiotensin II infusion. In the angiotensin II AT1 receptor blockade, telmisartan was administered by gastric gavage (10 mg/kg/day during angiotensin II infusion. The heart and aorta were isolated for Western blot analysis and immunohistochemistry. Results: Angiotensin II infusion caused a significant reduction in the number of monocytes in the spleen through the AT1 receptor-activated monocyte chemoattractant protein-1. Comparison of angiotensin II infusion, splenectomy and telmisartan comparatively reduced the recruitment of macrophages into the heart. Associated with this change, transforming growth factor β1 expression and myofibroblast proliferation were inhibited, and Smad2/3 and collagen I/III were downregulated. Furthermore, interstitial/perivascular fibrosis was attenuated. These modifications occurred in coincidence with reduced blood pressure. At week 4, invasion of macrophages and myofibroblasts in the thoracic aorta was attenuated and expression of endothelial nitric oxide synthase was upregulated, along with a reduction in aortic fibrosis. Conclusions: These results suggest that macrophages when recruited into the heart and aorta from the spleen potentially contribute to angiotensin II-induced cardiac fibrosis and hypertension.

  12. Receptor binding kinetics equations: Derivation using the Laplace transform method.

    Science.gov (United States)

    Hoare, Sam R J

    Measuring unlabeled ligand receptor binding kinetics is valuable in optimizing and understanding drug action. Unfortunately, deriving equations for estimating kinetic parameters is challenging because it involves calculus; integration can be a frustrating barrier to the pharmacologist seeking to measure simple rate parameters. Here, a well-known tool for simplifying the derivation, the Laplace transform, is applied to models of receptor-ligand interaction. The method transforms differential equations to a form in which simple algebra can be applied to solve for the variable of interest, for example the concentration of ligand-bound receptor. The goal is to provide instruction using familiar examples, to enable investigators familiar with handling equilibrium binding equations to derive kinetic equations for receptor-ligand interaction. First, the Laplace transform is used to derive the equations for association and dissociation of labeled ligand binding. Next, its use for unlabeled ligand kinetic equations is exemplified by a full derivation of the kinetics of competitive binding equation. Finally, new unlabeled ligand equations are derived using the Laplace transform. These equations incorporate a pre-incubation step with unlabeled or labeled ligand. Four equations for measuring unlabeled ligand kinetics were compared and the two new equations verified by comparison with numerical solution. Importantly, the equations have not been verified with experimental data because no such experiments are evident in the literature. Equations were formatted for use in the curve-fitting program GraphPad Prism 6.0 and fitted to simulated data. This description of the Laplace transform method will enable pharmacologists to derive kinetic equations for their model or experimental paradigm under study. Application of the transform will expand the set of equations available for the pharmacologist to measure unlabeled ligand binding kinetics, and for other time

  13. Two signaling molecules share a phosphotyrosine-containing binding site in the platelet-derived growth factor receptor.

    Science.gov (United States)

    Nishimura, R; Li, W; Kashishian, A; Mondino, A; Zhou, M; Cooper, J; Schlessinger, J

    1993-11-01

    Autophosphorylation sites of growth factor receptors with tyrosine kinase activity function as specific binding sites for Src homology 2 (SH2) domains of signaling molecules. This interaction appears to be a crucial step in a mechanism by which receptor tyrosine kinases relay signals to downstream signaling pathways. Nck is a widely expressed protein consisting exclusively of SH2 and SH3 domains, the overexpression of which causes cell transformation. It has been shown that various growth factors stimulate the phosphorylation of Nck and its association with autophosphorylated growth factor receptors. A panel of platelet-derived growth factor (PDGF) receptor mutations at tyrosine residues has been used to identify the Nck binding site. Here we show that mutation at Tyr-751 of the PDGF beta-receptor eliminates Nck binding both in vitro and in living cells. Moreover, the Y751F PDGF receptor mutant failed to mediate PDGF-stimulated phosphorylation of Nck in intact cells. A phosphorylated Tyr-751 is also required for binding of phosphatidylinositol-3 kinase to the PDGF receptor. Hence, the SH2 domains of p85 and Nck share a binding site in the PDGF receptor. Competition experiments with different phosphopeptides derived from the PDGF receptor suggest that binding of Nck and p85 is influenced by different residues around Tyr-751. Thus, a single tyrosine autophosphorylation site is able to link the PDGF receptor to two distinct SH2 domain-containing signaling molecules.

  14. Simultaneous Multiple MS Binding Assays Addressing D1 and D2 Dopamine Receptors.

    Science.gov (United States)

    Schuller, Marion; Höfner, Georg; Wanner, Klaus T

    2017-10-09

    MS Binding Assays are a label-free alternative to radioligand binding assays. They provide basically the same capabilities as the latter, but use a non-labeled reporter ligand instead of a radioligand. In contrast to radioligand binding assays, MS Binding Assays offer-owing to the selectivity of mass spectrometric detection-the opportunity to monitor the binding of different reporter ligands at different targets simultaneously. The present study shows a proof of concept for this strategy as exemplified for MS Binding Assays selectively addressing D 1 and D 2 dopamine receptors in a single binding experiment. A highly sensitive, rapid and robust LC-ESI-MS/MS quantification method capable of quantifying both SCH23390 and raclopride, selectively addressing D 1 and D 2 receptors, respectively, was established and validated for this purpose. Based thereon, simultaneous saturation and competition experiments with SCH23390 and raclopride in the presence of both D 1 and D 2 receptors were performed and analyzed by LC-MS/MS within a single chromatographic cycle. The present study thus demonstrates the feasibility of this strategy and the high versatility of MS Binding Assays that appears to surpass that common for conventional radioligand binding assays. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Management of hyperkalaemia consequent to mineralocorticoid-receptor antagonist therapy

    NARCIS (Netherlands)

    Roscioni, Sara S.; de Zeeuw, Dick; Bakker, Stephan J. L.; Lambers Heerspink, Hiddo J.

    2012-01-01

    Mineralocorticoid-receptor antagonists (MRAs) reduce blood pressure and albuminuria in patients treated with angiotensin-converting-enzyme inhibitors or angiotensin-II-receptor blockers. The use of MRAs, however, is limited by the occurrence of hyperkalaemia, which frequently occurs in patients

  16. Dual Blockade of the Renin-angiotensin-aldosterone System in Type 2 Diabetic Kidney Disease

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    Yan-Huan Feng

    2016-01-01

    Full Text Available Objective: To examine the efficacy and safety of dual blockade of the renin-angiotensin-aldosterone system (RAAS among patients with type 2 diabetic kidney disease. Data Sources: We searched the major literature repositories, including the Cochrane Central Register of Controlled Trials, MEDLINE and EMBASE, for randomized clinical trials published between January 1990 and October 2015 that compared the efficacy and safety of the use of dual blockade of the RAAS versus the use of monotherapy, without applying any language restrictions. Keywords for the searches included "diabetic nephropathy," "chronic kidney disease," "chronic renal insufficiency," "diabetes mellitus," "dual therapy," "combined therapy," "dual blockade," "renin-angiotensin system," "angiotensin-converting enzyme inhibitor," "angiotensin-receptor blocker," "aldosterone blockade," "selective aldosterone blockade," "renin inhibitor," "direct renin inhibitor," "mineralocorticoid receptor blocker," etc. Study Selection: The selected articles were carefully reviewed. We excluded randomized clinical trials in which the kidney damage of patients was related to diseases other than diabetes mellitus. Results: Combination treatment with an angiotensin-converting enzyme inhibitor supplemented by an angiotensin II receptor blocking agent is expected to provide a more complete blockade of the RAAS and a better control of hypertension. However, existing literature has presented mixed results, in particular, related to patient safety. In view of this, we conducted a comprehensive literature review in order to explain the rationale for dual blockade of the RAAS, and to discuss the pros and cons. Conclusions: Despite the negative results of some recent large-scale studies, it may be immature to declare that the dual blockade is a failure because of the complex nature of the RAAS surrounding its diversified functions and utility. Further trials are warranted to study the combination therapy as an

  17. Site-directed alkylation of multiple opioid receptors. I. Binding selectivity

    International Nuclear Information System (INIS)

    James, I.F.; Goldstein, A.

    1984-01-01

    A method for measuring and expressing the binding selectivity of ligands for mu, delta, and kappa opioid binding sites is reported. Radioligands are used that are partially selective for these sites in combination with membrane preparations enriched in each site. Enrichment was obtained by treatment of membranes with the alkylating agent beta-chlornaltrexamine in the presence of appropriate protecting ligands. After enrichment for mu receptors, [ 3 H] dihydromorphine bound to a single type of site as judged by the slope of competition binding curves. After enrichment for delta or kappa receptors, binding sites for [ 3 H] [D-Ala2, D-Leu5]enkephalin and [3H]ethylketocyclazocine, respectively, were still not homogeneous. There were residual mu sites in delta-enriched membranes but no evidence for residual mu or delta sites in kappa-enriched membranes were found. This method was used to identify ligands that are highly selective for each of the three types of sites

  18. Investigation into the Mechanism of Homo- and Heterodimerization of Angiotensin-Converting Enzyme.

    Science.gov (United States)

    Abrie, J Albert; Moolman, Wessel J A; Cozier, Gyles E; Schwager, Sylva L; Acharya, K Ravi; Sturrock, Edward D

    2018-04-01

    Angiotensin-converting enzyme (ACE) plays a central role in the renin-angiotensin system (RAS), which is primarily responsible for blood pressure homeostasis. Studies have shown that ACE inhibitors yield cardiovascular benefits that cannot be entirely attributed to the inhibition of ACE catalytic activity. It is possible that these benefits are due to interactions between ACE and RAS receptors that mediate the protective arm of the RAS, such as angiotensin II receptor type 2 (AT 2 R) and the receptor MAS. Therefore, in this study, we investigated the molecular interactions of ACE, including ACE homodimerization and heterodimerization with AT 2 R and MAS, respectively. Molecular interactions were assessed by fluorescence resonance energy transfer and bimolecular fluorescence complementation in human embryonic kidney 293 cells and Chinese hamster ovary-K1 cells transfected with vectors encoding fluorophore-tagged proteins. The specificity of dimerization was verified by competition experiments using untagged proteins. These techniques were used to study several potential requirements for the germinal isoform of angiotensin-converting enzyme expressed in the testes (tACE) dimerization as well as the effect of ACE inhibitors on both somatic isoforms of angiotensin-converting enzyme expressed in the testes (sACE) and tACE dimerization. We demonstrated constitutive homodimerization of sACE and of both of its domains separately, as well as heterodimerization of both sACE and tACE with AT 2 R, but not MAS. In addition, we investigated both soluble sACE and the sACE N domain using size-exclusion chromatography-coupled small-angle X-ray scattering and we observed dimers in solution for both forms of the enzyme. Our results suggest that ACE homo- and heterodimerization does occur under physiologic conditions. Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.

  19. Evidence of changes in alpha-1/AT1 receptor function generated by diet-induced obesity.

    Science.gov (United States)

    Juarez, Esther; Tufiño, Cecilia; Querejeta, Enrique; Bracho-Valdes, Ismael; Bobadilla-Lugo, Rosa A

    2017-11-01

    To study whether hypercaloric diet-induced obesity deteriorates vascular contractility of rat aorta through functional changes in α 1 adrenergic and/or AT1 Angiotensin II receptors. Angiotensin II- or phenylephrine-induced contraction was tested on isolated aorta rings with and without endothelium from female Wistar rats fed for 7 weeks with hypercaloric diet or standard diet. Vascular expression of Angiotensin II Receptor type 1 (AT1R), Angiotensin II Receptor type 2 (AT2R), Cyclooxygenase-1 (COX-1), Cyclooxygenase-2 (COX-2), inducible Nitric Oxide Synthase (iNOS) and endothelial Nitric Oxide Synthase (eNOS), as well as blood pressure, glucose, insulin and angiotensin II blood levels were measured. Diet-induced obesity did not significantly change agonist-induced contractions (Emax and pD 2 hypercaloric diet vs standard diet n.s.d.) of both intact (e+) or endothelium free (e-) vessels but significantly decrease both phenylephrine and angiotensin II contraction (Emax p obesity did not change angiotensin II AT1, AT2 receptor proteins expression but reduced COX-1 and NOS2 ( p obesity produces alterations in vascular adrenergic and angiotensin II receptor dynamics that suggest an endothelium-dependent adrenergic/angiotensin II crosstalk. These changes reflect early-stage vascular responses to obesity.

  20. Two Differential Binding Mechanisms of FG-Nucleoporins and Nuclear Transport Receptors

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    Piau Siong Tan

    2018-03-01

    Full Text Available Summary: Phenylalanine-glycine-rich nucleoporins (FG-Nups are intrinsically disordered proteins, constituting the selective barrier of the nuclear pore complex (NPC. Previous studies showed that nuclear transport receptors (NTRs were found to interact with FG-Nups by forming an “archetypal-fuzzy” complex through the rapid formation and breakage of interactions with many individual FG motifs. Here, we use single-molecule studies combined with atomistic simulations to show that, in sharp contrast, FG-Nup214 undergoes a coupled reconfiguration-binding mechanism when interacting with the export receptor CRM1. Association and dissociation rate constants are more than an order of magnitude lower than in the archetypal-fuzzy complex between FG-Nup153 and NTRs. Unexpectedly, this behavior appears not to be encoded selectively into CRM1 but rather into the FG-Nup214 sequence. The same distinct binding mechanisms are unperturbed in O-linked β-N-acetylglucosamine-modified FG-Nups. Our results have implications for differential roles of distinctly spatially distributed FG-Nup⋅NTR interactions in the cell. : Archetypal-fuzzy complexes found in most FG-Nucleoporin⋅nuclear transport receptor complexes allow fast yet specific nuclear transport. Tan et al. show that FG-Nup214, located at the periphery of the nuclear pore complex, binds to CRM1⋅RanGTP via a coupled reconfiguration-binding mechanism, which can enable different functionalities e.g., cargo release. Keywords: intrinsically disordered protein, glycosylation, FG-Nup, nuclear transport receptors, binding mechanism, single-molecule FRET, molecular dynamics simulations

  1. Muscarinic and alpha 1-adrenergic receptor binding characteristics of saw palmetto extract in rat lower urinary tract.

    Science.gov (United States)

    Suzuki, Mayumi; Oki, Tomomi; Sugiyama, Tomomi; Umegaki, Keizo; Uchida, Shinya; Yamada, Shizuo

    2007-06-01

    To elucidate the in vitro and ex vivo effects of saw palmetto extract (SPE) on autonomic receptors in the rat lower urinary tract. The in vitro binding affinities for alpha 1-adrenergic, muscarinic, and purinergic receptors in the rat prostate and bladder were measured by radioligand binding assays. Rats received vehicle or SPE (0.6 to 60 mg/kg/day) orally for 4 weeks, and alpha 1-adrenergic and muscarinic receptor binding in tissues of these rats were measured. Saw palmetto extract inhibited specific binding of [3H]prazosin and [N-methyl-3H]scopolamine methyl chloride (NMS) but not alpha, beta-methylene adenosine triphosphate [2,8-(3)H]tetrasodium salt in the rat prostate and bladder. The binding activity of SPE for muscarinic receptors was four times greater than that for alpha 1-adrenergic receptors. Scatchard analysis revealed that SPE significantly reduced the maximal number of binding sites (Bmax) for each radioligand in the prostate and bladder under in vitro condition. Repeated oral administration of SPE to rats brought about significant alteration in Bmax for prostatic [3H]prazosin binding and for bladder [3H]NMS binding. Such alteration by SPE was selective to the receptors in the lower urinary tract. Saw palmetto extract exerts significant binding activity on autonomic receptors in the lower urinary tract under in vitro and in vivo conditions.

  2. Discovery of novel, potent and low-toxicity angiotensin II receptor type 1 (AT1) blockers: Design, synthesis and biological evaluation of 6-substituted aminocarbonyl benzimidazoles with a chiral center.

    Science.gov (United States)

    Han, Xiao-Feng; He, Xing; Wang, Miao; Xu, Di; Hao, Li-Ping; Liang, Ai-Hua; Zhang, Jun; Zhou, Zhi-Ming

    2015-10-20

    Novel angiotensin II receptor type 1 (AT1) blockers bearing 6-substituted carbamoyl benzimidazoles with a chiral center were designed and synthesized as the first step to develop new antihypertensive agents and understand their pharmacodynamic and pharmacokinetic properties. The newly synthesized compounds were tested for their potential ability to displace [(125)I] Sar(1) Ile(8)-Ang II, which was specifically bound to human AT1 receptor. Radioligand binding assays revealed nanomolar affinity in several compounds under study. The IC50 values of nine ligands were higher than those of Losartan. The screening of decreased blood pressure in spontaneous hypertensive rats displayed that compound 8S (IC₅₀ = 5.0 nM) was equipotent with Losartan, whereas compounds 13R (IC₅₀ = 7.3 nM), 14R (IC₅₀ = 6.3 nM), and 14S (IC₅₀ = 3.5 nM) were slightly ahead of Losartan, and the most significant activity was demonstrated by compound 8R (IC₅₀ = 1.1 nM). Candidate 8R was identified for its excellent efficacy in antihypertension and fairly low toxicity based on plasma analyses, toxicology studies, and chronic oral tests. Finally, compound 8R exhibited strong and multiple interactions with target active sites of the theoretical AT1 receptor model in docking study. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  3. Synthetic Receptors Induce Anti Angiogenic and Stress Signaling on Human First Trimester Cytotrophoblast Cells

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    Ahmed F. Pantho

    2017-05-01

    Full Text Available The cytotrophoblast (CTB cells of the human placenta have membrane receptors that bind certain cardiotonic steroids (CTS found in blood plasma. One of these, marinobufagenin, is a key factor in the etiology of preeclampsia. Herein, we used synthetic receptors (SR to study their effectiveness on the angiogenic profile of human first trimester CTB cells. The humanextravillous CTB cells (Sw.71 used in this study were derived from first trimester chorionic villus tissue. Culture media of CTB cells treated with ≥1 nM SR level revealed sFlt-1 (Soluble fms-like tyrosine kinase-1 was significantly increased while VEGF (vascular endothelial growth factor was significantly decreased in the culture media (* p < 0.05 for each The AT2 receptor (Angiotensin II receptor type 2 expression was significantly upregulated in ≥1 nM SR-treated CTB cells as compared to basal; however, the AT1 (Angiotensin II receptor, type 1 and VEGFR-1 (vascular endothelial growth factor receptor 1 receptor expression was significantly downregulated (* p < 0.05 for each. Our results show that the anti-proliferative and anti-angiogenic effects of SR on CTB cells are similar to the effects of CTS. The observed anti angiogenic activity of SR on CTB cells demonstrates that the functionalized-urea/thiourea molecules may be useful as potent inhibitors to prevent CTS-induced impairment of CTB cells.

  4. [Role of angiotensin II receptor type 2 in predicting biochemical recurrence in the treatment of prostate cancer].

    Science.gov (United States)

    Chibichyan, M B; Kogan, M I; Chernogubova, E A; Pavlenko, I A; Matishov, D G

    2016-12-01

    To identify markers for predicting aggressive forms of prostate cancer. The study retrospectively evaluated expression of angiotensin II type 2 receptors (AT2-R) in prostate needle biopsy tissue from patients with and without biochemical recurrence after combined hormone and radiation therapy. The study findings showed that low expression of AT2-R in prostate tissue was associated with a high risk of biochemical recurrence. The data on the nature of AT2-R expression in prostate tissue of prostate cancer patients may be considered as a tool for predicting biochemical recurrence after combined hormone and radiation therapy. The test has a sensitivity of 87.5% and specificity of 85.71%.

  5. Management of hypertension: Insights into prescribing behavior with focus on angiotensin receptor blockers

    Directory of Open Access Journals (Sweden)

    S Ramakrishnan

    2017-01-01

    Full Text Available Introduction: Angiotensin receptor blockers (ARBs are emerging as an attractive first choice antihypertensive as recommended by various guidelines. However, choice among the first-line antihypertensive classes and among ARBs differs between practicing physicians. Aims: This survey aimed to understand the usage preferences of ARBs and its place in for treating hypertension (HTN among physicians from various clinical settings in India. Methods: A cross-sectional survey was conducted with a prevalidated survey questionnaire consisting of 25 questions for HTN management. Practicing general physicians and cardiologists were approached for seeking their perception, opinions, and prescribing behavior. Results: Responses of 594 physicians and cardiologists were received. As opined by 90.1% of physicians, newly diagnosed HTN represented more than 10% of their overall patient load. As a monotherapy, 59.9% of the physicians preferred ARB as the first choice in newly diagnosed HTN patients, followed by calcium channel blocker (12.3% and angiotensin-converting-enzyme inhibitor (8.1%. Of all ARBs, telmisartan is preferred by 73% of physicians. Most physicians prefer telmisartan among all ARBs for 24 h blood pressure (BP control, including morning BP surge (76.4% and for prevention of cardiovascular morbidity and mortality (78.8% followed by olmesartan and losartan. Predominantly, majority of physicians (89.1% agreed for the beneficial role of telmisartan in preventing onset of microalbuminuria and nephropathy. Conclusion: Indian physicians prefer ARBs as the first choice in most hypertensive patients, which shows agreement with the guideline recommendations followed globally. Telmisartan has emerged as the most preferred ARB among all, for most of the HTN patients including those with comorbidities.

  6. Selectivity in progesterone and androgen receptor binding of progestagens used in oral contraceptives

    International Nuclear Information System (INIS)

    Kloosterboer, H.J.; Vonk-Noordegraaf, C.A.; Turpijn, E.W.

    1988-01-01

    The relative binding affinities (RBAs) of four progestational compounds (norethisterone, levonorgestrel, 3-keto-desogestrel and gestodene) for the human progesterone and androgen receptors were measured in MCF-7 cytosol and intact MCF-7 cells. For the binding to the progesterone receptor, both Org 2058 and Org 3236 (or 3-keto-desogestrel) were used as labelled ligands. The following ranking (low to high) for the RBA of the nuclear (intact cells) progesterone receptor irrespective of the ligand used is found: norethisterone much less than levonorgestrel less than 3-keto-destogestrel less than gestodene. The difference between the various progestagens is significant with the exception of that between 3-keto-desogestrel and gestodene, when Org 2058 is used as ligand. For the cytosolic progesterone receptor, the same order is found with the exception that similar RBAs are found for gestodene and 3-keto-desogestrel. The four progestagens clearly differ with respect to binding to the androgen receptor using dihydrotestosterone as labelled ligand in intact cells; the ranking (low to high) is: norethisterone less than 3 keto-desogestrel less than levonorgestrel and gestodene. The difference between 3-keto-desogestrel and levonorgestrel or gestodene is significant. The selectivity indices (ratio of the mean RBA for the progesterone receptor to that of androgen receptor) in intact cells are significantly higher for 3-keto-desogestrel and gestodene than for levonorgestrel and norethisterone. From these results we conclude that the introduction of the 18-methyl in norethisterone (levonorgestel) increases both the binding to the progesterone and androgen receptors

  7. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

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    Kratochwil Nicole A

    2007-10-01

    Full Text Available Abstract Background Differences in sweet taste perception among species depend on structural variations of the sweet taste receptor. The commercially used isovanillyl sweetener neohesperidin dihydrochalcone activates the human but not the rat sweet receptor TAS1R2+TAS1R3. Analysis of interspecies combinations and chimeras of rat and human TAS1R2+TAS1R3 suggested that the heptahelical domain of human TAS1R3 is crucial for the activation of the sweet receptor by neohesperidin dihydrochalcone. Results By mutational analysis combined with functional studies and molecular modeling we identified a set of different amino acid residues within the heptahelical domain of human TAS1R3 that forms the neohesperidin dihydrochalcone binding pocket. Sixteen amino acid residues in the transmembrane domains 2 to 7 and one in the extracellular loop 2 of hTAS1R3 influenced the receptor's response to neohesperidin dihydrochalcone. Some of these seventeen residues are also part of the binding sites for the sweetener cyclamate or the sweet taste inhibitor lactisole. In line with this observation, lactisole inhibited activation of the sweet receptor by neohesperidin dihydrochalcone and cyclamate competitively, whereas receptor activation by aspartame, a sweetener known to bind to the N-terminal domain of TAS1R2, was allosterically inhibited. Seven of the amino acid positions crucial for activation of hTAS1R2+hTAS1R3 by neohesperidin dihydrochalcone are thought to play a role in the binding of allosteric modulators of other class C GPCRs, further supporting our model of the neohesperidin dihydrochalcone pharmacophore. Conclusion From our data we conclude that we identified the neohesperidin dihydrochalcone binding site at the human sweet taste receptor, which overlaps with those for the sweetener cyclamate and the sweet taste inhibitor lactisole. This readily delivers a molecular explanation of our finding that lactisole is a competitive inhibitor of the receptor

  8. Agonist and antagonist binding to rat brain muscarinic receptors: influence of aging

    International Nuclear Information System (INIS)

    Gurwitz, D.; Egozi, Y.; Henis, Y.I.; Kloog, Y.; Sokolovsky, M.

    1987-01-01

    The objective of the present study was to determine the binding properties of muscarinic receptors in six brain regions in mature and old rats of both sexes by employing direct binding of [ 3 H]-antagonist as well as of the labeled natural neurotransmitter, [ 3 H]-acetylcholine [( 3 H]-AcCh). In addition, age-related factors were evaluated in the modulation processes involved in agonist binding. The results indicate that as the rat ages the density of the muscarinic receptors is altered differently in the various brain regions: it is decreased in the cerebral cortex, hippocampus, striatum and olfactory bulb of both male and female rats, but is increased (58%) in the brain stem of senescent males while no significant change is observed for females. The use of the highly sensitive technique measuring direct binding of [ 3 H]-AcCh facilitated the separate detection of age-related changes in the two classes (high- and low-affinity) of muscarinic agonist binding sites. In old female rats the density of high-affinity [ 3 H]-AcCh binding sites was preserved in all tissues studied, indicating that the decreases in muscarinic receptor density observed with [ 3 H]-antagonist represent a loss of low-affinity agonist binding sites. In contrast, [ 3 H]-AcCh binding is decreased in the hypothalamus and increased in the brain stem of old male rats. These data imply sexual dimorphism of the aging process in central cholinergic mechanisms

  9. Enhanced Human-Type Receptor Binding by Ferret-Transmissible H5N1 with a K193T Mutation.

    Science.gov (United States)

    Peng, Wenjie; Bouwman, Kim M; McBride, Ryan; Grant, Oliver C; Woods, Robert J; Verheije, Monique H; Paulson, James C; de Vries, Robert P

    2018-05-15

    All human influenza pandemics have originated from avian influenza viruses. Although multiple changes are needed for an avian virus to be able to transmit between humans, binding to human-type receptors is essential. Several research groups have reported mutations in H5N1 viruses that exhibit specificity for human-type receptors and promote respiratory droplet transmission between ferrets. Upon detailed analysis, we have found that these mutants exhibit significant differences in fine receptor specificity compared to human H1N1 and H3N2 and retain avian-type receptor binding. We have recently shown that human influenza viruses preferentially bind to α2-6-sialylated branched N-linked glycans, where the sialic acids on each branch can bind to receptor sites on two protomers of the same hemagglutinin (HA) trimer. In this binding mode, the glycan projects over the 190 helix at the top of the receptor-binding pocket, which in H5N1 would create a stearic clash with lysine at position 193. Thus, we hypothesized that a K193T mutation would improve binding to branched N-linked receptors. Indeed, the addition of the K193T mutation to the H5 HA of a respiratory-droplet-transmissible virus dramatically improves both binding to human trachea epithelial cells and specificity for extended α2-6-sialylated N-linked glycans recognized by human influenza viruses. IMPORTANCE Infections by avian H5N1 viruses are associated with a high mortality rate in several species, including humans. Fortunately, H5N1 viruses do not transmit between humans because they do not bind to human-type receptors. In 2012, three seminal papers have shown how these viruses can be engineered to transmit between ferrets, the human model for influenza virus infection. Receptor binding, among others, was changed, and the viruses now bind to human-type receptors. Receptor specificity was still markedly different compared to that of human influenza viruses. Here we report an additional mutation in ferret

  10. Potent haloperidol derivatives covalently binding to the dopamine D2 receptor.

    Science.gov (United States)

    Schwalbe, Tobias; Kaindl, Jonas; Hübner, Harald; Gmeiner, Peter

    2017-10-01

    The dopamine D 2 receptor (D 2 R) is a common drug target for the treatment of a variety of neurological disorders including schizophrenia. Structure based design of subtype selective D 2 R antagonists requires high resolution crystal structures of the receptor and pharmacological tools promoting a better understanding of the protein-ligand interactions. Recently, we reported the development of a chemically activated dopamine derivative (FAUC150) designed to covalently bind the L94C mutant of the dopamine D 2 receptor. Using FAUC150 as a template, we elaborated the design and synthesis of irreversible analogs of the potent antipsychotic drug haloperidol forming covalent D 2 R-ligand complexes. The disulfide- and Michael acceptor-functionalized compounds showed significant receptor affinity and an irreversible binding profile in radioligand depletion experiments. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Some Aspects of the Renin-Angiotensin-System in Hemodialysis Patients

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    Umar Malik

    2015-11-01

    Full Text Available Understanding of the renin-angiotensin system (RAS has changed remarkably over the past decade. Renin, angiotensin converting enzyme (ACE, angiotensin II (Ang II, and Ang II receptors are the main components of the RAS. Recent studies identified the ACE2/Ang 1-7/Mas receptor axis, which counter-regulates the classical RAS. Many studies have examined the effects of the RAS on the progression of cardiovascular disease and chronic kidney disease (CKD. In addition, many studies have documented increased levels of ACE in hemodialysis (HD patients, raising concerns about the negative effects of RAS activation on the progression of renal disease. Elevated ACE increases the level of Ang II, leading to vasoconstriction and cell proliferation. Ang II stimulation of the sympathetic system leads to renal and cardiovascular complications that are secondary to uncontrolled hypertension. This review provides an overview of the RAS, evaluates new research on the role of ACE2 in dialysis, and reviews the evidence for potentially better treatments for patients undergoing HD. Further understanding of the role of ACE and ACE2 in HD patients may aid the development of targeted therapies that slow the progression of CKD and cardiovascular disease.

  12. Increased Angiotensin II Sensitivity Contributes to Microvascular Dysfunction in Women Who Have Had Preeclampsia.

    Science.gov (United States)

    Stanhewicz, Anna E; Jandu, Sandeep; Santhanam, Lakshmi; Alexander, Lacy M

    2017-08-01

    Women who have had preeclampsia have increased cardiovascular disease risk; however, the mechanism(s) responsible for this association remain unclear. Microvascular damage sustained during a preeclamptic pregnancy may persist postpartum. The putative mechanisms mediating this dysfunction include a reduction in NO-dependent dilation and an increased sensitivity to angiotensin II. In this study, we evaluated endothelium-dependent dilation, angiotensin II sensitivity, and the therapeutic effect of angiotensin II receptor blockade (losartan) on endothelium-dependent dilation in vivo in the microvasculature of women with a history of preeclampsia (n=12) and control women who had a healthy pregnancy (n=12). We hypothesized that preeclampsia would have (1) reduced endothelium-dependent dilation, (2) reduced NO-mediated dilation, and (3) increased sensitivity to angiotensin II. We further hypothesized that localized losartan would increase endothelium-dependent vasodilation in preeclampsia. We assessed microvascular endothelium-dependent vasodilator function by measurement of cutaneous vascular conductance responses to graded infusion of acetylcholine (acetylcholine; 10 -7 -102 mmol/L) and a standardized local heating protocol in control sites and sites treated with 15 mmol/L L-NAME ( N G -nitro-l-arginine methyl ester; NO-synthase inhibitor) or 43 µmol/L losartan. Further, we assessed microvascular vasoconstrictor sensitivity to angiotensin II (10 -20 -10 -4 mol/L). Preeclampsia had significantly reduced endothelium-dependent dilation (-0.3±0.5 versus -1.0±0.4 log EC50 ; P Preeclampsia also had augmented vasoconstrictor sensitivity to angiotensin II (-10.2±1.3 versus -8.3±0.5; P =0.006). Angiotensin II type I receptor inhibition augmented endothelium-dependent vasodilation and NO-dependent dilation in preeclampsia but had no effect in healthy pregnancy. These data suggest that women who have had preeclampsia have persistent microvascular dysfunction postpartum

  13. Ondansetron and granisetron binding orientation in the 5-HT(3) receptor determined by unnatural amino acid mutagenesis.

    Science.gov (United States)

    Duffy, Noah H; Lester, Henry A; Dougherty, Dennis A

    2012-10-19

    The serotonin type 3 receptor (5-HT(3)R) is a ligand-gated ion channel found in the central and peripheral nervous systems. The 5-HT(3)R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT(3)A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket.

  14. The discovery of new potent non-peptide Angiotensin II AT1 receptor blockers: A concise synthesis, molecular docking studies and biological evaluation of N-substituted 5-butylimidazole derivatives

    Czech Academy of Sciences Publication Activity Database

    Agelis, G.; Resvani, A.; Durdagi, S.; Spyridaki, K.; Tůmová, Tereza; Slaninová, Jiřina; Giannopoulos, P.; Vlahakos, D.; Liapakis, G.; Mavromoustakos, T.; Matsoukas, J.

    2012-01-01

    Roč. 55, Sep (2012), s. 358-374 ISSN 0223-5234 Institutional research plan: CEZ:AV0Z40550506 Keywords : synthesis * angiotensin II receptor blockers * N-substituted 5-butylimidazole derivatives * antihypertensive activity * molecular docking Subject RIV: CC - Organic Chemistry Impact factor: 3.499, year: 2012

  15. Increased methylglyoxal formation with upregulation of renin angiotensin system in fructose fed Sprague Dawley rats.

    Directory of Open Access Journals (Sweden)

    Indu Dhar

    Full Text Available The current epidemic of obesity and type 2 diabetes is attributed to a high carbohydrate diet, containing mainly high fructose corn syrup and sucrose. More than two thirds of diabetic patients have hypertension. Methylglyoxal is a highly reactive dicarbonyl generated during glucose and fructose metabolism, and a major precursor of advanced glycation end products (AGEs. Plasma methylglyoxal levels are increased in hypertensive rats and diabetic patients. Our aim was to examine the levels of methylglyoxal, mediators of the renin angiotensin system and blood pressure in male Sprague-Dawley rats treated with a high fructose diet (60% of total calories for 4 months. The thoracic aorta and kidney were used for molecular studies, along with cultured vascular smooth muscle cells (VSMCs. HPLC, Western blotting and Q-PCR were used to measure methylglyoxal and reduced glutathione (GSH, proteins and mRNA, respectively. Fructose treated rats developed a significant increase in blood pressure. Methylglyoxal level and protein and mRNA for angiotensin II, AT1 receptor, adrenergic α1D receptor and renin were significantly increased, whereas GSH levels were decreased, in the aorta and/or kidney of fructose fed rats. The protein expression of the receptor for AGEs (RAGE and NF-κB were also significantly increased in the aorta of fructose fed rats. MG treated VSMCs showed increased protein for angiotensin II, AT1 receptor, and α1D receptor. The effects of methylglyoxal were attenuated by metformin, a methylglyoxal scavenger and AGEs inhibitor. In conclusion, we report a strong association between elevated levels of methylglyoxal, RAGE, NF-κB, mediators of the renin angiotensin system and blood pressure in high fructose diet fed rats.

  16. Increased methylglyoxal formation with upregulation of renin angiotensin system in fructose fed Sprague Dawley rats.

    Science.gov (United States)

    Dhar, Indu; Dhar, Arti; Wu, Lingyun; Desai, Kaushik M

    2013-01-01

    The current epidemic of obesity and type 2 diabetes is attributed to a high carbohydrate diet, containing mainly high fructose corn syrup and sucrose. More than two thirds of diabetic patients have hypertension. Methylglyoxal is a highly reactive dicarbonyl generated during glucose and fructose metabolism, and a major precursor of advanced glycation end products (AGEs). Plasma methylglyoxal levels are increased in hypertensive rats and diabetic patients. Our aim was to examine the levels of methylglyoxal, mediators of the renin angiotensin system and blood pressure in male Sprague-Dawley rats treated with a high fructose diet (60% of total calories) for 4 months. The thoracic aorta and kidney were used for molecular studies, along with cultured vascular smooth muscle cells (VSMCs). HPLC, Western blotting and Q-PCR were used to measure methylglyoxal and reduced glutathione (GSH), proteins and mRNA, respectively. Fructose treated rats developed a significant increase in blood pressure. Methylglyoxal level and protein and mRNA for angiotensin II, AT1 receptor, adrenergic α1D receptor and renin were significantly increased, whereas GSH levels were decreased, in the aorta and/or kidney of fructose fed rats. The protein expression of the receptor for AGEs (RAGE) and NF-κB were also significantly increased in the aorta of fructose fed rats. MG treated VSMCs showed increased protein for angiotensin II, AT1 receptor, and α1D receptor. The effects of methylglyoxal were attenuated by metformin, a methylglyoxal scavenger and AGEs inhibitor. In conclusion, we report a strong association between elevated levels of methylglyoxal, RAGE, NF-κB, mediators of the renin angiotensin system and blood pressure in high fructose diet fed rats.

  17. Angiotensin II Type 1 Receptor Mechanoactivation Involves RGS5 (Regulator of G Protein Signaling 5) in Skeletal Muscle Arteries: Impaired Trafficking of RGS5 in Hypertension.

    Science.gov (United States)

    Hong, Kwangseok; Li, Min; Nourian, Zahra; Meininger, Gerald A; Hill, Michael A

    2017-12-01

    Studies suggest that arteriolar pressure-induced vasoconstriction can be initiated by GPCRs (G protein-coupled receptors), including the AT 1 R (angiotensin II type 1 receptor). This raises the question, are such mechanisms regulated by negative feedback? The present studies examined whether RGS (regulators of G protein signaling) proteins in vascular smooth muscle cells are colocalized with the AT 1 R when activated by mechanical stress or angiotensin II and whether this modulates AT 1 R-mediated vasoconstriction. To determine whether activation of the AT 1 R recruits RGS5, an in situ proximity ligation assay was performed in primary cultures of cremaster muscle arteriolar vascular smooth muscle cells treated with angiotensin II or hypotonic solution in the absence or presence of candesartan (an AT 1 R blocker). Proximity ligation assay results revealed a concentration-dependent increase in trafficking/translocation of RGS5 toward the activated AT 1 R, which was attenuated by candesartan. In intact arterioles, knockdown of RGS5 enhanced constriction to angiotensin II and augmented myogenic responses to increased intraluminal pressure. Myogenic constriction was attenuated to a higher degree by candesartan in RGS5 siRNA-transfected arterioles, consistent with RGS5 contributing to downregulation of AT 1 R-mediated signaling. Further, translocation of RGS5 was impaired in vascular smooth muscle cells of spontaneously hypertensive rats. This is consistent with dysregulated (RGS5-mediated) AT 1 R signaling that could contribute to excessive vasoconstriction in hypertension. In intact vessels, candesartan reduced myogenic vasoconstriction to a greater extent in spontaneously hypertensive rats compared with controls. Collectively, these findings suggest that AT 1 R activation results in translocation of RGS5 toward the plasma membrane, limiting AT 1 R-mediated vasoconstriction through its role in G q/11 protein-dependent signaling. © 2017 American Heart Association, Inc.

  18. Molecular conformation, receptor binding, and hormone action of natural and synthetic estrogens and antiestrogens.

    Science.gov (United States)

    Duax, W L; Griffin, J F; Weeks, C M; Korach, K S

    1985-01-01

    The X-ray crystallographic structural determinations of synthetic estrogens and antiestrogens provide reliable information on the global minimum energy conformation of these molecules or a local minimum energy conformation that is within 1 or 2 kcal/mole of the global minimum. In favorable cases, state-of-the-art molecular mechanics calculations provide quantitative agreement with X-ray results and information on the relative energy of other local minimum energy conformations not observed crystallographically. Because the conformation of diethylstilbestrol (DES) observed in solvated crystals has an overall conformation and dipole moment more similar to estradiol it is the form more likely to bind to the receptor and produce hormone activity. Either phenol ring of DES can successfully mimic the estradiol A-ring in binding to the receptor. Indenestrol A (INDA) and indenestrol B (INDB) have nearly identical fully extended planar conformations. Either the alpha or gamma rings of these compounds may mimic the A ring of estradiol and compete for the estrogen receptor. Although there are eight distinct ways in which molecules of a racemic mixture of INDA or INDB can bind to the receptor, not all of them may be able to elicit a hormonal response. This may account for the reduced biological activity of the compounds despite their successful competition for receptor binding. The minimum energy conformations of Z-pseudodiethylstilbestrol (ZPD) and E-pseudodiethylstilbestrol (EPD) are bent in a fashion similar to that of indanestrol (INDC). These molecules have good binding affinity suggesting that the receptor does not require a flat molecule. Therefore these conformations would appear to be compatible with receptor binding, but only the Z isomer has an energetically allowed extended conformation that accounts for its observed biological activity relative to DES. PMID:3905370

  19. Sequence similarity between the erythrocyte binding domain 1 of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals binding residues for the Duffy Antigen Receptor for Chemokines

    Directory of Open Access Journals (Sweden)

    Garry Robert F

    2011-01-01

    Full Text Available Abstract Background The surface glycoprotein (SU, gp120 of the human immunodeficiency virus (HIV must bind to a chemokine receptor, CCR5 or CXCR4, to invade CD4+ cells. Plasmodium vivax uses the Duffy Binding Protein (DBP to bind the Duffy Antigen Receptor for Chemokines (DARC and invade reticulocytes. Results Variable loop 3 (V3 of HIV-1 SU and domain 1 of the Plasmodium vivax DBP share a sequence similarity. The site of amino acid sequence similarity was necessary, but not sufficient, for DARC binding and contained a consensus heparin binding site essential for DARC binding. Both HIV-1 and P. vivax can be blocked from binding to their chemokine receptors by the chemokine, RANTES and its analog AOP-RANTES. Site directed mutagenesis of the heparin binding motif in members of the DBP family, the P. knowlesi alpha, beta and gamma proteins abrogated their binding to erythrocytes. Positively charged residues within domain 1 are required for binding of P. vivax and P. knowlesi erythrocyte binding proteins. Conclusion A heparin binding site motif in members of the DBP family may form part of a conserved erythrocyte receptor binding pocket.

  20. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    Energy Technology Data Exchange (ETDEWEB)

    Daughaday, W.H.; Trivedi, B.

    1987-07-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of /sup 125/I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound /sup 125/I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor.

  1. Absence of serum growth hormone binding protein in patients with growth hormone receptor deficiency (Laron dwarfism)

    International Nuclear Information System (INIS)

    Daughaday, W.H.; Trivedi, B.

    1987-01-01

    It has recently been recognized that human serum contains a protein that specifically binds human growth hormone (hGH). This protein has the same restricted specificity for hGH as the membrane-bound GH receptor. To determine whether the GH-binding protein is a derivative of, or otherwise related to, the GH receptor, the authors have examined the serum of three patients with Laron-type dwarfism, a condition in which GH refractoriness has been attributed to a defect in the GH receptor. The binding of 125 I-labeled hGH incubated with serum has been measured after gel filtration of the serum through an Ultrogel AcA 44 minicolumn. Results are expressed as percent of specifically bound 125 I-hGH and as specific binding relative to that of a reference serum after correction is made for endogenous GH. The mean +/- SEM of specific binding of sera from eight normal adults (26-46 years of age) was 21.6 +/- 0.45%, and the relative specific binding was 101.1 +/- 8.6%. Sera from 11 normal children had lower specific binding of 12.5 +/- 1.95% and relative specific binding of 56.6 +/- 9.1%. Sera from three children with Laron-type dwarfism lacked any demonstrable GH binding, whereas sera from 10 other children with other types of nonpituitary short stature had normal relative specific binding. They suggest that the serum GH-binding protein is a soluble derivative of the GH receptor. Measurement of the serum GH-binding protein may permit recognition of other abnormalities of the GH receptor

  2. Direct labelling of the human P2X7 receptor and identification of positive and negative cooperativity of binding.

    Science.gov (United States)

    Michel, A D; Chambers, L J; Clay, W C; Condreay, J P; Walter, D S; Chessell, I P

    2007-05-01

    The P2X(7) receptor exhibits complex pharmacological properties. In this study, binding of a [(3)H]-labelled P2X(7) receptor antagonist to human P2X(7) receptors has been examined to further understand ligand interactions with this receptor. The P2X(7) receptor antagonist, N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17), was radiolabelled with tritium and binding studies were performed using membranes prepared from U-2 OS or HEK293 cells expressing human recombinant P2X(7) receptors. Binding of [(3)H]-compound-17 was higher in membranes prepared from cells expressing P2X(7) receptors than from control cells and was inhibited by ATP suggesting labelled sites represented human P2X(7) receptors. Binding was reversible, saturable and modulated by P2X(7) receptor ligands (Brilliant Blue G, KN62, ATP, decavanadate). Furthermore, ATP potency was reduced in the presence of divalent cations or NaCl. Radioligand binding exhibited both positive and negative cooperativity. Positive cooperativity was evident from bell shaped Scatchard plots, reduction in radioligand dissociation rate by unlabelled compound-17 and enhancement of radioligand binding by KN62 and unlabelled compound-17. ATP and decavanadate inhibited binding in a negative cooperative manner as they enhanced radioligand dissociation. These data demonstrate that human P2X(7) receptors can be directly labelled and provide novel insights into receptor function. The positive cooperativity observed suggests that binding of compound-17 to one subunit in the P2X(7) receptor complex enhances subsequent binding to other P2X(7) subunits in the same complex. The negative cooperative effects of ATP suggest that ATP and compound-17 bind at separate, interacting, sites on the P2X(7) receptor.

  3. Structures of Receptor Complexes of a North American H7N2 Influenza Hemagglutinin with a Loop Deletion in the Receptor Binding Site

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Hua; Chen, Li-Mei; Carney, Paul J.; Donis, Ruben O.; Stevens, James (CDC)

    2012-02-21

    Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107), including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN) and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb). Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering) are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type ({alpha}2-3) receptor binding profile, with only moderate binding to human-type ({alpha}2-6) receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

  4. Structures of receptor complexes of a North American H7N2 influenza hemagglutinin with a loop deletion in the receptor binding site.

    Directory of Open Access Journals (Sweden)

    Hua Yang

    2010-09-01

    Full Text Available Human infections with subtype H7 avian influenza viruses have been reported as early as 1979. In 1996, a genetically stable 24-nucleotide deletion emerged in North American H7 influenza virus hemagglutinins, resulting in an eight amino acid deletion in the receptor-binding site. The continuous circulation of these viruses in live bird markets, as well as its documented ability to infect humans, raises the question of how these viruses achieve structural stability and functionality. Here we report a detailed molecular analysis of the receptor binding site of the North American lineage subtype H7N2 virus A/New York/107/2003 (NY107, including complexes with an avian receptor analog (3'-sialyl-N-acetyllactosamine, 3'SLN and two human receptor analogs (6'-sialyl-N-acetyllactosamine, 6'SLN; sialyllacto-N-tetraose b, LSTb. Structural results suggest a novel mechanism by which residues Arg220 and Arg229 (H3 numbering are used to compensate for the deletion of the 220-loop and form interactions with the receptor analogs. Glycan microarray results reveal that NY107 maintains an avian-type (alpha2-3 receptor binding profile, with only moderate binding to human-type (alpha2-6 receptor. Thus despite its dramatically altered receptor binding site, this HA maintains functionality and confirms a need for continued influenza virus surveillance of avian and other animal reservoirs to define their zoonotic potential.

  5. Apparent non-statistical binding in a ditopic receptor for guanosine

    NARCIS (Netherlands)

    Likhitsup, Asawin; Deeth, Robert J.; Otto, Sijbren; Marsh, Andrew

    2009-01-01

    Analysis of stepwise association constants for guests binding to more than one site in a receptor is expected to give a ratio of the first association constant to the second of about 4 : 1 on statistical grounds (since a second guest should have an equal chance of binding to a different site on the

  6. Renin angiotensin system and gender differences in dopaminergic degeneration

    Directory of Open Access Journals (Sweden)

    Rodriguez-Perez Ana I

    2011-08-01

    Full Text Available Abstract Background There are sex differences in dopaminergic degeneration. Men are approximately two times as likely as premenopausal women of the same age to develop Parkinson's disease (PD. It has been shown that the local renin angiotensin system (RAS plays a prominent role in sex differences in the development of chronic renal and cardiovascular diseases, and there is a local RAS in the substantia nigra and dopaminergic cell loss is enhanced by angiotensin via type 1 (AT1 receptors. Results In the present study, we observed that intrastriatal injection of 6-hydroxydopamine induced a marked loss of dopaminergic neurons in the substantia nigra of male rats, which was significantly higher than the loss induced in ovariectomized female rats given estrogen implants (i.e. rats with estrogen. However, the loss of dopaminergic neurons was significantly lower in male rats treated with the AT1 antagonist candesartan, and similar to that observed in female rats with estrogen. The involvement of the RAS in gender differences in dopaminergic degeneration was confirmed with AT1a-null mice lesioned with the dopaminergic neurotoxin MPTP. Significantly higher expression of AT1 receptors, angiotensin converting enzyme activity, and NADPH-oxidase complex activity, and much lower levels of AT2 receptors were observed in male rats than in female rats with estrogen. Conclusions The results suggest that brain RAS plays a major role in the increased risk of developing PD in men, and that manipulation of brain RAS may be an efficient approach for neuroprotective treatment of PD in men, without the feminizing effects of estrogen.

  7. Upregulation of [125I] CGP42112 binding in the rat brainstem following nodose ganglionectomy

    International Nuclear Information System (INIS)

    Roulston, C.L.; Lawrence, A.J.; Jarrott, B.; Widdop, R.E.

    1999-01-01

    Full text: [ 125 I] CGP42112 is a specific ligand which has been used to demonstrate the presence of angiotensin AT 2 receptors in peripheral and brain tissue, although [ 125 I] CGP42112 also binds to a non-angiotensin II (Ang II) site. In the present study we have examined [ 125 I] CGP42112 binding in the brainstem following nodose ganglionectomy using autoradiography. Male spontaneously hypertensive rats (SHR) and Wistar Kyoto (WKY) rats underwent unilateral nodose ganglionectomy or sham operation (anaesthetised with methohexitone, 60mg/kg ip). Following a 14 day recovery period, animals were killed and slide mounted brainstem sections (14μm) were prepared and incubated in the presence of either [ 125 I] (Sar 1 Ile 8 )Ang II (0.5nM), [ 125 I] CGP42112 (0.3nM) or [ 3 H] PKI11195 (3nM, a marker for activated microglia). Following incubation, slides were apposed to film for 10 days. Specific binding was determined in adjacent sections by co-incubations with either the AT 1 receptor antagonist losartan, the AT 2 receptor antagonist PD 123319, Ang II or PKI11195 (all at 10μM). In the ganglionectomised groups, there were significant reductions in [ 125 I] (Sar 1 Ile 8 )Ang II binding on the denervated side of the nucleus of the solitary tract (NTS) by 30 ± 2 % (n=5, P 125 I] CGP42112 binding in the NTS revealed an AT 2 receptor component which was displaceable by PD 123319 and Ang II (∼60%), and a non-Ang II component (∼40%). Nodose ganglionectomy increased the density of [ 125 I] CGP42112 binding on the denervated side of the NTS by 55 ± 3 % (n=5, P 125 I] CGP42112 binding in the NTS of the ganglionectomised groups was comprised of a greater non-Ang II component than in the sham group, since only ∼30% was displaced by PD123319 and Ang II. [ 125 I] CGP42112 also revealed high binding density in the dorsal motor nucleus and the nucleus ambiguus on the denervated side in both SHR and WKY rats. This binding was absent in shams and was only ∼30-40% displaceable

  8. Preliminary X-ray crystallographic study of the receptor-binding domain of the D/C mosaic neurotoxin from Clostridium botulinum

    International Nuclear Information System (INIS)

    Nuemket, Nipawan; Tanaka, Yoshikazu; Tsukamoto, Kentaro; Tsuji, Takao; Nakamura, Keiji; Kozaki, Shunji; Yao, Min; Tanaka, Isao

    2010-01-01

    To determine the binding mechanism of BoNT/OFD05 and its ganglioside receptors on neuronal cells, recombinant BoNT/OFD05 receptor-binding domain has been expressed, purified and crystallized. Botulinum toxin (BoNT) from Clostridium botulinum OFD05, isolated from bovine botulism, is a D/C mosaic-type BoNT. BoNTs possess binding, translocation and catalytic domains. The BoNT/OFD05 binding domain exhibits significant sequence identity to BoNT/C, which requires a single ganglioside as a binding receptor on neuronal cells, while BoNT/A and BoNT/B require two receptors for specific binding. To determine the binding mechanism of BoNT/OFD05 and its ganglioside receptors on neuronal cells, recombinant BoNT/OFD05 receptor-binding domain has been expressed, purified and crystallized. Native and SeMet-derivative crystals showed X-ray diffraction to 2.8 and 3.1 Å resolution, respectively. The crystals belonged to space group P2 1 2 1 2 1

  9. Reporter mouse strain provides a novel look at angiotensin type-2 receptor distribution in the central nervous system.

    Science.gov (United States)

    de Kloet, Annette D; Wang, Lei; Ludin, Jacob A; Smith, Justin A; Pioquinto, David J; Hiller, Helmut; Steckelings, U Muscha; Scheuer, Deborah A; Sumners, Colin; Krause, Eric G

    2016-03-01

    Angiotensin-II acts at its type-1 receptor (AT1R) in the brain to regulate body fluid homeostasis, sympathetic outflow and blood pressure. However, the role of the angiotensin type-2 receptor (AT2R) in the neural control of these processes has received far less attention, largely because of limited ability to effectively localize these receptors at a cellular level in the brain. The present studies combine the use of a bacterial artificial chromosome transgenic AT2R-enhanced green fluorescent protein (eGFP) reporter mouse with recent advances in in situ hybridization (ISH) to circumvent this obstacle. Dual immunohistochemistry (IHC)/ISH studies conducted in AT2R-eGFP reporter mice found that eGFP and AT2R mRNA were highly co-localized within the brain. Qualitative analysis of eGFP immunoreactivity in the brain then revealed localization to neurons within nuclei that regulate blood pressure, metabolism, and fluid balance (e.g., NTS and median preoptic nucleus [MnPO]), as well as limbic and cortical areas known to impact stress responding and mood. Subsequently, dual IHC/ISH studies uncovered the phenotype of specific populations of AT2R-eGFP cells. For example, within the NTS, AT2R-eGFP neurons primarily express glutamic acid decarboxylase-1 (80.3 ± 2.8 %), while a smaller subset express vesicular glutamate transporter-2 (18.2 ± 2.9 %) or AT1R (8.7 ± 1.0 %). No co-localization was observed with tyrosine hydroxylase in the NTS. Although AT2R-eGFP neurons were not observed within the paraventricular nucleus (PVN) of the hypothalamus, eGFP immunoreactivity is localized to efferents terminating in the PVN and within GABAergic neurons surrounding this nucleus. These studies demonstrate that central AT2R are positioned to regulate blood pressure, metabolism, and stress responses.

  10. Mimicking of Arginine by Functionalized N(ω)-Carbamoylated Arginine As a New Broadly Applicable Approach to Labeled Bioactive Peptides: High Affinity Angiotensin, Neuropeptide Y, Neuropeptide FF, and Neurotensin Receptor Ligands As Examples.

    Science.gov (United States)

    Keller, Max; Kuhn, Kilian K; Einsiedel, Jürgen; Hübner, Harald; Biselli, Sabrina; Mollereau, Catherine; Wifling, David; Svobodová, Jaroslava; Bernhardt, Günther; Cabrele, Chiara; Vanderheyden, Patrick M L; Gmeiner, Peter; Buschauer, Armin

    2016-03-10

    Derivatization of biologically active peptides by conjugation with fluorophores or radionuclide-bearing moieties is an effective and commonly used approach to prepare molecular tools and diagnostic agents. Whereas lysine, cysteine, and N-terminal amino acids have been mostly used for peptide conjugation, we describe a new, widely applicable approach to peptide conjugation based on the nonclassical bioisosteric replacement of the guanidine group in arginine by a functionalized carbamoylguanidine moiety. Four arginine-containing peptide receptor ligands (angiotensin II, neurotensin(8-13), an analogue of the C-terminal pentapeptide of neuropeptide Y, and a neuropeptide FF analogue) were subject of this proof-of-concept study. The N(ω)-carbamoylated arginines, bearing spacers with a terminal amino group, were incorporated into the peptides by standard Fmoc solid phase peptide synthesis. The synthesized chemically stable peptide derivatives showed high receptor affinities with Ki values in the low nanomolar range, even when bulky fluorophores had been attached. Two new tritiated tracers for angiotensin and neurotensin receptors are described.

  11. 1-Methyl-beta-carboline (harmane), a potent endogenous inhibitor of benzodiazepine receptor binding.

    Science.gov (United States)

    Rommelspacher, H; Nanz, C; Borbe, H O; Fehske, K J; Müller, W E; Wollert, U

    1980-10-01

    The interaction of several beta-carbolines with specific [3H]-flunitrazepam binding to benzodiazepine receptors in rat brain membranes was investigated. Out of the investigated compounds, harmane and norharmane were the most potent inhibitors of specific [3H]-flunitrazepam binding, with IC50-values in the micromolar range. All other derivatives, including harmine, harmaline, and several tetrahydroderivatives were at least ten times less potent. Harmane has been previously found in rat brain and human urine, so it is the most potent endogenous inhibitor of specific [3H]-flunitrazepam binding known so far, with a several fold higher affinity for the benzodiazepine receptor than inosine and hypoxanthine. Thus, we suggest that harmane or other related beta-carbolines could be potential candidates as endogenous ligands of the benzodiazepine receptor.

  12. Ondansetron and Granisetron Binding Orientation in the 5-HT3 Receptor Determined by Unnatural Amino Acid Mutagenesis

    Science.gov (United States)

    Duffy, Noah H.; Lester, Henry A.; Dougherty, Dennis A.

    2012-01-01

    The serotonin type 3 receptor (5-HT3R) is a ligand-gated ion channel that mediates fast synaptic transmission in the central and peripheral nervous systems. The 5-HT3R is a therapeutic target, and the clinically available drugs ondansetron and granisetron inhibit receptor activity. Their inhibitory action is through competitive binding to the native ligand binding site, although the binding orientation of the drugs at the receptor has been a matter of debate. Here we heterologously express mouse 5-HT3A receptors in Xenopus oocytes and use unnatural amino acid mutagenesis to establish a cation-π interaction for both ondansetron and granisetron to tryptophan 183 in the ligand binding pocket. This cation-π interaction establishes a binding orientation for both ondansetron and granisetron within the binding pocket. PMID:22873819

  13. Calcium channel blockers, angiotensin receptor blockers, and angiotensin-converting enzyme inhibitors: Effectiveness in combination with diuretics or β-blockers for treating hypertension

    Directory of Open Access Journals (Sweden)

    John D Bisognano

    2007-11-01

    Full Text Available John D Bisognano1, Trent McLaughlin2, Craig S Roberts3, Simon SK Tang31Internal Medicine Department, Cardiology Division, the University of Rochester Medical Center, Rochester, NY, USA; 2NDC Health, Phoenix, Arizona, USA; 3Pfizer Inc, New York, NY, USAAbstract: This retrospective database analysis compared the effectiveness of dihydropyridine calcium channel blockers (DHPs, angiotensin-converting enzyme (ACE inhibitors, and angiotensin receptor blockers (ARBs added to diuretics or β-blockers. Adults with hypertension treated with diuretic or β-blocker monotherapy between 1998 and 2001 were identified from a large US electronic medical records database of primary care practices. Patients were required to have a baseline blood pressure (BP ≥140/90 mmHg (≥130/80 mmHg for diabetes mellitus and recorded BP measurements within 6 months before and 1–12 months following index date. Patients were matched 1:1:1 by propensity score to correct for differences in baseline characteristics. 1875 patients met study criteria and 660 (220 in each cohort were matched based on propensity scores. Matched cohorts had no significant differences in baseline characteristics. Mean changes in systolic/diastolic BP were –17.5/–8.8, –15.7/–6.3, and –13.0/–8.0 mmHg with DHPs, ACE inhibitors, and ARBs, respectively. Joint National Committee on the Prevention, Detection, Evaluation, and Treatment of High BP 6/7 goal attainment for each regimen was 47.3%, 40.0%, and 32.2%, respectively. DHPs, ACE inhibitors, and ARBs improved BP when added to patients’ β-blocker or diuretic therapy. The greatest benefits were observed with DHPs, followed by ACE inhibitors, then ARBs.Keywords: hypertension, amlodipine besylate, lisinopril, valsartan, Joint National Committee (JNC 6 and 7

  14. Palmitoylethanolamide treatment reduces blood pressure in spontaneously hypertensive rats: involvement of cytochrome p450-derived eicosanoids and renin angiotensin system.

    Directory of Open Access Journals (Sweden)

    Giuseppina Mattace Raso

    Full Text Available Palmitoylethanolamide (PEA, a peroxisome proliferator-activated receptor-α agonist, has been demonstrated to reduce blood pressure and kidney damage secondary to hypertension in spontaneously hypertensive rat (SHR. Currently, no information is available concerning the putative effect of PEA on modulating vascular tone. Here, we investigate the mechanisms underpinning PEA blood pressure lowering effect, exploring the contribution of epoxyeicosatrienoic acids, CYP-dependent arachidonic acid metabolites, as endothelium-derived hyperpolarizing factors (EDHF, and renin angiotensin system (RAS modulation. To achieve this aim SHR and Wistar-Kyoto rats were treated with PEA (30 mg/kg/day for five weeks. Functional evaluations on mesenteric bed were performed to analyze EDHF-mediated vasodilation. Moreover, mesenteric bed and carotid were harvested to measure CYP2C23 and CYP2J2, the key isoenzymes in the formation of epoxyeicosatrienoic acids, and the soluble epoxide hydrolase, which is responsible for their degradation in the corresponding diols. Effect of PEA on RAS modulation was investigated by analyzing angiotensin converting enzyme and angiotensin receptor 1 expression. Here, we showed that EDHF-mediated dilation in response to acetylcholine was increased in mesenteric beds of PEA-treated SHR. Western blot analysis revealed that the increase in CYP2C23 and CYP2J2 observed in SHR was significantly attenuated in mesenteric beds of PEA-treated SHR, but unchanged in the carotids. Interestingly, in both vascular tissues, PEA significantly decreased the soluble epoxide hydrolase protein level, accompanied by a reduced serum concentration of its metabolite 14-15 dihydroxyeicosatrienoic acid, implying a reduction in epoxyeicosatrienoic acid hydrolisis. Moreover, PEA treatment down-regulated angiotensin receptor 1 and angiotensin converting enzyme expression, indicating a reduction in angiotensin II-mediated effects. Consistently, a damping of the

  15. D-2 dopamine receptor activation reduces free [3H]arachidonate release induced by hypophysiotropic peptides in anterior pituitary cells

    International Nuclear Information System (INIS)

    Canonico, P.L.

    1989-01-01

    Dopamine reduces the stimulation of intracellular [ 3 H]arachidonate release produced by the two PRL-stimulating peptides angiotensin-II and TRH. This effect is concentration dependent and is mediated by stimulation of D-2 dopamine receptors. D-2 receptor agonists (bromocriptine, dihydroergocryptine, and dihydroergocristine) inhibit the release of fatty acid induced by angiotensin-II with a potency that parallels their ability to inhibit PRL release in vitro. Conversely, the selective D-2 receptor antagonist L-sulpiride completely prevents dopamine's effect, whereas SCH 23390 (a D-1 receptor antagonist) is ineffective. The inhibitory action of dopamine does not seem to be consequent to an action on the adenylate cyclase-cAMP system, as 8-bromo-cAMP (1 mM) does not affect either basal or dopamine-inhibited [ 3 H]arachidonate release. However, a 24-h pertussis toxin pretreatment significantly reduces the action of dopamine on fatty acid release. Collectively, these results suggest that D-2 dopamine receptor-mediated inhibition of intracellular [ 3 H]arachidonate release requires the action of a GTP-binding protein, but is not a consequence of an inhibitory action on cAMP levels

  16. Angiotensin type 1a receptors in the paraventricular nucleus of the hypothalamus protect against diet-induced obesity

    Science.gov (United States)

    de Kloet, Annette D.; Pati, Dipanwita; Wang, Lei; Hiller, Helmut; Sumners, Colin; Frazier, Charles J.; Seeley, Randy J.; Herman, James P.; Woods, Stephen C.; Krause, Eric G.

    2013-01-01

    Obesity is associated with increased levels of angiotensin-II (Ang-II), which activates angiotensin type-1a receptors (AT1a) to influence cardiovascular function and energy homeostasis. To test the hypothesis that specific AT1a within the brain control these processes, we utilized the Cre/lox system to delete AT1a from the paraventricular nucleus of the hypothalamus (PVN) of mice. PVN AT1a deletion did not affect body mass or adiposity when mice were maintained on standard chow. However, maintenance on a high-fat diet revealed a gene by environment interaction whereby mice lacking AT1a in the PVN had increased food intake and decreased energy expenditure that augmented body mass and adiposity relative to controls. Despite this increased adiposity, PVN AT1a deletion reduced systolic blood pressure, suggesting that this receptor population mediates the positive correlation between adiposity and blood pressure. Gene expression studies revealed that PVN AT1a deletion decreased hypothalamic expression of corticotrophin-releasing hormone and oxytocin, neuropeptides known to control food intake and sympathetic nervous system activity. Whole cell patch clamp recordings confirmed that PVN AT1a deletion eliminates responsiveness of PVN parvocellular neurons to Ang-II, and suggest that Ang-II responsiveness is increased in obese wild-type mice. Central inflammation is associated with metabolic and cardiovascular disorders and PVN AT1a deletion reduced indices of hypothalamic inflammation. Collectively, these studies demonstrate that PVN AT1a regulate energy balance during environmental challenges that promote metabolic and cardiovascular pathologies. The implication is that the elevated Ang-II that accompanies obesity serves as a negative feedback signal that activates PVN neurons to alleviate weight gain. PMID:23486953

  17. Functional and biochemical responses of cultured heart cells to angiotensin II

    International Nuclear Information System (INIS)

    Allen, I.; Gaa, S.; Rogers, T.B.

    1986-01-01

    The authors have utilized a cultured neonatal rat heart myocyte system to study the molecular mechanisms involved in the stimulation of heart cells by angiotensin II (AII). The intact cultured cells, and membranes from these cells, have specific, high affinity receptors for 125 I-AII and for an AII antagonist, 125 I-Sar 1 ,Leu 8 -AII. Binding affinity was in the nanomolar range and was inhibited by guanine nucleotides. Functional studies on intact, beating cells revealed a maximal increase in contractile frequency of 50%, observed at 5 nM AII, with half maximal effects noted at around 1 nM. These responses were reversible and specific as the antagonist, Sar 1 , Ala 8 -AII, inhibited AII-induced chronotropic stimulation. AII (100 nM) had no effect on basal adenylate cyclase activity (20 pmoles cAMP/mg prot/min at 2.5mM Mg 2+ ) in cell membranes. Further, in membranes where cyclase activity was stimulated with isoproterenol (290 pmoles cAMP/mg prot/min at 2.5mM Mg 2+ ), addition of AII had no effect. The cyclase-inhibitory muscarinic agonist, carbachol, also failed to reduce isoproterenol-stimulated activity. In preliminary work with the intact cells, AII again did not alter basal cAMP levels (3-10 pmoles cAMP/mg prot). However, the hormone increased isoproterenol-stimulated cAMP levels by almost 50%. These cells are an excellent system for correlating AII receptor binding with functional and biochemical responses

  18. Resveratrol prevents angiotensin II-induced hypertrophy of vascular smooth muscle cells through the transactivation of growth factor receptors.

    Science.gov (United States)

    Hossain, Ekhtear; Anand-Srivastava, Madhu B

    2017-08-01

    We previously showed that augmented levels of endogenous angiotensin II (AngII) contribute to vascular smooth muscle cell (VSMC) hypertrophy through the transactivation of growth factor receptors in spontaneously hypertensive rats. Resveratrol (RV), a polyphenolic component of red wine, has also been shown to attenuate AngII-evoked VSMC hypertrophy; however, the molecular mechanism mediating this response is obscure. The present study was therefore undertaken to examine whether RV could prevent AngII-induced VSMC hypertrophy through the transactivation of growth factor receptor and associated signaling pathways. AngII treatment of VSMC enhanced the protein synthesis that was attenuated towards control levels by RV pretreatment as well as by the inhibitors of NADPH oxidase, c-Src, and growth factor receptors. Furthermore, RV pretreatment also inhibited enhanced levels of superoxide anion, NADPH oxidase activity, increased expression of NADPH oxidase subunits, and phosphorylation of c-Src, EGF-R, PDGE-R, ERK1/2, and AKT1/2. In conclusion, these results indicate that RV attenuates AngII-induced VSMC hypertrophy through the inhibition of enhanced oxidative stress and activation of c-Src, growth factor receptors, and MAPK/AKT signaling. We suggest that RV could be used as a therapeutic agent in the treatment of vascular complications associated with hypertension and hypertrophy.

  19. Effects of common anesthetic agents on [(18)F]flumazenil binding to the GABAA receptor

    DEFF Research Database (Denmark)

    Palner, Mikael; Beinat, Corinne; Banister, Sam

    2016-01-01

    in preclinical imaging studies and clinical imaging studies involving patient populations that do not tolerate relatively longer scan times. The objective of this study was to examine the effects of anesthesia on the binding of [(18)F]flumazenil to GABAA receptors in mice. METHODS: Brain and whole blood...... mice. CONCLUSIONS: Anesthesia has pronounced effects on the binding and blood-brain distribution of [(18)F]flumazenil. Consequently, considerable caution must be exercised in the interpretation of preclinical and clinical PET studies of GABAA receptors involving the use of anesthesia.......BACKGROUND: The availability of GABAA receptor binding sites in the brain can be assessed by positron emission tomography (PET) using the radioligand, [(18)F]flumazenil. However, the brain uptake and binding of this PET radioligand are influenced by anesthetic drugs, which are typically needed...

  20. Field flow fractionation for assessing neonatal Fc receptor and Fcγ receptor binding to monoclonal antibodies in solution.

    Science.gov (United States)

    Pollastrini, Joey; Dillon, Thomas M; Bondarenko, Pavel; Chou, Robert Y-T

    2011-07-01

    Analysis of the strength and stoichiometry of immunoglobulin G (IgG) binding to neonatal Fc receptor (FcRn) and Fcγ receptor (FcγR) is important for evaluating the pharmacokinetics and effector functions of therapeutic monoclonal antibody (mAb) products, respectively. The current standard for assessing FcγR and FcRn binding is composed of cell-based and surface plasmon resonance (SPR) assays. In this work, asymmetrical flow field flow fractionation (AF4) was evaluated to establish the true stoichiometry of IgG binding in solution. AF4 and liquid chromatography-mass spectrometry (LC-MS) were applied to directly observe IgG/FcγR and IgG/FcRn complexes, which were not observed using nonequilibrium size exclusion chromatography (SEC) analysis. Human serum albumin (HSA), an abundant component of human blood and capable of binding FcRn, was studied in combination with FcRn and IgG. AF4 demonstrated that the majority of large complexes of IgG/FcRn/HSA were at an approximate 1:2:1 molar ratio. In addition, affinity measurements of the complex were performed in the sub-micromolar affinity range. A significant decrease in binding was detected for IgG molecules with increased oxidation in the Fc region. AF4 was useful in detecting weak binding between full-length IgG/Fc fragments and Fc receptors and the effect of chemical modifications on binding. AF4 is a useful technique in the assessment of mAb product quality attributes. Copyright © 2011 Elsevier Inc. All rights reserved.

  1. Changes in 5-HT4 receptor and 5-HT transporter binding in olfactory bulbectomized and glucocorticoid receptor heterozygous mice

    DEFF Research Database (Denmark)

    Licht, Cecilie L; Kirkegaard, Lisbeth; Zueger, Maha

    2010-01-01

    . The olfactory bulbectomized mice displayed increased activity in the open field test, a characteristic depression-like feature of this model. After bulbectomy, 5-HT(4) receptor binding was increased in the ventral hippocampus (12%) but unchanged in the dorsal hippocampus, frontal and caudal caudate putamen......]citalopram in two murine models of depression-related states, olfactory bulbectomy and glucocorticoid receptor heterozygous (GR(+/-)) mice. The olfactory bulbectomy model is characterized by 5-HT system changes, while the GR(+/-) mice have a deficit in hypothalamic-pituitary-adrenal (HPA) system control....... Among post hoc analyzed regions, there was a 14% decrease in 5-HT(4) receptor binding in the olfactory tubercles. The 5-HTT binding was unchanged in the hippocampus and caudate putamen of bulbectomized mice but post hoc analysis showed small decreases in lateral septum and lateral globus pallidus...

  2. Gene expression profiling associated with angiotensin II type 2 receptor-induced apoptosis in human prostate cancer cells.

    Directory of Open Access Journals (Sweden)

    Nana Pei

    Full Text Available Increased expression of angiotensin II type 2 receptor (AT2R induces apoptosis in numerous tumor cell lines, with either Angiotensin II-dependent or Angiotensin II-independent regulation, but its molecular mechanism remains poorly understood. Here, we used PCR Array analysis to determine the gene and microRNA expression profiles in human prostate cancer cell lines transduced with AT2R recombinant adenovirus. Our results demonstrated that AT2R over expression leads to up-regulation of 6 apoptosis-related genes (TRAIL-R2, BAG3, BNIPI, HRK, Gadd45a, TP53BP2, 2 cytokine genes (IL6 and IL8 and 1 microRNA, and down-regulation of 1 apoptosis-related gene TNFSF10 and 2 cytokine genes (BMP6, BMP7 in transduced DU145 cells. HRK was identified as an up-regulated gene in AT2R-transduced PC-3 cells by real-time RT-PCR. Next, we utilized siRNAs to silence the up-regulated genes to further determine their roles on AT2R overexpression mediated apoptosis. The results showed downregulation of Gadd45a reduced the apoptotic effect by ∼30% in DU145 cells, downregulation of HRK reduced AT2R-mediated apoptosis by more than 50% in PC-3 cells, while downregulation of TRAIL-R2 enhanced AT2R-mediated apoptosis more than 4 times in DU145 cells. We also found that the effects on AT2R-mediated apoptosis caused by downregulation of Gadd45a, TRAIL-R2 and HRK were independent in activation of p38 MAPK, p44/42 MAPK and p53. Taken together, our results demonstrated that TRAIL-R2, Gadd45a and HRK may be novel target genes for further study of the mechanism of AT2R-mediated apoptosis in prostate cancer cells.

  3. Skeletal muscle wasting: new role of nonclassical renin-angiotensin system.

    Science.gov (United States)

    Cabello-Verrugio, Claudio; Rivera, Juan C; Garcia, Dominga

    2017-05-01

    Skeletal muscle can be affected by many physiological and pathological conditions that contribute to the development of muscle weakness, including skeletal muscle loss, inflammatory processes, or fibrosis. Therefore, research into therapeutic treatment alternatives or alleviation of these effects on skeletal muscle is of great importance. Recent studies have shown that angiotensin (1-7) [Ang-(1-7)] - a vasoactive peptide of the nonclassical axis in the renin-angiotensin system (RAS) - and its Mas receptor are expressed in skeletal muscle. Ang-(1-7), through its Mas receptor, prevents or diminishes deleterious effects induced by skeletal muscle disease or injury. Specifically, the Ang-(1-7)-Mas receptor axis modulates molecular mechanisms involved in muscle mass regulation, such as the ubiquitin proteasome pathway, the insulin-like growth factor type 1/Akt (protein kinase B) pathway, or myonuclear apoptosis, and also inflammation and fibrosis pathways. Although further research into this topic and the possible side effects of Ang-(1-7) is necessary, these findings are promising, and suggest that the Ang-(1-7)-Mas axis can be considered a possible therapeutic target for treating patients with muscular disorders.

  4. Cortical and subcortical 5-HT2A receptor binding in neuroleptic-naive first-episode schizophrenic patients

    DEFF Research Database (Denmark)

    Erritzoe, David; Rasmussen, Hans; Kristiansen, Klaus Nyegaard

    2008-01-01

    MRIs and PET images. The cerebellum was used as a reference region. The binding potential of specific tracer binding (BP(p)) was used as the outcome measure. No significant difference was seen in cortical receptor distribution between patients and controls. An increase in 5-HT(2A) receptor binding...

  5. Epac is required for exogenous and endogenous stimulation of adenosine A2B receptor for inhibition of angiotensin II-induced collagen synthesis and myofibroblast differentiation.

    Science.gov (United States)

    Phosri, Sarawuth; Bunrukchai, Kwanchai; Parichatikanond, Warisara; Sato, Vilasinee H; Mangmool, Supachoke

    2018-01-10

    Angiotensin II (Ang II) plays an important role on the pathogenesis of cardiac fibrosis. Prolong and overstimulation of angiotensin II type 1 receptor with Ang II-induced collagen synthesis and myofibroblast differentiation in cardiac fibroblasts, leading to cardiac fibrosis. Although adenosine and its analogues are known to have cardioprotective effects, the mechanistic by which adenosine A 2 receptors (A 2 Rs) inhibit Ang II-induced cardiac fibrosis is not clearly understood. In the present study, we examined the effects of exogenous adenosine and endogenous adenosine on Ang II-induced collagen and myofibroblast differentiation determined by α-smooth muscle action (α-SMA) overexpression and their underlying signal transduction. Elevation of endogenous adenosine levels resulted in the inhibition of Ang II-induced collagen type I and III and α-SMA synthesis in cardiac fibroblasts. Moreover, treatment with exogenous adenosine which selectively stimulated A 2 Rs also suppressed Ang II-induced collagen synthesis and α-SMA production. These antifibrotic effects of both endogenous and exogenous adenosines are mediated through the A 2B receptor (A 2B R) subtype. Stimulation of A 2B R exhibited antifibrotic effects via the cAMP-dependent and Epac-dependent pathways. Our results provide new mechanistic insights regarding the role for cAMP and Epac on A 2B R-mediated antifibrotic effects. Thus, A 2B R is one of the potential therapeutic targets against cardiac fibrosis.

  6. Flow Cytometry-Based Bead-Binding Assay for Measuring Receptor Ligand Specificity

    NARCIS (Netherlands)

    Sprokholt, Joris K.; Hertoghs, Nina; Geijtenbeek, Teunis B. H.

    2016-01-01

    In this chapter we describe a fluorescent bead-binding assay, which is an efficient and feasible method to measure interaction between ligands and receptors on cells. In principle, any ligand can be coated on fluorescent beads either directly or via antibodies. Binding between ligand-coated beads

  7. Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

    Energy Technology Data Exchange (ETDEWEB)

    Peterson, K.M.; Alderete, J.F.

    1984-08-01

    Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites.

  8. Iron uptake and increased intracellular enzyme activity follow host lactoferrin binding by Trichomonas vaginalis receptors

    International Nuclear Information System (INIS)

    Peterson, K.M.; Alderete, J.F.

    1984-01-01

    Lactoferrin acquisition and iron uptake by pathogenic Trichomonas vaginalis was examined. Saturation binding kinetics were obtained for trichomonads using increasing amounts of radioiodinated lactoferrin, while no significant binding by transferrin under similar conditions was achieved. Only unlabeled lactoferrin successfully and stoichiometrically competed with 125I-labeled lactoferrin binding. Time course studies showed maximal lactoferrin binding by 30 min at 37 degrees C. Data suggest no internalization of bound lactoferrin. The accumulation of radioactivity in supernatants after incubation of T. vaginalis with 125I-labeled lactoferrin and washing in PBS suggested the presence of low affinity sites for this host macromolecule. Scatchard analysis indicated the presence of 90,000 receptors per trichomonad with an apparent Kd of 1.0 microM. Two trichomonad lactoferrin binding proteins were identified by affinity chromatography and immunoprecipitation of receptor-ligand complexes. A 30-fold accumulation of iron was achieved using 59Fe-lactoferrin when compared to the steady state concentration of bound lactoferrin. The activity of pyruvate/ferrodoxin oxidoreductase, an enzyme involved in trichomonal energy metabolism, increased more than sixfold following exposure of the parasites to lactoferrin, demonstrating a biologic response to the receptor-mediated binding of lactoferrin. These data suggest that T. vaginalis possesses specific receptors for biologically relevant host proteins and that these receptors contribute to the metabolic processes of the parasites

  9. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    Directory of Open Access Journals (Sweden)

    Jenkins Jeremy L

    2001-10-01

    Full Text Available Abstract Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm aminopeptidase N (APN and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM, and unusually tight binding to the cadherin-like receptor (2.6 nM, which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research.

  10. Binding specificity of Bacillus thuringiensis Cry1Aa for purified, native Bombyx mori aminopeptidase N and cadherin-like receptors

    Science.gov (United States)

    Jenkins, Jeremy L; Dean, Donald H

    2001-01-01

    Background To better understand the molecular interactions of Bt toxins with non-target insects, we have examined the real-time binding specificity and affinity of Cry1 toxins to native silkworm (Bombyx mori) midgut receptors. Previous studies on B. mori receptors utilized brush border membrane vesicles or purifed receptors in blot-type assays. Results The Bombyx mori (silkworm) aminopeptidase N (APN) and cadherin-like receptors for Bacillus thuringiensis insecticidal Cry1Aa toxin were purified and their real-time binding affinities for Cry toxins were examined by surface plasmon resonance. Cry1Ab and Cry1Ac toxins did not bind to the immobilized native receptors, correlating with their low toxicities. Cry1Aa displayed moderate affinity for B. mori APN (75 nM), and unusually tight binding to the cadherin-like receptor (2.6 nM), which results from slow dissociation rates. The binding of a hybrid toxin (Aa/Aa/Ac) was identical to Cry1Aa. Conclusions These results indicate domain II of Cry1Aa is essential for binding to native B. mori receptors and for toxicity. Moreover, the high-affinity binding of Cry1Aa to native cadherin-like receptor emphasizes the importance of this receptor class for Bt toxin research. PMID:11722800

  11. Signaling-sensitive amino acids surround the allosteric ligand binding site of the thyrotropin receptor.

    Science.gov (United States)

    Kleinau, Gunnar; Haas, Ann-Karin; Neumann, Susanne; Worth, Catherine L; Hoyer, Inna; Furkert, Jens; Rutz, Claudia; Gershengorn, Marvin C; Schülein, Ralf; Krause, Gerd

    2010-07-01

    The thyrotropin receptor [thyroid-stimulating hormone receptor (TSHR)], a G-protein-coupled receptor (GPCR), is endogenously activated by thyrotropin, which binds to the extracellular region of the receptor. We previously identified a low-molecular-weight (LMW) agonist of the TSHR and predicted its allosteric binding pocket within the receptor's transmembrane domain. Because binding of the LMW agonist probably disrupts interactions or leads to formation of new interactions among amino acid residues surrounding the pocket, we tested whether mutation of residues at these positions would lead to constitutive signaling activity. Guided by molecular modeling, we performed site-directed mutagenesis of 24 amino acids in this spatial region, followed by functional characterization of the mutant receptors in terms of expression and signaling, measured as cAMP accumulation. We found that mutations V421I, Y466A, T501A, L587V, M637C, M637W, S641A, Y643F, L645V, and Y667A located in several helices exhibit constitutive activity. Of note is mutation M637W at position 6.48 in transmembrane helix 6, which has a significant effect on the interaction of the receptor with the LMW agonist. In summary, we found that a high proportion of residues in several helices surrounding the allosteric binding site of LMW ligands in the TSHR when mutated lead to constitutively active receptors. Our findings of signaling-sensitive residues in this region of the transmembrane bundle may be of general importance as this domain appears to be evolutionarily retained among GPCRs.

  12. Spectrum of mutations in the renin-angiotensin system genes in autosomal recessive renal tubular dysgenesis

    DEFF Research Database (Denmark)

    Gribouval, Olivier; Morinière, Vincent; Pawtowski, Audrey

    2012-01-01

    , pulmonary hypoplasia, and refractory arterial hypotension. The disease is linked to mutations in the genes encoding several components of the renin-angiotensin system (RAS): AGT (angiotensinogen), REN (renin), ACE (angiotensin-converting enzyme), and AGTR1 (angiotensin II receptor type 1). Here, we review...... the series of 54 distinct mutations identified in 48 unrelated families. Most of them are novel and ACE mutations are the most frequent, observed in two-thirds of families (64.6%). The severity of the clinical course was similar whatever the mutated gene, which underlines the importance of a functional RAS...

  13. Increasing brain angiotensin converting enzyme 2 activity decreases anxiety-like behavior in male mice by activating central Mas receptors.

    Science.gov (United States)

    Wang, Lei; de Kloet, Annette D; Pati, Dipanwita; Hiller, Helmut; Smith, Justin A; Pioquinto, David J; Ludin, Jacob A; Oh, S Paul; Katovich, Michael J; Frazier, Charles J; Raizada, Mohan K; Krause, Eric G

    2016-06-01

    Over-activation of the brain renin-angiotensin system (RAS) has been implicated in the etiology of anxiety disorders. Angiotensin converting enzyme 2 (ACE2) inhibits RAS activity by converting angiotensin-II, the effector peptide of RAS, to angiotensin-(1-7), which activates the Mas receptor (MasR). Whether increasing brain ACE2 activity reduces anxiety by stimulating central MasR is unknown. To test the hypothesis that increasing brain ACE2 activity reduces anxiety-like behavior via central MasR stimulation, we generated male mice overexpressing ACE2 (ACE2 KI mice) and wild type littermate controls (WT). ACE2 KI mice explored the open arms of the elevated plus maze (EPM) significantly more than WT, suggesting increasing ACE2 activity is anxiolytic. Central delivery of diminazene aceturate, an ACE2 activator, to C57BL/6 mice also reduced anxiety-like behavior in the EPM, but centrally administering ACE2 KI mice A-779, a MasR antagonist, abolished their anxiolytic phenotype, suggesting that ACE2 reduces anxiety-like behavior by activating central MasR. To identify the brain circuits mediating these effects, we measured Fos, a marker of neuronal activation, subsequent to EPM exposure and found that ACE2 KI mice had decreased Fos in the bed nucleus of stria terminalis but had increased Fos in the basolateral amygdala (BLA). Within the BLA, we determined that ∼62% of GABAergic neurons contained MasR mRNA and expression of MasR mRNA was upregulated by ACE2 overexpression, suggesting that ACE2 may influence GABA neurotransmission within the BLA via MasR activation. Indeed, ACE2 overexpression was associated with increased frequency of spontaneous inhibitory postsynaptic currents (indicative of presynaptic release of GABA) onto BLA pyramidal neurons and central infusion of A-779 eliminated this effect. Collectively, these results suggest that ACE2 may reduce anxiety-like behavior by activating central MasR that facilitate GABA release onto pyramidal neurons within the

  14. Cholinergic Receptor Binding in Alzheimer Disease and Healthy Aging: Assessment In Vivo with Positron Emission Tomography Imaging.

    Science.gov (United States)

    Sultzer, David L; Melrose, Rebecca J; Riskin-Jones, Hannah; Narvaez, Theresa A; Veliz, Joseph; Ando, Timothy K; Juarez, Kevin O; Harwood, Dylan G; Brody, Arthur L; Mandelkern, Mark A

    2017-04-01

    To compare regional nicotinic cholinergic receptor binding in older adults with Alzheimer disease (AD) and healthy older adults in vivo and to assess relationships between receptor binding and clinical symptoms. Using cross-sectional positron emission tomography (PET) neuroimaging and structured clinical assessment, outpatients with mild to moderate AD (N = 24) and healthy older adults without cognitive complaints (C group; N = 22) were studied. PET imaging of α4β2* nicotinic cholinergic receptor binding using 2-[ 18 F]fluoro-3-(2(S)azetidinylmethoxy)pyridine (2FA) and clinical measures of global cognition, attention/processing speed, verbal memory, visuospatial memory, and neuropsychiatric symptoms were used. 2FA binding was lower in the AD group compared with the C group in the medial thalamus, medial temporal cortex, anterior cingulate, insula/opercula, inferior caudate, and brainstem (p healthy older adults, lower receptor binding may be associated with slower processing speed. Cholinergic receptor binding in vivo may reveal links to other key brain changes associated with aging and AD and may provide a potential molecular treatment target. Published by Elsevier Inc.

  15. Post-stroke angiotensin II type 2 receptor activation provides long-term neuroprotection in aged rats

    DEFF Research Database (Denmark)

    Bennion, Douglas M; Isenberg, Jacob D; Harmel, Allison T

    2017-01-01

    Activation of the angiotensin II type 2 receptor (AT2R) by administration of Compound 21 (C21), a selective AT2R agonist, induces neuroprotection in models of ischemic stroke in young adult animals. The mechanisms of this neuroprotective action are varied, and may include direct and indirect....... These findings demonstrate that the neuroprotection previously characterized only during earlier time points using stroke models in young animals is sustained long-term in aged rats, implying even greater clinical relevance for the study of AT2R agonists for the acute treatment of ischemic stroke in human....... Intraperitoneal injections of C21 (0.03mg/kg) after ischemic stroke induced by transient monofilament middle cerebral artery occlusion resulted in protective effects that were sustained for up to at least 3-weeks post-stroke. These included improved neurological function across multiple assessments...

  16. Characterization of the hormone-binding domain of the chicken c-erbA/thyroid hormone receptor protein

    DEFF Research Database (Denmark)

    Muñoz, A; Zenke, M; Gehring, U

    1988-01-01

    mutations present in the carboxy-terminal half of P75gag-v-erbA co-operate in abolishing hormone binding, and that the ligand-binding domain resides in a position analogous to that of steroid receptors. Furthermore, a point mutation that is located between the putative DNA and ligand-binding domains of P75......To identify and characterize the hormone-binding domain of the thyroid hormone receptor, we analyzed the ligand-binding capacities of proteins representing chimeras between the normal receptor and P75gag-v-erbA, the retrovirus-encoded form deficient in binding ligand. Our results show that several......gag-v-erbA and that renders it biologically inactive fails to affect hormone binding by the c-erbA protein. These results suggest that the mutation changed the ability of P75gag-v-erbA to affect transcription since it also had no effect on DNA binding. Our data also suggest that hormone...

  17. Estrogen Receptor Binding Affinity of Food Contact Material Components Estimated by QSAR.

    Science.gov (United States)

    Sosnovcová, Jitka; Rucki, Marián; Bendová, Hana

    2016-09-01

    The presented work characterized components of food contact materials (FCM) with potential to bind to estrogen receptor (ER) and cause adverse effects in the human organism. The QSAR Toolbox, software application designed to identify and fill toxicological data gaps for chemical hazard assessment, was used. Estrogen receptors are much less of a lock-and-key interaction than highly specific ones. The ER is nonspecific enough to permit binding with a diverse array of chemical structures. There are three primary ER binding subpockets, each with different requirements for hydrogen bonding. More than 900 compounds approved as of FCM components were evaluated for their potential to bind on ER. All evaluated chemicals were subcategorized to five groups with respect to the binding potential to ER: very strong, strong, moderate, weak binder, and no binder to ER. In total 46 compounds were characterized as potential disturbers of estrogen receptor. Among the group of selected chemicals, compounds with high and even very high affinity to the ER binding subpockets were found. These compounds may act as gene activators and cause adverse effects in the organism, particularly during pregnancy and breast-feeding. It should be considered to carry out further in vitro or in vivo tests to confirm their potential to disturb the regulation of physiological processes in humans by abnormal ER signaling and subsequently remove these chemicals from the list of approved food contact materials. Copyright© by the National Institute of Public Health, Prague 2016

  18. Impact of Angiotensin Type 1A Receptors in Principal Cells of the Collecting Duct on Blood Pressure and Hypertension.

    Science.gov (United States)

    Chen, Daian; Stegbauer, Johannes; Sparks, Matthew A; Kohan, Donald; Griffiths, Robert; Herrera, Marcela; Gurley, Susan B; Coffman, Thomas M

    2016-06-01

    The main actions of the renin-angiotensin system to control blood pressure (BP) are mediated by the angiotensin type 1 receptors (AT1Rs). The major murine AT1R isoform, AT1AR, is expressed throughout the nephron, including the collecting duct in both principal and intercalated cells. Principal cells play the major role in sodium and water reabsorption. Although aldosterone is considered to be the dominant regulator of sodium reabsorption by principal cells, recent studies suggest a role for direct actions of AT1R. To specifically examine the contributions of AT1AR in principal cells to BP regulation and the development of hypertension in vivo, we generated inbred 129/SvEv mice with deletion of AT1AR from principal cells (PCKO). At baseline, we found that BPs measured by radiotelemetry were similar between PCKOs and controls. During 1-week of low-salt diet (hypertension, there was a modest but significant attenuation of hypertension in PCKOs (163±6 mm Hg) compared with controls (178±2 mm Hg; Phypertension and epithelial sodium channel activation. © 2016 American Heart Association, Inc.

  19. An Angiotensin II Type 1 Receptor Blocker Prevents Renal Injury via Inhibition of the Notch Pathway in Ins2 Akita Diabetic Mice

    Directory of Open Access Journals (Sweden)

    Masaya Koshizaka

    2012-01-01

    Full Text Available Recently, it has been reported that the Notch pathway is involved in the pathogenesis of diabetic nephropathy. In this study, we investigated the activation of the Notch pathway in Ins2 Akita diabetic mouse (Akita mouse and the effects of telmisartan, an angiotensin II type1 receptor blocker, on the Notch pathway. The intracellular domain of Notch1 (ICN1 is proteolytically cleaved from the cell plasma membrane in the course of Notch activation. The expression of ICN1 and its ligand, Jagged1, were increased in the glomeruli of Akita mice, especially in the podocytes. Administration of telmisartan significantly ameliorated the expression of ICN1 and Jagged1. Telmisartan inhibited the angiotensin II-induced increased expression of transforming growth factor β and vascular endothelial growth factor A which could directly activate the Notch signaling pathway in cultured podocytes. Our results indicate that the telmisartan prevents diabetic nephropathy through the inhibition of the Notch pathway.

  20. Risperidone treatment increases CB1 receptor binding in rat brain

    DEFF Research Database (Denmark)

    Secher, Anna; Husum, Henriette; Holst, Birgitte

    2010-01-01

    , the ghrelin receptor, neuropeptide Y, adiponectin and proopiomelanocortin. We investigated whether the expression of these factors was affected in rats chronically treated with the antipsychotic risperidone. METHODS: Male Sprague-Dawley rats were treated with risperidone (1.0 mg/kg/day) or vehicle (20...... showed that risperidone treatment altered CB(1) receptor binding in the rat brain. Risperidone-induced adiposity and metabolic dysfunction in the clinic may be explained by increased CB(1) receptor density in brain regions involved in appetite and regulation of metabolic function....

  1. β-adrenergic receptor binding characteristics and responsiveness in cultured Wistar-Kyoto rat arterial smooth muscle cells

    International Nuclear Information System (INIS)

    Jazayeri, A.; Meyer, W.J. III

    1988-01-01

    The tone of arterial blood vessels is regulated by the catecholamines through their receptors on arterial smooth muscle cells (ASMC). β- 2 -adrenergic receptors of ASMC mediate vasodilation through agonist mediated c-AMP production. Previous reports have described these receptors on freshly isolated blood vessels. This study demonstrates the presence of β 2 -adrenergic receptors on cultured rat ASMC and that these receptors are functional. β-adrenergic receptor binding was measured using [ 3 H]-dihydroalprenolol (DHA) binding to the membrane of cultured ASMC from normotensive Wistar-Kyoto rats. The ASMC β-adrenergic receptors have a Kd of 0.56 +/- 0.16 nM and a Bmax of 57.2 +/- 21.7 fmol/mg protein. Competition binding studies revealed a much greater affinity of these receptors for epinephrine than norepinephrine, indicating the preponderance of a β 2 -adrenergic receptor subtype. Isoproterenol stimulation of cultured ASMC resulted in a 14 +/- 7 fold increase in intracellular c-AMP content of these cells indicating these receptors are functional. β-adrenergic receptors of cultured ASMC provide an excellent system in which the association between hypertension and observed β-adrenergic receptor differences can be further explored

  2. Binding interactions of convulsant and anticonvulsant gamma-butyrolactones and gamma-thiobutyrolactones with the picrotoxin receptor

    International Nuclear Information System (INIS)

    Holland, K.D.; McKeon, A.C.; Covey, D.F.; Ferrendelli, J.A.

    1990-01-01

    Alkyl-substituted gamma-butyrolactones (GBLs) and gamma-thiobutyrolactones (TBLs) are neuroactive chemicals. beta-Substituted compounds are convulsant, whereas alpha-alkyl substituted GBLs and TBLs are anticonvulsant. The structural similarities between beta-alkyl GBLs and the convulsant picrotoxinin suggested that alkyl substituted GBLs and TBLs act at the picrotoxin receptor. To test this hypothesis we examined the interactions of convulsant and anticonvulsant GBLs and TBLs with the picrotoxin, benzodiazepine and gamma-aminobutyric acid (GABA) binding sites of the GABA receptor complex. All of these convulsants and anticonvulsants studied competitively displaced 35S-t-butylbicyclophosphorothionate (35S-TBPS), a ligand that binds to the picrotoxin receptor. This inhibition of 35S-TBPS binding was not blocked by the GABA antagonist bicuculline methobromide. The convulsant GBLs and TBLs also partially inhibited [3H]muscimol binding to the GABA site and [3H]flunitrazepam binding to the benzodiazepine site, but they did so at concentrations substantially greater than those that inhibited 35S-TBPS binding. The anticonvulsant GBLs and TBLs had no effect on either [3H]muscimol or [3H]flunitrazepam binding. In contrast to the GBLs and TBLs, pentobarbital inhibited TBPS binding in a manner that was blocked by bicuculline methobromide, and it enhanced both [3H]flunitrazepam and [3H]muscimol binding. Both ethosuximide and tetramethylsuccinimide, neuroactive compounds structurally similar to GBLs, competitively displaced 35S-TBPS from the picrotoxin receptor and both compounds were weak inhibitors of [3H] muscimol binding. In addition, ethosuximide also partially diminished [3H]flunitrazepam binding. These data demonstrate that the site of action of alkyl-substituted GBLs and TBLs is different from that of GABA, barbiturates and benzodiazepines

  3. Angiotensin Converting-Enzyme Inhibitors, Angiotensin Receptor Blockers, and Calcium Channel Blockers Are Associated with Prolonged Vascular Access Patency in Uremic Patients Undergoing Hemodialysis.

    Directory of Open Access Journals (Sweden)

    Fu-An Chen

    Full Text Available Vascular access failure is a huge burden for patients undergoing hemodialysis. Many efforts have been made to maintain vascular access patency, including pharmacotherapy. Angiotensin converting enzyme inhibitor (ACE-I, angiotensin receptor blocker (ARB, and calcium channel blocker (CCB are known for their antihypertensive and cardio-protective effects, however, their effects on long-term vascular access patency are still inconclusive.We retrospectively enrolled patients commencing maintenance hemodialysis between January 1, 2000, and December 31, 2006 by using National Health Insurance Research Database in Taiwan. Primary patency was defined as the date of first arteriovenous fistula (AVF or arteriovenous graft (AVG creation to the time of access thrombosis or any intervention aimed to maintain or re-establish vascular access patency. Cox proportional hazards models were used to adjust the influences of patient characteristics, co-morbidities and medications.Total 42244 patients were enrolled in this study, 37771 (89.4% used AVF, 4473 (10.6% used AVG as their first long term dialysis access. ACE-I, ARB, and CCB use were all associated with prolonged primary patency of AVF [hazard ratio (HR 0.586, 95% confidence interval (CI 0.557-0.616 for ACE-I use; HR 0.532, CI 0.508-0.556 for ARB use; HR 0.485, CI 0.470-0.501 for CCB use] and AVG (HR 0.557, CI 0.482-0.643 for ACE-I use, HR 0.536, CI 0.467-0.614 for ARB use, HR 0.482, CI 0.442-0.526 for CCB use.In our analysis, ACE-I, ARB, and CCB were strongly associated with prolonged primary patency of both AVF and AVG. Further prospective randomized studies are still warranted to prove the causality.

  4. Dietary Sodium Suppresses Digestive Efficiency via the Renin-Angiotensin System.

    Science.gov (United States)

    Weidemann, Benjamin J; Voong, Susan; Morales-Santiago, Fabiola I; Kahn, Michael Z; Ni, Jonathan; Littlejohn, Nicole K; Claflin, Kristin E; Burnett, Colin M L; Pearson, Nicole A; Lutter, Michael L; Grobe, Justin L

    2015-06-11

    Dietary fats and sodium are both palatable and are hypothesized to synergistically contribute to ingestive behavior and thereby obesity. Contrary to this hypothesis, C57BL/6J mice fed a 45% high fat diet exhibited weight gain that was inhibited by increased dietary sodium content. This suppressive effect of dietary sodium upon weight gain was mediated specifically through a reduction in digestive efficiency, with no effects on food intake behavior, physical activity, or resting metabolism. Replacement of circulating angiotensin II levels reversed the effects of high dietary sodium to suppress digestive efficiency. While the AT1 receptor antagonist losartan had no effect in mice fed low sodium, the AT2 receptor antagonist PD-123,319 suppressed digestive efficiency. Correspondingly, genetic deletion of the AT2 receptor in FVB/NCrl mice resulted in suppressed digestive efficiency even on a standard chow diet. Together these data underscore the importance of digestive efficiency in the pathogenesis of obesity, and implicate dietary sodium, the renin-angiotensin system, and the AT2 receptor in the control of digestive efficiency regardless of mouse strain or macronutrient composition of the diet. These findings highlight the need for greater understanding of nutrient absorption control physiology, and prompt more uniform assessment of digestive efficiency in animal studies of energy balance.

  5. An automated system for the analysis of G protein-coupled receptor transmembrane binding pockets: alignment, receptor-based pharmacophores, and their application.

    Science.gov (United States)

    Kratochwil, Nicole A; Malherbe, Pari; Lindemann, Lothar; Ebeling, Martin; Hoener, Marius C; Mühlemann, Andreas; Porter, Richard H P; Stahl, Martin; Gerber, Paul R

    2005-01-01

    G protein-coupled receptors (GPCRs) share a common architecture consisting of seven transmembrane (TM) domains. Various lines of evidence suggest that this fold provides a generic binding pocket within the TM region for hosting agonists, antagonists, and allosteric modulators. Here, a comprehensive and automated method allowing fast analysis and comparison of these putative binding pockets across the entire GPCR family is presented. The method relies on a robust alignment algorithm based on conservation indices, focusing on pharmacophore-like relationships between amino acids. Analysis of conservation patterns across the GPCR family and alignment to the rhodopsin X-ray structure allows the extraction of the amino acids lining the TM binding pocket in a so-called ligand binding pocket vector (LPV). In a second step, LPVs are translated to simple 3D receptor pharmacophore models, where each amino acid is represented by a single spherical pharmacophore feature and all atomic detail is omitted. Applications of the method include the assessment of selectivity issues, support of mutagenesis studies, and the derivation of rules for focused screening to identify chemical starting points in early drug discovery projects. Because of the coarseness of this 3D receptor pharmacophore model, however, meaningful scoring and ranking procedures of large sets of molecules are not justified. The LPV analysis of the trace amine-associated receptor family and its experimental validation is discussed as an example. The value of the 3D receptor model is demonstrated for a class C GPCR family, the metabotropic glutamate receptors.

  6. Biological activity of cloned mammary tumor virus DNA fragments that bind purified glucocorticoid receptor protein in vitro

    International Nuclear Information System (INIS)

    Yamamoto, K.R.; Payvar, F.; Firestone, G.L.; Maler, B.A.; Wrange, O.; Carlstedt-Duke, J.; Gustafsson, J.A.; Chandler, V.L.; Karolinska Institutet, Stockholm, Sweden)

    1983-01-01

    To test whether high-affinity receptor:DNA interactions can be correlated with receptor effects on promoter function in vivo, we have mapped in greater detail the receptor-binding regions on murine mammary tumor virus DNA, using both nitrocellulose-filter binding and electron microscopy. Recombinant plasmids bearing these receptor-binding domains have been transfected into cultured cells, and the expression of the plasmid sequences has been monitored for hormonal regulation. The results are considered in terms of a speculative proposal that the glucocorticoid receptor may effect changes in promoter activity via specific alteration of chromatin and/or DNA structure. 37 references, 6 figures, 2 tables

  7. Serotoninergic receptors in brain tissue: properties and identification of various 3H-ligand binding sites in vitro

    International Nuclear Information System (INIS)

    Leysen, J.E.

    1981-01-01

    In vitro binding studies to serotoninergic receptors were performed using 3 H-LSD, 3 H-5-HT and 3 H-spiperone. An overwiew is given on findings using these three ligands with respect to the following: localization of specific binding sites, in various animal species, the regional distribution in the brain and periphery, the subcellular and cellular distribution. Properties of the binding sites, influence of the composition of the assay medium, binding kinetic properties, receptor regulation in vivo. Identity of the binding sites, differences between site for various 3 H-ligands, pharmacological specificity of the membranous binding sites, chemical composition of the macromolecular complex constituting the binding site. Function of the receptor. Binding affinities of 44 compounds were measured in binding assays using 3 H-spiperone and 3 H-LSD with rat frontal cortex membrane preparations and using 3 H-5-HT and 3 H-LSD with rat hippocampal membrane preparations

  8. Association of dopamine D(3) receptors with actin-binding protein 280 (ABP-280).

    Science.gov (United States)

    Li, Ming; Li, Chuanyu; Weingarten, Paul; Bunzow, James R; Grandy, David K; Zhou, Qun Yong

    2002-03-01

    Proteins that bind to G protein-coupled receptors have been identified as regulators of receptor localization and signaling. In our previous studies, a cytoskeletal protein, actin-binding protein 280 (ABP-280), was found to associate with the third cytoplasmic loop of dopamine D(2) receptors. In this study, we demonstrate that ABP-280 also interacts with dopamine D(3) receptors, but not with D(4) receptors. Similar to the dopamine D(2) receptor, the D(3)/ABP-280 association is of signaling importance. In human melanoma M2 cells lacking ABP-280, D(3) receptors were unable to inhibit forskolin-stimulated cyclic AMP (cAMP) production significantly. D(4) receptors, however, exhibited a similar degree of inhibition of forskolin-stimulated cAMP production in ABP-280-deficient M2 cells and ABP-280-replent M2 subclones (A7 cells). Further experiments revealed that the D(3)/ABP-280 interaction was critically dependent upon a 36 amino acid carboxyl domain of the D(3) receptor third loop, which is conserved in the D(2) receptor but not in the D(4) receptor. Our results demonstrate a subtype-specific regulation of dopamine D(2)-family receptor signaling by the cytoskeletal protein ABP-280.

  9. Human fetal malformations associated with the use of an angiotensin II receptor antagonist: Case Report

    Directory of Open Access Journals (Sweden)

    Henri Augusto Korkes

    2014-09-01

    Full Text Available Introduction: The potential risks related to drug exposure during pregnancy represent a vast chapter in modern obstetrics and data regarding the safety of antihypertensive drugs during pregnancy are relatively scarce. Case report: A 37-year-old patient discovered her fifth pregnancy at our hospital after 26 weeks and 4 days of gestation. She reported a history of hypertension and was currently being treated with Losartan. Hospitalization was recommended for the patient and further evaluation of fetal vitality was performed. On the fourth day an ultrasound was performed, resulting in a severe oligohydramnios, fetal centralization and abnormal ductus venosus. After 36 hours, the newborn died. Pathologic evaluation: At autopsy, the skullcap had large fontanels and deficient ossification. The kidneys were slightly enlarged. A microscopic examination detected underdevelopment of the tubules and the presence of some dilated lumens. Immunohistochemical detection of epithelial membrane antigen was positive. Immunoreactivity of CD 15 was also assayed to characterize the proximal tubules, and lumen collapse was observed in some regions. Discussion: Angiotensin-converting enzyme inhibitors (ACEIs and angiotensin receptor antagonists (ARAs are among the most widely prescribed drugs for hypertension. They are often used by hypertensive women who are considering become pregnant. While their fetal toxicity in the second or third trimesters has been documented, their teratogenic effect during the first trimester has only recently been demonstrated. Conclusion: Constant awareness by physicians and patients should be encouraged, particularly in regard to the prescription of antihypertensive drugs in women of childbearing age who are or intend to become pregnant.

  10. Role of Mas receptor in renal blood flow response to angiotensin-(1-7) in ovariectomized estradiol treated rats.

    Science.gov (United States)

    Saberi, Shadan; Dehghani, Aghdas; Nematbakhsh, Mehdi

    2016-01-01

    The angiotensin 1-7 (Ang 1-7), is abundantly produced in kidneys and antagonizes the function of angiotensin II through Mas receptor (MasR) or other unknown mechanisms. In the current study, the role of MasR and steroid hormone estrogen on renal blood flow response to Ang 1-7 administration was investigated in ovariectomized (OV) female rats. OV female Wistar-rats received estradiol (500 μg/kg/week) or vehicle for two weeks. In the day of the experiment, the animals were anesthetized, cannulated, and the responses including mean arterial pressure, renal blood flow (RBF), and renal vascular resistance at the constant level of renal perfusion pressure to graded infusion of Ang 1-7 at 0, 100 and 300 ng/kg/min were determined in OV and OV estradiol-treated (OVE) rats, treated with vehicle or MasR antagonist; A779. RBF response to Ang 1-7 infusion increased dose-dependently in vehicle (Pdose <0.001) and A779-treated (Pdose <0.01) animals. However, when MasR was blocked, the RBF response to Ang 1-7 significantly increased in OV animals compared with OVE rats (P<0.05). When estradiol was limited by ovariectomy, A779 increased RBF response to Ang 1-7 administration, while this response was attenuated in OVE animals.

  11. Regulation of formyl peptide receptor binding to rabbit neutrophil plasma membranes. Use of monovalent cations, guanine nucleotides, and bacterial toxins to discriminate among different states of the receptor

    International Nuclear Information System (INIS)

    Feltner, D.E.; Marasco, W.A.

    1989-01-01

    The regulation by monovalent cations, guanine nucleotides, and bacterial toxins of [3H]FMLP binding to rabbit neutrophil plasma membranes was studied by using dissociation techniques to identify regulatory effects on separate receptor states. Under conditions of low receptor occupancy (1 nM [3H]FMLP) and in both Na+ and K+ buffers, dissociation is heterogenous, displaying two distinct, statistically significant off rates. [3H]FMLP binding was enhanced by substituting other monovalent cations for Na+. In particular, enhanced binding in the presence of K+ relative to Na+ was caused by additional binding to both rapidly and slowly dissociating receptors. Three receptor dissociation rates, two of which appear to correspond to the two affinity states detected in equilibrium binding studies, were defined by specific GTP and pertussis toxin (PT) treatments. Neither GTP, nor PT or cholera toxins (CT) had an effect on the rate of dissociation of [3H]FMLP from the rapidly dissociating form of the receptor. Both 100 microM GTP and PT treatments increased the percentage of rapidly dissociating receptors, correspondingly decreasing the percentage of slowly dissociating receptors. The observed changes in the rapidly and slowly dissociating receptors after GTP, PT, and CT treatments were caused by an absolute decrease in the amount of binding to the slowly dissociating receptors. However, complete inhibition of slowly dissociating receptor binding by GTP, PT, or both was never observed. Both GTP and PT treatments, but not CT treatment, increased by two-fold the rate of dissociation of 1 nM [3H]FMLP from the slowly dissociating form of the receptor, resulting in a third dissociation rate. Thus, slowly dissociating receptors comprise two different receptor states, a G protein-associated guanine nucleotide and PT-sensitive state and a guanine nucleotide-insensitive state

  12. Homology-modeled ligand-binding domains of medaka estrogen receptors and androgen receptors: A model system for the study of reproduction

    International Nuclear Information System (INIS)

    Cui Jianzhou; Shen Xueyan; Yan Zuowei; Zhao Haobin; Nagahama, Yoshitaka

    2009-01-01

    Estrogen and androgen and their receptors play critical roles in physiological processes such as sexual differentiation and development. Using the available structural models for the human estrogen receptors alpha and beta and androgen receptor as templates, we designed in silico agonist and antagonist models of medaka estrogen receptor (meER) alpha, beta-1, and beta-2, and androgen receptor (meAR) alpha and beta. Using these models, we studied (1) the structural relationship between the ligand-binding domains (LBDs) of ERs and ARs of human and medaka, and (2) whether medaka ER and AR can be potential models for studying the ligand-binding activities of various agonists and antagonists of these receptors by docking analysis. A high level of conservation was observed between the sequences of the ligand-binding domains of meERα and huERα, meERβ1 and huERβ, meERβ2, and huERβ with 62.8%, 66.4%, and 65.1% identity, respectively. The sequence conservation between meARα and huAR, meARβ, and huAR was found with 70.1% and 61.0% of identity, respectively. Thirty-three selected endocrine disrupting chemicals (EDCs), including both agonists and antagonists, were docked into the LBD of ER and AR, and the corresponding docking score for medaka models and human templates were calculated. In order to confirm the conservation of the overall geometry and the binding pocket, the backbone root mean square deviation (RMSD) for Cα atoms was derived from the structure superposition of all 10 medaka homology models to the six human templates. Our results suggested conformational conservation between the ERs and ARs of medaka and human, Thus, medaka could be highly useful as a model system for studies involving estrogen and androgen interaction with their receptors.

  13. Kinetics of leptin binding to the Q223R leptin receptor.

    Directory of Open Access Journals (Sweden)

    Hans Verkerke

    Full Text Available Studies in human populations and mouse models of disease have linked the common leptin receptor Q223R mutation to obesity, multiple forms of cancer, adverse drug reactions, and susceptibility to enteric and respiratory infections. Contradictory results cast doubt on the phenotypic consequences of this variant. We set out to determine whether the Q223R substitution affects leptin binding kinetics using surface plasmon resonance (SPR, a technique that allows sensitive real-time monitoring of protein-protein interactions. We measured the binding and dissociation rate constants for leptin to the extracellular domain of WT and Q223R murine leptin receptors expressed as Fc-fusion proteins and found that the mutant receptor does not significantly differ in kinetics of leptin binding from the WT leptin receptor. (WT: ka 1.76×106±0.193×106 M-1 s-1, kd 1.21×10-4±0.707×10-4 s-1, KD 6.47×10-11±3.30×10-11 M; Q223R: ka 1.75×106±0.0245×106 M-1 s-1, kd 1.47×10-4±0.0505×10-4 s-1, KD 8.43×10-11±0.407×10-11 M. Our results support earlier findings that differences in affinity and kinetics of leptin binding are unlikely to explain mechanistically the phenotypes that have been linked to this common genetic variant. Future studies will seek to elucidate the mechanism by which this mutation influences susceptibility to metabolic, infectious, and malignant pathologies.

  14. A urokinase receptor-associated protein with specific collagen binding properties

    DEFF Research Database (Denmark)

    Behrendt, N; Jensen, O N; Engelholm, L H

    2000-01-01

    membrane-bound lectin with hitherto unknown function. The human cDNA was cloned and sequenced. The protein, designated uPARAP, is a member of the macrophage mannose receptor protein family and contains a putative collagen-binding (fibronectin type II) domain in addition to 8 C-type carbohydrate recognition...... domains. It proved capable of binding strongly to a single type of collagen, collagen V. This collagen binding reaction at the exact site of plasminogen activation on the cell may lead to adhesive functions as well as a contribution to cellular degradation of collagen matrices....

  15. The absence of protective effect of candesartan and angiotensin IV in the moderate brain injury in rats

    International Nuclear Information System (INIS)

    Nasser, M.; Botelle, L.; Javellaud, J.; Oudart, N.; Achard, J-M

    2012-01-01

    Background: angiotensin receptor blockers (ARB) are protective in various models of experimental ischemic stroke. This protective effect is mediated by the stimulation of non-AT1 receptors by angiotensin II and angiotensin IV. Since traumatic brain injury shares with ischemic cerebral injury several common mechanisms, we examined if a pretreatment with the ARB candesartan, or a post-treatment with angiotensin IV are also protective in a rat model of blunt traumatic brain injury (TBI). Methods :adults Sprague Dawley rats were treated for five days with candesartan (0.5 mg/kg/day) or saline by gavage prior to the induction of diffuse moderate TBI using the impact-acceleration model. Two others groups of rats were treated by a daily intraperitoneal injection of angiotensin IV (1.5 mg/kg/day) or saline for five days following TBI. Overall neurological insult were assessed daily by measuring the neurological score. Sensitive deficits (scotch test) and sensorimotor deficits (beam-walking test) were evaluated daily from day 1 to 7 and at day 15; cognitive impairment (object recognition test) was evaluated at day 15. Results : TBI induced significant sensitive and sensorimotor deficits that were maximal at day 1 and spontaneously improved with time. At day 15, traumatised animals had a marked alteration of the working memory. Neither treatment with candesartan, angiotensin IV or with erythropoietin decreased the severity of the initial sensorimotor deficits, nor accelerate the recovery rate. Candesartan, angiotensin IV had likewise no protective effect on the cognitive deficit evaluated to day 15. Conclusion: pretreatment with candesartan and post-treatment with angiotensin IV are both ineffective to protect against sensorimotor and c ognitive impairment in a rat model of impact-acceleration TBI. (author)

  16. Structural analysis of binding functionality of folic acid-PEG dendrimers against folate receptor.

    Science.gov (United States)

    Sampogna-Mireles, Diana; Araya-Durán, Ingrid D; Márquez-Miranda, Valeria; Valencia-Gallegos, Jesús A; González-Nilo, Fernando D

    2017-03-01

    Dendrimers functionalized with folic acid (FA) are drug delivery systems that can selectively target cancer cells with folate receptors (FR-α) overexpression. Incorporation of polyethylene glycol (PEG) can enhance dendrimers solubility and pharmacokinetics, but ligand-receptor binding must not be affected. In this work we characterized, at atomic level, the binding functionality of conventional site-specific dendrimers conjugated with FA with PEG 750 or PEG 3350 as a linker. After Molecular Dynamics simulation, we observed that both PEG's did not interfere over ligand-receptor binding functionality. Although binding kinetics could be notably affected, the folate fragment from both dendrimers remained exposed to the solvent before approaching selectively to FR-α. PEG 3350 provided better solubility and protection from enzymatic degradation to the dendrimer than PEG 750. Also, FA-PEG3350 dendrimer showed a slightly better interaction with FR-α than FA-PEG750 dendrimer. Therefore, theoretical evidence supports that both dendrimers are suitable as drug delivery systems for cancer therapies. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. Is the isolated ligand binding domain a good model of the domain in the native receptor?

    Science.gov (United States)

    Deming, Dustin; Cheng, Qing; Jayaraman, Vasanthi

    2003-05-16

    Numerous studies have used the atomic level structure of the isolated ligand binding domain of the glutamate receptor to elucidate the agonist-induced activation and desensitization processes in this group of proteins. However, no study has demonstrated the structural equivalence of the isolated ligand binding fragments and the protein in the native receptor. In this report, using visible absorption spectroscopy we show that the electronic environment of the antagonist 6-cyano-7-nitro-2,3-dihydroxyquinoxaline is identical for the isolated protein and the native glutamate receptors expressed in cells. Our results hence establish that the local structure of the ligand binding site is the same in the two proteins and validate the detailed structure-function relationships that have been developed based on a comparison of the structure of the isolated ligand binding domain and electrophysiological consequences in the native receptor.

  18. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Science.gov (United States)

    Hardison, D Ransom; Holland, William C; McCall, Jennifer R; Bourdelais, Andrea J; Baden, Daniel G; Darius, H Taiana; Chinain, Mireille; Tester, Patricia A; Shea, Damian; Quintana, Harold A Flores; Morris, James A; Litaker, R Wayne

    2016-01-01

    Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs). One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R)). However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R) in certain labs. A fluorescence based receptor binding assay (RBA(F)) was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2) for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C) with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1). Fish (N = 61) of six different species were screened using the RBA(F). Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a) correlated well (R2 = 0.71) with those of the RBA(F), given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F) affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F), which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F) advantages include the long-term (> 5 years) stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R). The RBA(F) is cost-effective, allows high sample

  19. Fluorescent Receptor Binding Assay for Detecting Ciguatoxins in Fish.

    Directory of Open Access Journals (Sweden)

    D Ransom Hardison

    Full Text Available Ciguatera fish poisoning is an illness suffered by > 50,000 people yearly after consumption of fish containing ciguatoxins (CTXs. One of the current methodologies to detect ciguatoxins in fish is a radiolabeled receptor binding assay (RBA(R. However, the license requirements and regulations pertaining to radioisotope utilization can limit the applicability of the RBA(R in certain labs. A fluorescence based receptor binding assay (RBA(F was developed to provide an alternative method of screening fish samples for CTXs in facilities not certified to use radioisotopes. The new assay is based on competition binding between CTXs and fluorescently labeled brevetoxin-2 (BODIPY®-PbTx-2 for voltage-gated sodium channel receptors at site 5 instead of a radiolabeled brevetoxin. Responses were linear in fish tissues spiked from 0.1 to 1.0 ppb with Pacific ciguatoxin-3C (P-CTX-3C with a detection limit of 0.075 ppb. Carribean ciguatoxins were confirmed in Caribbean fish by LC-MS/MS analysis of the regional biomarker (C-CTX-1. Fish (N = 61 of six different species were screened using the RBA(F. Results for corresponding samples analyzed using the neuroblastoma cell-based assay (CBA-N2a correlated well (R2 = 0.71 with those of the RBA(F, given the low levels of CTX present in positive fish. Data analyses also showed the resulting toxicity levels of P-CTX-3C equivalents determined by CBA-N2a were consistently lower than the RBA(F affinities expressed as % binding equivalents, indicating that a given amount of toxin bound to the site 5 receptors translates into corresponding lower cytotoxicity. Consequently, the RBA(F, which takes approximately two hours to perform, provides a generous estimate relative to the widely used CBA-N2a which requires 2.5 days to complete. Other RBA(F advantages include the long-term (> 5 years stability of the BODIPY®-PbTx-2 and having similar results as the commonly used RBA(R. The RBA(F is cost-effective, allows high sample

  20. Evaluation of the In Vivo and Ex Vivo Binding of Novel BC1 Cannabinoid Receptor Radiotracers

    Energy Technology Data Exchange (ETDEWEB)

    Miller, A.; Gatley, J.; Gifford, A.

    2002-01-01

    The primary active ingredient of marijuana, 9-tetrahydrocannabinol, exerts its psychoactive effects by binding to cannabinoid CB1 receptors. These receptors are found throughout the brain with high concentrations in the hippocampus and cerebellum. The current study was conducted to evaluate the binding of a newly developed putative cannabinoid antagonist, AM630, and a classical cannabinoid 8-tetrahydrocannabinol as potential PET and/or SPECT imaging agents for brain CB1 receptors. For both of these ligands in vivo and ex vivo studies in mice were conducted. AM630 showed good overall brain uptake (as measure by %IA/g) and a moderately rapid clearance from the brain with a half-clearance time of approximately 30 minutes. However, AM630 did not show selective binding to CB1 cannabinoid receptors. Ex vivo autoradiography supported the lack of selective binding seen in the in vivo study. Similar to AM630, 8-tetrahydrocanibol also failed to show selective binding to CB1 receptor rich brain areas. The 8-tetrahydrocanibol showed moderate overall brain uptake and relatively slow brain clearance as compared to AM630. Further studies were done with AM2233, a cannabinoid ligand with a similar structure as AM630. These studies were done to develop an ex vivo binding assay to quantify the displacement of [131I]AM2233 binding by other ligands in Swiss-Webster and CB1 receptor knockout mice. By developing this assay we hoped to determine the identity of an unknown binding site for AM2233 present in the hippocampus of CB1 knockout mice. Using an approach based on incubation of brain slices prepared from mice given intravenous [131I]AM2233 in either the presence or absence of AM2233 (unlabelled) it was possible to demonstrate a significant AM2233-displacable binding in the Swiss-Webster mice. Future studies will determine if this assay is appropriate for identifying the unknown binding site for AM2233 in the CB1 knockout mice.

  1. Ligand binding to G protein-coupled receptors in tethered cell membranes

    DEFF Research Database (Denmark)

    Martinez, Karen L.; Meyer, Bruno H.; Hovius, Ruud

    2003-01-01

    for the surface immobilization of membrane proteins was developed using the prototypic seven transmembrane neurokinin-1 receptor. The receptor was expressed as a biotinylated protein in mammalian cells. Membranes from cell homogenates were selectively immobilized on glass surfaces covered with streptavidin. TIRF...... measurements showed that a fluorescent agonist binds to the receptor on the sensor surface with similar affinity as to the receptor in live cells. This approach offers the possibility to investigate minute amounts of membrane protein in an active form and in its native environment without purification....

  2. Overexpression of ß-Arrestin1 in the Rostral Ventrolateral Medulla Downregulates Angiotensin Receptor and Lowers Blood Pressure in Hypertension.

    Science.gov (United States)

    Sun, Jia-Cen; Liu, Bing; Zhang, Ru-Wen; Jiao, Pei-Lei; Tan, Xing; Wang, Yang-Kai; Wang, Wei-Zhong

    2018-01-01

    Background: Hypertension is characterized by sympathetic overactivity, which is associated with an enhancement in angiotensin receptor type I (AT1R) in the rostral ventrolateral medulla (RVLM). β-arrestin1, a canonical scaffold protein, has been suggested to show a negative effect on G protein-coupled receptors via its internalization and desensitization and/or the biased signaling pathway. The major objectives of the present study were to observe the effect of β-arrestin1 overexpression in the RVLM on cardiovascular regulation in spontaneously hypertensive rats (SHR), and further determine the effect of β-arrestin1 on AT1R expression in the RVLM. Methods: The animal model of β-arrestin1 overexpression was induced by bilateral injection of adeno-associated virus containing Arrb1 gene (AAV-Arrb1) into the RVLM of WKY and SHR. Results: β-arrestin1 was expressed on the pre-sympathetic neurons in the RVLM, and its expression in the RVLM was significantly ( P Overexpression of β-arrestin1 in SHR significantly decreased baseline levels of blood pressure and renal sympathetic nerve activity, and attenuated cardiovascular effects induced by RVLM injection of angiotensin II (100 pmol). Furthermore, β-arrestin1 overexpression in the RVLM significantly reduced the expression of AT1R by 65% and NF-κB p65 phosphorylation by 66% in SHR. It was confirmed that β-arrestin1 overexpression in the RVLM led to an enhancement of interaction between β-arrestin1 and IκB-α. Conclusion: Overexpression of β-arrestin1 in the RVLM reduces BP and sympathetic outflow in hypertension, which may be associated with NFκB-mediated AT1R downregulation.

  3. Severe hypoglycaemia in type 1 diabetes: impact of the renin-angiotensin system and other risk factors

    DEFF Research Database (Denmark)

    Pedersen-Bjergaard, Ulrik

    2009-01-01

    this thesis was conducted to assess the significance of severe hypoglycaemia as a clinical problem in the type 1 diabetic population, to evaluate the impact of known risk factors on occurrence of severe hypoglycaemia, and to identify new markers that could contribute to improved prediction of, and inspire...... targets and thereby open for prevention of severe hypoglycaemia. Furthermore, subjects with elevated renin-angiotensin system activity and a high rate of severe hypoglycaemia might benefit from pharmacological blockade of the renin-angiotensin system by ACE inhibitors or angiotensin II receptor blockers...

  4. The importance of short-term off-target effects in estimating the long-term renal and cardiovascular protection of angiotensin receptor blockers

    DEFF Research Database (Denmark)

    Smink, P A; Miao, Y; Eijkemans, M J C

    2014-01-01

    Angiotensin receptor blockers (ARBs) have multiple effects that may contribute to their efficacy on renal/cardiovascular outcomes. We developed and validated a risk score that incorporated short-term changes in multiple risk markers to predict the ARB effect on renal/cardiovascular outcomes.......98), in addition to being markedly more accurate than predicted RRRs based on changes in single markers. The score was validated in an independent ARB trial. Predictions of long-term renal/cardiovascular ARB effects are more accurate when considering short-term changes in multiple risk markers, challenging the use...

  5. In vivo [3H]spiperone binding: evidence for accumulation in corpus striatum by agonist-mediated receptor internalization

    International Nuclear Information System (INIS)

    Chugani, D.C.; Ackermann, R.F.; Phelps, M.E.

    1988-01-01

    The processes of receptor internalization and recycling have been well-documented for receptors for hormones, growth factors, lysosomal enzymes, and cellular substrates. Evidence also exists that these processes also occur for beta-adrenergic, muscarinic cholinergic, and delta-opiate receptors in frog erythrocytes or cultured nervous tissue. In this study, evidence is presented that agonist-mediated receptor internalization and recycling occurs at the dopamine receptor in rat corpus striatum. First, the in vivo binding of the dopamine antagonist [3H]spiperone was increased by both electrical stimulation and pharmacologically induced increases of dopamine release. Conversely, depletion of dopamine with reserpine decreased in vivo [3H]spiperone binding, but the same reserpine treatment did not alter its in vitro binding. Second, the rate of dissociation of [3H]spiperone from microsomal membranes prepared from rat striatum following in vivo binding was fivefold slower than its dissociation following in vitro equilibrium binding. Mild detergent treatment, employed to disrupt endocytic vesicle membranes, increased the rate of dissociation of in vivo bound [3H]spiperone from microsomal membranes to values not significantly different from its in vitro bound dissociation rate. Third, treatment of rats with chloroquine, a drug that prevents receptor recycling but not internalization, prior to [3H]spiperone injection resulted in a selective increase of in vivo [3H]spiperone binding in the light microsome membranes. The existence of mechanisms that rapidly alter the number of neurotransmitter receptors at synapses provides dynamic regulation of receptors in response to varied acute stimulation states

  6. Identification of Fc Gamma Receptor Glycoforms That Produce Differential Binding Kinetics for Rituximab.

    Science.gov (United States)

    Hayes, Jerrard M; Frostell, Asa; Karlsson, Robert; Müller, Steffen; Martín, Silvia Míllan; Pauers, Martin; Reuss, Franziska; Cosgrave, Eoin F; Anneren, Cecilia; Davey, Gavin P; Rudd, Pauline M

    2017-10-01

    Fc gamma receptors (FcγR) bind the Fc region of antibodies and therefore play a prominent role in antibody-dependent cell-based immune responses such as ADCC, CDC and ADCP. The immune effector cell activity is directly linked to a productive molecular engagement of FcγRs where both the protein and glycan moiety of antibody and receptor can affect the interaction and in the present study we focus on the role of the FcγR glycans in this interaction. We provide a complete description of the glycan composition of Chinese hamster ovary (CHO) expressed human Fcγ receptors RI (CD64), RIIa Arg131/His131 (CD32a), RIIb (CD32b) and RIIIa Phe158/Val158 (CD16a) and analyze the role of the glycans in the binding mechanism with IgG. The interactions of the monoclonal antibody rituximab with each FcγR were characterized and we discuss the CHO-FcγRIIIa Phe158/Val158 and CHO-FcγRI interactions and compare them to the equivalent interactions with human (HEK293) and murine (NS0) produced receptors. Our results reveal clear differences in the binding profiles of rituximab, which we attribute in each case to the differences in host cell-dependent FcγR glycosylation. The glycan profiles of CHO expressed FcγRI and FcγRIIIa Phe158/Val158 were compared with the glycan profiles of the receptors expressed in NS0 and HEK293 cells and we show that the glycan type and abundance differs significantly between the receptors and that these glycan differences lead to the observed differences in the respective FcγR binding patterns with rituximab. Oligomannose structures are prevalent on FcγRI from each source and likely contribute to the high affinity rituximab interaction through a stabilization effect. On FcγRI and FcγRIIIa large and sialylated glycans have a negative impact on rituximab binding, likely through destabilization of the interaction. In conclusion, the data show that the IgG1-FcγR binding kinetics differ depending on the glycosylation of the FcγR and further support a

  7. Reduced 5-HT2A receptor binding in patients with mild cognitive impairment

    DEFF Research Database (Denmark)

    Hasselbalch, S G; Madsen, K; Svarer, C

    2008-01-01

    cerebral 5-HT(2A) receptor binding in patients with mild cognitive impairment (MCI) and related 5-HT(2A) receptor binding to clinical symptoms. Sixteen patients with MCI of the amnestic type (mean age 73, mean MMSE 26.1) and 17 age and sex matched control subjects were studied with MRI and [(18)F......Previous studies of patients with Alzheimer's disease (AD) have described reduced brain serotonin 2A (5-HT(2A)) receptor density. It is unclear whether this abnormality sets in early in the course of the disease and whether it is related to early cognitive and neuropsychiatric symptoms. We assessed...

  8. Structural and mutational analyses of the receptor binding domain of botulinum D/C mosaic neurotoxin: Insight into the ganglioside binding mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Nuemket, Nipawan [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Yoshikazu [Creative Research Institution ' Sousei,' Hokkaido University, Sapporo 001-0021 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tsukamoto, Kentaro; Tsuji, Takao [Department of Microbiology, Fujita Health University School of Medicine, Toyoake, Aichi 470-1192 (Japan); Nakamura, Keiji; Kozaki, Shunji [Department of Veterinary Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, Osaka 598-8531 (Japan); Yao, Min [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan); Tanaka, Isao, E-mail: tanaka@castor.sci.hokudai.ac.jp [Graduate School of Life Sciences, Hokkaido University, Sapporo 060-0810 (Japan); Faculty of Advanced Life Science, Hokkaido University, Sapporo 060-0810 (Japan)

    2011-07-29

    Highlights: {yields} We determined the crystal structure of the receptor binding domain of BoNT in complex with 3'-sialyllactose. {yields} An electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. {yields} Alanine site-directed mutagenesis showed that GBS and GBL are important for ganglioside binding. {yields} A cell binding mechanism, which involves cooperative contribution of two sites, was proposed. -- Abstract: Clostridium botulinum type D strain OFD05, which produces the D/C mosaic neurotoxin, was isolated from cattle killed by the recent botulism outbreak in Japan. The D/C mosaic neurotoxin is the most toxic of the botulinum neurotoxins (BoNT) characterized to date. Here, we determined the crystal structure of the receptor binding domain of BoNT from strain OFD05 in complex with 3'-sialyllactose at a resolution of 3.0 A. In the structure, an electron density derived from the 3'-sialyllactose was confirmed at the cleft in the C-terminal subdomain. Alanine site-directed mutagenesis showed the significant contribution of the residues surrounding the cleft to ganglioside recognition. In addition, a loop adjoining the cleft also plays an important role in ganglioside recognition. In contrast, little effect was observed when the residues located around the surface previously identified as the protein receptor binding site in other BoNTs were substituted. The results of cell binding analysis of the mutants were significantly correlated with the ganglioside binding properties. Based on these observations, a cell binding mechanism of BoNT from strain OFD05 is proposed, which involves cooperative contribution of two ganglioside binding sites.

  9. Takifugu rubripes cation independent mannose 6-phosphate receptor: Cloning, expression and functional characterization of the IGF-II binding domain.

    Science.gov (United States)

    A, Ajith Kumar; Nadimpalli, Siva Kumar

    2018-07-01

    Mannose 6-phosphate/IGF-II receptor mediated lysosomal clearance of insulin-like growth factor-II is significantly associated with the evolution of placental mammals. The protein is also referred to as the IGF-II receptor. Earlier studies suggested relatively low binding affinity between the receptor and ligand in prototherian and metatherian mammals. In the present study, we cloned the IGF-II binding domain of the early vertebrate fugu fish and expressed it in bacteria. A 72000Da truncated receptor containing the IGF-II binding domain was obtained. Analysis of this protein (covering domains 11-13 of the CIMPR) for its affinity to fish and human IGF-II by ligand blot assays and ELISA showed that the expressed receptor can specifically bind to both fish and human IGF-II. Additionally, a peptide-specific antibody raised against the region of the IGF-II binding domain also was able to recognize the IGF-II binding regions of mammalian and non-mammalian cation independent MPR protein. These interactions were further characterized by Surface Plasma resonance support that the receptor binds to fish IGF-II, with a dissociation constant of 548nM. Preliminary analysis suggests that the binding mechanism as well as the affinity of the fish and human receptor for IGF-II may have varied according to different evolutionary pressures. Copyright © 2018. Published by Elsevier B.V.

  10. Definition of the G protein-coupled receptor transmembrane bundle binding pocket and calculation of receptor similarities for drug design

    DEFF Research Database (Denmark)

    Gloriam, David Erik Immanuel; Foord, Steven M; Blaney, Frank E

    2009-01-01

    currently available crystal structures. This was used to characterize pharmacological relationships of Family A/Rhodopsin family GPCRs, minimizing evolutionary influence from parts of the receptor that do not generally affect ligand binding. The resultant dendogram tended to group receptors according...

  11. Imidazole derivatives as angiotensin II AT1 receptor blockers: Benchmarks, drug-like calculations and quantitative structure-activity relationships modeling

    Science.gov (United States)

    Alloui, Mebarka; Belaidi, Salah; Othmani, Hasna; Jaidane, Nejm-Eddine; Hochlaf, Majdi

    2018-03-01

    We performed benchmark studies on the molecular geometry, electron properties and vibrational analysis of imidazole using semi-empirical, density functional theory and post Hartree-Fock methods. These studies validated the use of AM1 for the treatment of larger systems. Then, we treated the structural, physical and chemical relationships for a series of imidazole derivatives acting as angiotensin II AT1 receptor blockers using AM1. QSAR studies were done for these imidazole derivatives using a combination of various physicochemical descriptors. A multiple linear regression procedure was used to design the relationships between molecular descriptor and the activity of imidazole derivatives. Results validate the derived QSAR model.

  12. Enhancement of bradykinin and resensitization of its B2 receptor.

    Science.gov (United States)

    Marcic, B; Deddish, P A; Jackman, H L; Erdös, E G

    1999-03-01

    We studied the enhancement of the effects of bradykinin B2 receptor agonists by agents that react with active centers of angiotensin-converting enzyme (ACE) independent of enzymatic inactivation. The potentiation and the desensitization and resensitization of B2 receptor were assessed by measuring [3H]arachidonic acid release and [Ca2+]i mobilization in Chinese hamster ovary cells transfected to express human ACE and B2 receptor, or in endothelial cells with constitutively expressed ACE and receptor. Administration of bradykinin or its ACE-resistant analogue desensitized the receptor, but it was resensitized (arachidonic acid release or [Ca2+]i mobilization) by agents such as enalaprilat (1 micromol/L). Enalaprilat was inactive in the absence of ACE expression. La3+ (100 micromol/L) inhibited the apparent resensitization, probably by blocking the entry of extracellular calcium. Enalaprilat resensitized the receptor via ACE to release arachidonic acid by bradykinin at a lower concentration (5 nmol/L) than required to mobilize [Ca2+]i (1 micromol/L). Monoclonal antibodies inhibiting the ACE N-domain active center and polyclonal antiserum potentiated bradykinin. The snake venom peptide BPP5a and metabolites of angiotensin and bradykinin (angiotensin-[1-9], angiotensin-[1-7], bradykinin-[1-8]; 1 micromol/L) enhanced arachidonic acid release by bradykinin. Angiotensin-(1-9) and -(1-7) also resensitized the receptor. Enalaprilat potentiated the bradykinin effect in cells expressing a mutant ACE with a single N-domain active site. Agents that reacted with a single active site, on the N-domain or on the C-domain, potentiated bradykinin not by blocking its inactivation but by inducing crosstalk between ACE and the receptor. Enalaprilat enhanced signaling via ACE by Galphai in lower concentration than by Galphaq-coupled receptor.

  13. Beta 2-adrenergic receptors on eosinophils. Binding and functional studies

    International Nuclear Information System (INIS)

    Yukawa, T.; Ukena, D.; Kroegel, C.; Chanez, P.; Dent, G.; Chung, K.F.; Barnes, P.J.

    1990-01-01

    We have studied the binding characteristics and functional effects of beta-adrenoceptors on human and guinea pig eosinophils. We determined the binding of the beta-antagonist radioligand [125I]pindolol (IPIN) to intact eosinophils obtained from the peritoneal cavity of guinea pigs and from blood of patients with eosinophilia. Specific binding was saturable, and Scatchard analysis showed a single binding site with a dissociation constant (Kd) of 24.6 pM and maximal number of binding sites (Bmax) of 7,166 per cell. ICI 118,551, a beta 2-selective antagonist, inhibited IPIN binding with a Ki value of 0.28 nM and was approximately 5,000-fold more effective than the beta 1-selective antagonist, atenolol. Isoproterenol increased cAMP levels about 5.5-fold above basal levels (EC50 = 25 microM); albuterol, a beta 2-agonist, behaved as a partial agonist with a maximal stimulation of 80%. Binding to human eosinophils gave similar results with a Kd of 25.3 pM and a Bmax corresponding to 4,333 sites per cell. Incubation of both human and guinea pig eosinophils with opsonized zymosan (2 mg/ml) or with phorbol myristate acetate (PMA) (10(-8) and 10(-6) M) resulted in superoxide anion generation and the release of eosinophil peroxidase; albuterol (10(-7) to 10(-5) M) had no inhibitory effect on the release of these products. Thus, eosinophils from patients with eosinophilia and from the peritoneal cavity of guinea pigs possess beta-receptors of the beta 2-subtype that are coupled to adenylate cyclase; however, these receptors do not modulate oxidative metabolism or degranulation. The possible therapeutic consequences of these observations to asthma are discussed

  14. Pharmacokinetic/Pharmacodynamic Modeling of Renin-Angiotensin Aldosterone Biomarkers Following Angiotensin-Converting Enzyme (ACE) Inhibition Therapy with Benazepril in Dogs.

    Science.gov (United States)

    Mochel, Jonathan P; Fink, Martin; Peyrou, Mathieu; Soubret, Antoine; Giraudel, Jérôme M; Danhof, Meindert

    2015-06-01

    The objective of this research was to provide a comprehensive description of the effect of benazepril on the dynamics of the renin-angiotensin aldosterone system (RAAS) in dogs. Blood specimens for renin activity (RA), angiotensin II (AII), and aldosterone (ALD) quantitation in plasma were drawn from 12 healthy adult beagle dogs randomly allocated to 2 treatment groups: (i) benazepril 5 mg PO, q24 h (n: 6) and (ii) placebo (n: 6), in a cross-over design. A mechanism-based pharmacokinetic/pharmacodynamic model, which includes the periodic nature of RA, AII, and ALD during placebo treatment and the subsequent changes in dynamics following repeated dosing with benazepril, was developed. The disposition kinetics of benazepril active metabolite, benazeprilat, was characterized using a saturable binding model to the angiotensin converting enzyme. The modulatory effect of benazeprilat on the RAAS was described using a combination of immediate response models. Our data show that benazepril noticeably influences the dynamics of the renin cascade, resulting in a substantial decrease in AII and ALD, while increasing RA throughout the observation span. The model provides a quantitative framework for better understanding the effect of ACE inhibition on the dynamics of the systemic RAAS in dogs.

  15. Phosphorylation inhibits DNA-binding of alternatively spliced aryl hydrocarbon receptor nuclear translocator

    International Nuclear Information System (INIS)

    Kewley, Robyn J.; Whitelaw, Murray L.

    2005-01-01

    The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer

  16. Receptors for vasoactive intestinal peptide in rat anterior pituitary glands: Localization of binding to lactotropes

    International Nuclear Information System (INIS)

    Wanke, I.E.; Rorstad, O.P.

    1990-01-01

    Vasoactive intestinal peptide (VIP) has been implicated as a physiological PRL-releasing factor; however, characterization of VIP receptors on normal pituitaries using radioligand-binding methods has been problematic. In this study we demonstrated specific receptors for VIP in anterior pituitary glands of female rats using HPLC-purified monoiodinated [Tyr(125I)10]VIP. Binding of VIP was reversible, saturable to receptor and radioligand, regulated by guanine nucleotides, and dependent on time and temperature. Scatchard analysis of competitive binding studies indicated high and low affinity binding sites, with equilibrium dissociation constants (Kd) of 0.19 +/- 0.03 and 28 +/- 16 nM, respectively. The corresponding maximum numbers of binding sites were 158 +/- 34 fmol/mg and 11.7 +/- 6.9 pmol/mg. Binding was specific, as peptides with structural homology to VIP were less than 100th as potent as VIP. The rank order of potency of the peptides tested was VIP greater than rat (r) peptide histidine isoleucine = human (h) PHI greater than rGRF greater than bovine GRF = porcine PHI = VIP-(10-28) greater than hGRF greater than secretin greater than apamin greater than glucagon. Radioligand binding was associated primarily with lactotrope-enriched fractions prepared by unit gravity sedimentation of dispersed anterior pituitary cells. VIP stimulated PRL release from cultured rat anterior pituitary cells, with an ED50 of 1 nM. These results, comprising the first identification of specific VIP receptors in normal rat anterior pituitary tissue using radioligand-binding methods, provide additional support for a biological role of VIP in lactotrope function

  17. Quantitative characterization of glycan-receptor binding of H9N2 influenza A virus hemagglutinin.

    Directory of Open Access Journals (Sweden)

    Karunya Srinivasan

    Full Text Available Avian influenza subtypes such as H5, H7 and H9 are yet to adapt to the human host so as to establish airborne transmission between humans. However, lab-generated reassorted viruses possessing hemagglutinin (HA and neuraminidase (NA genes from an avian H9 isolate and other genes from a human-adapted (H3 or H1 subtype acquired two amino acid changes in HA and a single amino acid change in NA that confer respiratory droplet transmission in ferrets. We previously demonstrated for human-adapted H1, H2 and H3 subtypes that quantitative binding affinity of their HA to α2→6 sialylated glycan receptors correlates with respiratory droplet transmissibility of the virus in ferrets. Such a relationship remains to be established for H9 HA. In this study, we performed a quantitative biochemical characterization of glycan receptor binding properties of wild-type and mutant forms of representative H9 HAs that were previously used in context of reassorted viruses in ferret transmission studies. We demonstrate here that distinct molecular interactions in the glycan receptor-binding site of different H9 HAs affect the glycan-binding specificity and affinity. Further we show that α2→6 glycan receptor-binding affinity of a mutant H9 HA carrying Thr-189→Ala amino acid change correlates with the respiratory droplet transmission in ferrets conferred by this change. Our findings contribute to a framework for monitoring the evolution of H9 HA by understanding effects of molecular changes in HA on glycan receptor-binding properties.

  18. Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo

    Energy Technology Data Exchange (ETDEWEB)

    Rodriguez-Tebar, A.; Barde, Y.A.

    1988-09-01

    Brain-derived neurotrophic factor (BDNF), a protein known to support the survival of embryonic sensory neurons and retinal ganglion cells, was derivatized with 125I-Bolton-Hunter reagent and obtained in a biologically active, radioactive form (125I-BDNF). Using dorsal root ganglion neurons from chick embryos at 9 d of development, the basic physicochemical parameters of the binding of 125I-BDNF with its receptors were established. Two different classes of receptors were found, with dissociation constants of 1.7 x 10(-11) M (high-affinity receptors) and 1.3 x 10(-9) M (low-affinity receptors). Unlabeled BDNF competed with 125I-BDNF for binding to the high-affinity receptors with an inhibition constant essentially identical to the dissociation constant of the labeled protein: 1.2 x 10(-11) M. The association and dissociation rates from both types of receptors were also determined, and the dissociation constants calculated from these kinetic experiments were found to correspond to the results obtained from steady-state binding. The number of high-affinity receptors (a few hundred per cell soma) was 15 times lower than that of low-affinity receptors. No high-affinity receptors were found on sympathetic neurons, known not to respond to BDNF, although specific binding of 125I-BDNF to these cells was detected at a high concentration of the radioligand. These results are discussed and compared with those obtained with nerve growth factor on the same neuronal populations.

  19. Binding characteristics of brain-derived neurotrophic factor to its receptors on neurons from the chick embryo

    International Nuclear Information System (INIS)

    Rodriguez-Tebar, A.; Barde, Y.A.

    1988-01-01

    Brain-derived neurotrophic factor (BDNF), a protein known to support the survival of embryonic sensory neurons and retinal ganglion cells, was derivatized with 125I-Bolton-Hunter reagent and obtained in a biologically active, radioactive form (125I-BDNF). Using dorsal root ganglion neurons from chick embryos at 9 d of development, the basic physicochemical parameters of the binding of 125I-BDNF with its receptors were established. Two different classes of receptors were found, with dissociation constants of 1.7 x 10(-11) M (high-affinity receptors) and 1.3 x 10(-9) M (low-affinity receptors). Unlabeled BDNF competed with 125I-BDNF for binding to the high-affinity receptors with an inhibition constant essentially identical to the dissociation constant of the labeled protein: 1.2 x 10(-11) M. The association and dissociation rates from both types of receptors were also determined, and the dissociation constants calculated from these kinetic experiments were found to correspond to the results obtained from steady-state binding. The number of high-affinity receptors (a few hundred per cell soma) was 15 times lower than that of low-affinity receptors. No high-affinity receptors were found on sympathetic neurons, known not to respond to BDNF, although specific binding of 125I-BDNF to these cells was detected at a high concentration of the radioligand. These results are discussed and compared with those obtained with nerve growth factor on the same neuronal populations

  20. N-terminal truncation enables crystallization of the receptor-binding domain of the FedF bacterial adhesin

    Energy Technology Data Exchange (ETDEWEB)

    De Kerpel, Maia; Van Molle, Inge [Department of Ultrastructure, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium); Brys, Lea [Department of Cellular and Molecular Immunology, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium); Wyns, Lode; De Greve, Henri; Bouckaert, Julie, E-mail: bouckaej@vub.ac.be [Department of Ultrastructure, Vrije Universiteit Brussel (VUB), Flanders Interuniversity Institute for Biotechnology (VIB), Pleinlaan 2, 1050 Brussels (Belgium)

    2006-12-01

    The N-terminal receptor-binding domain of the FedF adhesin from enterotoxigenic E. coli has been crystallized. This required the deletion of its first 14 residues, which are also cleaved off naturally. FedF is the two-domain tip adhesin of F18 fimbriae from enterotoxigenic Escherichia coli. Bacterial adherence, mediated by the N-terminal receptor-binding domain of FedF to carbohydrate receptors on intestinal microvilli, causes diarrhoea and oedema disease in newly weaned piglets and induces the secretion of Shiga toxins. A truncate containing only the receptor-binding domain of FedF was found to be further cleaved at its N-terminus. Reconstruction of this N-terminal truncate rendered FedF amenable to crystallization, resulting in crystals with space group P2{sub 1}2{sub 1}2{sub 1} and unit-cell parameters a = 36.20, b = 74.64, c = 99.03 Å that diffracted to beyond 2 Å resolution. The binding specificity of FedF was screened for on a glycan array, exposing 264 glycoconjugates, to identify specific receptors for cocrystallization with FedF.

  1. Angiotensin II regulation of neuromodulation: downstream signaling mechanism from activation of mitogen-activated protein kinase.

    Science.gov (United States)

    Lu, D; Yang, H; Raizada, M K

    1996-12-01

    Angiotensin II (Ang II) stimulates expression of tyrosine hydroxylase and norepinephrine transporter genes in brain neurons; however, the signal-transduction mechanism is not clearly defined. This study was conducted to determine the involvement of the mitogen-activated protein (MAP) kinase signaling pathway in Ang II stimulation of these genes. MAP kinase was localized in the perinuclear region of the neuronal soma. Ang II caused activation of MAP kinase and its subsequent translocation from the cytoplasmic to nuclear compartment, both effects being mediated by AT1 receptor subtype. Ang II also stimulated SRE- and AP1-binding activities and fos gene expression and its translocation in a MAP kinase-dependent process. These observations are the first demonstration of a downstream signaling pathway involving MAP kinase in Ang II-mediated neuromodulation in noradrenergic neurons.

  2. Novel and validated titrimetric method for determination of selected angiotensin-II-receptor antagonists in pharmaceutical preparations and its comparison with UV spectrophotometric determination

    Directory of Open Access Journals (Sweden)

    Shrikant H. Patil

    2012-12-01

    Full Text Available A novel and simple titrimetric method for determination of commonly used angiotensin-II-receptor antagonists (ARA-IIs is developed and validated. The direct acid base titration of four ARA-IIs, namely eprosartan mesylate, irbesartan, telmisartan and valsartan, was carried out in the mixture of ethanol:water (1:1 as solvent using standardized sodium hydroxide aqueous solution as titrant, either visually using phenolphthalein as an indicator or potentiometrically using combined pH electrode. The method was found to be accurate and precise, having relative standard deviation of less than 2% for all ARA-IIs studied. Also, it was shown that the method could be successfully applied to the assay of commercial pharmaceuticals containing the above-mentioned ARA-IIs. The validity of the method was tested by the recovery studies of standard addition to pharmaceuticals and the results were found to be satisfactory. Results obtained by this method were found to be in good agreement with those obtained by UV spectrophotometric method. For UV spectrophotometric analysis ethanol was used as a solvent and wavelength of 233 nm, 246 nm, 296 nm, and 250 nm was selected for determination of eprosartan mesylate, irbesartan, telmisartan, and valsartan respectively. The proposed titrimetric method is simple, rapid, convenient and sufficiently precise for quality control purposes. Keywords: Angiotensin-II-receptor antagonists, Titrimetric assay, UV spectrophotometry, Validation

  3. Mortality benefit of long-term angiotensin-converting enzyme inhibitors or angiotensin receptor blockers after successful percutaneous coronary intervention in non-ST elevation acute myocardial infarction.

    Science.gov (United States)

    González-Cambeiro, María Cristina; López-López, Andrea; Abu-Assi, Emad; Raposeiras-Roubín, Sergio; Peña-Gil, Carlos; García-Acuña, José; González-Juanatey, Ramón

    2016-12-01

    Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) have been shown to reduce mortality after myocardial infarction (MI). Current guidelines recommend their prescription in all patients after MI. Limited data are available on whether ACEIs/ARBs still improve prognosis in the contemporary era of non-ST elevation MI (NSTEMI) management. We aimed to evaluate the mortality benefit of ACEIs/ARBs in NSTEMI patients treated successfully with percutaneous coronary intervention (PCI). We analyzed 2784 patients with NSTEMI treated successfully with in-hospital PCI. Two groups were formed based on ACEI/ARB prescription at discharge. Two propensity score (PS) analyses were performed to control for differences in covariates: one with adjustment among the entire cohort, and the other with PS matching (n=1626). The outcome variable was all-cause mortality at four-year follow-up. There were 1902 (68.3%) patients prescribed ACEIs/ARBs at discharge. When adjusted by PS, ACEI/ARB use was associated with a hazard ratio (HR) for mortality of 0.75 (0.60-0.94; absolute risk reduction [ARR] 4.0%) in the whole cohort (p=0.01). After one-to-one PS matching (n=813 in each group), the mortality rate was significantly lower in patients prescribed ACEIs/ARBs, with HR of 0.77 (0.63-0.94; ARR 3.8%) (p=0.03). In this observational study of patients with NSTEMI, all of them treated successfully by PCI, the use of ACEIs/ARBs was significantly associated with a lower risk of four-year all-cause mortality. Copyright © 2016 Sociedade Portuguesa de Cardiologia. Publicado por Elsevier España, S.L.U. All rights reserved.

  4. Characterization of the Binding Site of Aspartame in the Human Sweet Taste Receptor.

    Science.gov (United States)

    Maillet, Emeline L; Cui, Meng; Jiang, Peihua; Mezei, Mihaly; Hecht, Elizabeth; Quijada, Jeniffer; Margolskee, Robert F; Osman, Roman; Max, Marianna

    2015-10-01

    The sweet taste receptor, a heterodimeric G protein-coupled receptor comprised of T1R2 and T1R3, binds sugars, small molecule sweeteners, and sweet proteins to multiple binding sites. The dipeptide sweetener, aspartame binds in the Venus Flytrap Module (VFTM) of T1R2. We developed homology models of the open and closed forms of human T1R2 and human T1R3 VFTMs and their dimers and then docked aspartame into the closed form of T1R2's VFTM. To test and refine the predictions of our model, we mutated various T1R2 VFTM residues, assayed activity of the mutants and identified 11 critical residues (S40, Y103, D142, S144, S165, S168, Y215, D278, E302, D307, and R383) in and proximal to the binding pocket of the sweet taste receptor that are important for ligand recognition and activity of aspartame. Furthermore, we propose that binding is dependent on 2 water molecules situated in the ligand pocket that bridge 2 carbonyl groups of aspartame to residues D142 and L279. These results shed light on the activation mechanism and how signal transmission arising from the extracellular domain of the T1R2 monomer of the sweet receptor leads to the perception of sweet taste. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Binding of C-reactive protein to human polymorphonuclear leukocytes: evidence for association of binding sites with Fc receptors

    International Nuclear Information System (INIS)

    Mueller, H.; Fehr, J.

    1986-01-01

    The functional similarities between C-reactive protein (CRP) and IgG raised the question as to whether human phagocytes are stimulated by CRP in the same way as by binding of antigen-complexes or aggregated IgG to their Fc receptors. Studies with the use of highly purified 125 I-labeled CRP showed specific and saturable binding to human polymorphonuclear leukocytes (PNM) with a K/sub D/ of 10.5 +/- 5.7 x 10 -8 M only when carried out in heat-inactivated plasma. The number of specific binding sites per cell was estimated at 1 to 3 x 10 6 . Competitive inhibition of CRP binding by antigen-complexed or aggregated IgG suggests CRP binding sites to be associated IgG suggests CRP binding sites to be associated with PMN Fc receptors. Only when assayed in heat-inactivated plasma did CRP binding induce adherence of cells to tissue culture dishes. However, no metabolic and potentially cytotoxic simulation of PMN was detected during CRP plasma-dependent attachment to surfaces: induction of aggregation, release of secondary granule constituents, and activation of the hexose monophosphate pathway were not observed. These results imply that CRP-PMN interactions is dependent on an additional factor present in heat-inactivated plasma and is followed only by a complement-independent increase in PMN attachment to surfaces. Because CRP was found to be deposits at sites of tissue injury, the CRP-mediated adherence of PMN may be an important step in localizing an inflammatory focus

  6. Ligand binding and activation mechanism og the glucagon-like peptide-1 receptor

    DEFF Research Database (Denmark)

    Underwood, Christina Rye

    GLP-1R interacts with receptor agonists. The thesis includes four studies, which investigate different aspects of these interactions. The first study elucidates GLP-1 binding to the extracellular domain of GLP-1R (ECD) (Study I), whereas the second study identifies receptor domains important for small...

  7. Elevated glucocorticoid receptor binding in cultured human lymphoblasts following hydroxyurea treatment: lack of effect on steroid responsiveness

    International Nuclear Information System (INIS)

    Littlefield, B.A.; Hoagland, H.C.; Greipp, P.R.

    1986-01-01

    While studying the effects of chemotherapy on glucocorticoid receptor (GR) binding levels in hematological malignancies, we observed a sizable increase in nuclear GR binding of [ 3 H]dexamethasone in peripheral leukocytes from a chronic basophilic leukemia patient following treatment with hydroxyurea plus prednisone, but not after prednisone alone. This apparent clinical effect of hydroxyurea led to an examination of hydroxyurea effects on GR binding and sensitivity in the glucocorticoid-sensitive human lymphoblast cell line GM4672A. GR binding levels in GM4672A cells were measured following a 3-day exposure to 50 microM hydroxyurea, a concentration chosen to have a minimal but measurable effect on cellular growth rates with little or no effect on cellular viability. Under these conditions, nuclear [ 3 H]dexamethasone receptor binding measured by Scatchard analysis using a whole-cell assay was elevated 2.4-fold over control values (P less than 0.05), while cytosolic residual receptor binding (measured at 37 0 C) remained unchanged. Thus, the total cellular content of measurable GR was increased, and this increase was totally accounted for by GR capable of nuclear binding. Hydroxyurea treatment of GM4672A cells had no effect on the affinity of nuclear or cytosolic GR for [ 3 H]dexamethasone. The increase in measurable nuclear-bound receptors occurred in a time-dependent manner over a period of 3 days and was fully reversible within 3 days following removal of hydroxyurea. The increase in receptor binding could not be explained by the slight alterations in cell cycle kinetics which occur at this low level of hydroxyurea. Despite increased receptor binding, cellular glucocorticoid responsiveness was unaltered as assessed by dexamethasone inhibition of cell growth and dexamethasone inhibition of a urokinase-like plasminogen activator

  8. Interaction of alpha-conotoxin ImII and its analogs with nicotinic receptors and acetylcholine-binding proteins: additional binding sites on Torpedo receptor

    NARCIS (Netherlands)

    Kasheverov, I.E.; Zhmak, M.N.; Fish, A.; Rucktooa, P.; Khruschov, A.Y.; Osipov, A.V.; Ziganshin, R.H.; D'Hoedt, D.; Bertrand, D.; Sixma, T.K.; Smit, A.B.; Tsetlin, V.I.

    2009-01-01

    α-Conotoxins interact with nicotinic acetylcholine receptors (nAChRs) and acetylcholine-binding proteins (AChBPs) at the sites for agonists/competitive antagonists. α-Conotoxins blocking muscle-type or α7 nAChRs compete with α-bungarotoxin. However, α-conotoxin ImII, a close homolog of the α7

  9. Molecular-genetic risk assessement of determining angiotensin-converting enzyme hyperactivity in hemorrhagic fever with renal syndrome

    Directory of Open Access Journals (Sweden)

    Ildar R. Minniakhmetov

    2012-09-01

    Full Text Available The present study was designed to investigate changes in angiotensin-converting enzyme (ACE blood activity and angiotensin II type 1 receptor gene polymorphism as a possible disease predictor in hemorrhagic fever with renal syndrome (HFRS. Four hundred and nine patients (346 males and 63 females with HFRS serologic confirmation were enrolled in the study. Their age ranged from 15 to 65 years. ACE blood activity was assessed kinetically using the Bühlmann (Switzerland kit. Peripheral blood genomic DNA was isolated by a phenol-chloroform extraction. The genotyping of DNA loci was done using a polymerase chain reaction of DNA synthesis. Statistically, ACE blood activity was significantly higher throughout the entire HFRS course with diverse severity apart from the feverish phase of moderate-to-severe uncomplicated disease forms. *A1166 and *C1166 alleles, *A1166/*A1166 and *C1166/*C1166 genotypes of angiotensin II type 1 receptor gene were not associated with HFRS severity. The results of this study indicate that high ACE activity has not adaptive characteristics due to abnormalities in angiotensin II reception. It is an adequate metabolic response of the body to endotheliotropic virus activity.

  10. Brain serotonin 2A receptor binding: Relations to body mass index, tobacco and alcohol use

    DEFF Research Database (Denmark)

    Erritzoe, D.; Frokjaer, V. G.; Haugbol, S.

    2009-01-01

    receptor (5-HT(2A)) in humans, we tested in 136 healthy human subjects if body mass index (BMI), degree of alcohol consumption and tobacco smoking was associated to the cerebral in vivo 5-HT(2A) receptor binding as measured with (18)F-altanserin PET. The subjects' BMI's ranged from 18.4 to 42.8 (25.......2+/-4.3) kg/m(2). Cerebral cortex 5-HT(2A) binding was significantly positively correlated to BMI, whereas no association between cortical 5-HT(2A) receptor binding and alcohol or tobacco use was detected. We suggest that our observation is driven by a lower central 5-HT level in overweight people, leading...

  11. 125I-luteinizing hormone (LH) binding to soluble receptors from the primate (Macaca mulatta) corpus luteum: effects of ethanol exposure

    International Nuclear Information System (INIS)

    Danforth, D.R.; Stouffer, R.L.

    1988-01-01

    In the current study, we compared the effects of ethanol on gonadotropin receptors solubilized from macaque luteal membranes to those on receptors associated with the lipid bilayer. Treatment with 1% Triton X-100 for 30 min at 4C, followed by precipitation with polyethylene glycol, resulted in recovery of 50% more binding sites for 125 I-human luteinizing hormone (hLH) than were available in particulate preparations. However, the soluble receptors displayed a 3-fold lower affinity for 125 I-hLH. Conditions which enhanced LH binding to particulates, i.e., 1-8% ethanol at 25C, decreased specific 125 I-hLH binding to soluble receptors. Steady-state LH binding to soluble receptors during incubation at 4C was half of that observed at 25C. The presence of 8% ethanol at 4C restored LH binding to levels observed in the absence of ethanol at 25C. Thus, LH binding sites in the primate corpus luteum can be effectively solubilized with Triton X-100. The different binding characteristics of particulate and soluble receptors, including the response to ethanol exposure, suggest that the lipid environment in the luteal membrane modulates the availability and affinity of gonadotropin receptors

  12. Affinity of the enantiomers of. alpha. - and. beta. -cyclazocine for binding to the phencyclidine and. mu. opioid receptors

    Energy Technology Data Exchange (ETDEWEB)

    Todd, S.L.; Balster, R.L.; Martin, B.R. (Virginia Commonwealth Univ., Richmond (USA))

    1990-01-01

    The enantiomers in the {alpha} and {beta} series of cyclazocine were evaluated for their ability to bind to phencyclidine (PCP) and {mu}-opioid receptors in order to determine their receptor selectivity. The affinity of (-)-{beta}-cyclazocine for the PCP receptor was 1.5 greater than PCP itself. In contrast, (-)-{alpha}-cyclazocine, (+)-{alpha}-cyclazocine, and (+)-{beta}-cyclazocine were 3-, 5- and 138-fold less potent than PCP, respectively. Scatchard analysis of saturable binding of ({sup 3}H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) also exhibited a homogeneous population of binding sites with an apparent K{sub D} of 1.9 nM and an estimated Bmax of 117 pM. (3H)Tyr-D-Ala-Gly-N-MePhe-Gly-ol (DAMGO) binding studies revealed that (-)-{alpha}-cyclazocine (K{sub D} = 0.48 nM) was 31-, 1020- and 12,600-fold more potent than (-)-{beta}-cyclazocine, (+)-{alpha}-cyclazocine and (+)-{beta}-cyclazocine, respectively, for binding to the {mu}-opioid receptor. These data show that, although (-)-{beta}-cyclazocine is a potent PCP receptor ligand consistent with its potent PCP-like discriminative stimulus effects, it shows little selectivity for PCP receptor since it also potently displaces {mu}-opioid binding. However, these cyclazocine isomers, due to their extraordinary degree of stereoselectivity, may be useful in characterizing the structural requirements for benzomorphans having activity at the PCP receptor.

  13. A single mutation in Taiwanese H6N1 influenza hemagglutinin switches binding to human-type receptors

    Energy Technology Data Exchange (ETDEWEB)

    de Vries, Robert P.; Tzarum, Netanel; Peng, Wenjie; Thompson, Andrew J.; Ambepitiya Wickramasinghe, Iresha N.; de la Pena, Alba T. Torrents; van Breemen, Marielle J.; Bouwman, Kim M.; Zhu, Xueyong; McBride, Ryan; Yu, Wenli; Sanders, Rogier W.; Verheije, Monique H.; Wilson, Ian A.; Paulson, James C.

    2017-07-10

    In June 2013, the first case of human infection with an avian H6N1 virus was reported in a Taiwanese woman. Although this was a single non-fatal case, the virus continues to circulate in Taiwanese poultry. As with any emerging avian virus that infects humans, there is concern that acquisition of human-type receptor specificity could enable transmission in the human population. Despite mutations in the receptor-binding pocket of the human H6N1 isolate, it has retained avian-type (NeuAcα2-3Gal) receptor specificity. However, we show here that a single nucleotide substitution, resulting in a change from Gly to Asp at position 225 (G225D), completely switches specificity to human-type (NeuAcα2-6Gal) receptors. Significantly, G225D H6 loses binding to chicken trachea epithelium and is now able to bind to human tracheal tissue. Structural analysis reveals that Asp225 directly interacts with the penultimate Gal of the human-type receptor, stabilizing human receptor binding.

  14. Negative cooperativity in binding of muscarinic receptor agonists and GDP as a measure of agonist efficacy.

    Science.gov (United States)

    Jakubík, J; Janíčková, H; El-Fakahany, E E; Doležal, V

    2011-03-01

    Conventional determination of agonist efficacy at G-protein coupled receptors is measured by stimulation of guanosine-5'-γ-thiotriphosphate (GTPγS) binding. We analysed the role of guanosine diphosphate (GDP) in the process of activation of the M₂ muscarinic acetylcholine receptor and provide evidence that negative cooperativity between agonist and GDP binding is an alternative measure of agonist efficacy. Filtration and scintillation proximity assays measured equilibrium binding as well as binding kinetics of [³⁵S]GTPγS and [³H]GDP to a mixture of G-proteins as well as individual classes of G-proteins upon binding of structurally different agonists to the M₂ muscarinic acetylcholine receptor. Agonists displayed biphasic competition curves with the antagonist [³H]-N-methylscopolamine. GTPγS (1 µM) changed the competition curves to monophasic with low affinity and 50 µM GDP produced a similar effect. Depletion of membrane-bound GDP increased the proportion of agonist high-affinity sites. Carbachol accelerated the dissociation of [³H]GDP from membranes. The inverse agonist N-methylscopolamine slowed GDP dissociation and GTPγS binding without changing affinity for GDP. Carbachol affected both GDP association with and dissociation from G(i/o) G-proteins but only its dissociation from G(s/olf) G-proteins. These findings suggest the existence of a low-affinity agonist-receptor conformation complexed with GDP-liganded G-protein. Also the negative cooperativity between GDP and agonist binding at the receptor/G-protein complex determines agonist efficacy. GDP binding reveals differences in action of agonists versus inverse agonists as well as differences in activation of G(i/o) versus G(s/olf) G-proteins that are not identified by conventional GTPγS binding. © 2011 The Authors. British Journal of Pharmacology © 2011 The British Pharmacological Society.

  15. Why does the agonist [18F]FP-TZTP bind preferentially to the M2 muscarinic receptor?

    International Nuclear Information System (INIS)

    Ravasi, L.; Kiesewetter, D.O.; Shimoji, K.; Lucignani, G.; Eckelman, W.C.

    2006-01-01

    Preferential binding of FP-TZTP at the M 2 receptor in vivo led to investigation of [ 18 F]FP-TZTP as a potential PET tracer for Alzheimer's disease, in which a substantial reduction of M 2 receptors has been observed in autopsy studies. We hereby investigated in vitro the FP-TZTP behavior to further elucidate the properties of FP-TZTP that lead to its M 2 selectivity. Chinese hamster ovarian cells expressing the five subtypes of human muscarinic receptor as well as the wild type were harvested in culture to assess equilibrium binding. Specific binding was calculated by subtraction of non-specific binding from total binding. Internal specific binding was calculated by subtraction of external specific binding from the total specific binding. Saturation assays were also performed to calculate B max , K i , and IC 50 . In addition, equilibrium binding and dissociation kinetic studies were performed on rat brain tissue. Selected regions of interest were drawn on the digital autoradiograms and [ 18 F]FP-TZTP off-rates were determined by measurement of the rate of release into a buffer solution of [ 18 F]FP-TZTP from slide-bound cells that had been preincubated with [ 18 F]FP-TZTP. At equilibrium in vitro, M 2 subtype selectivity of [ 18 F]FP-TZTP was not evident. We demonstrated that ATP-dependent mechanisms are not responsible for FP-TZTP M 2 selectivity. In vitro off-rate studies from rat brain tissue showed that the off-rate of FP-TZTP varied with the percentage of M 2 subtype in the tissue region. The slower dissociation kinetics of FP-TZTP from M 2 receptors compared with the four other muscarinic receptor subtypes may be a factor in its M 2 selectivity. (orig.)

  16. Prediction of consensus binding mode geometries for related chemical series of positive allosteric modulators of adenosine and muscarinic acetylcholine receptors.

    Science.gov (United States)

    Sakkal, Leon A; Rajkowski, Kyle Z; Armen, Roger S

    2017-06-05

    Following insights from recent crystal structures of the muscarinic acetylcholine receptor, binding modes of Positive Allosteric Modulators (PAMs) were predicted under the assumption that PAMs should bind to the extracellular surface of the active state. A series of well-characterized PAMs for adenosine (A 1 R, A 2A R, A 3 R) and muscarinic acetylcholine (M 1 R, M 5 R) receptors were modeled using both rigid and flexible receptor CHARMM-based molecular docking. Studies of adenosine receptors investigated the molecular basis of the probe-dependence of PAM activity by modeling in complex with specific agonist radioligands. Consensus binding modes map common pharmacophore features of several chemical series to specific binding interactions. These models provide a rationalization of how PAM binding slows agonist radioligand dissociation kinetics. M 1 R PAMs were predicted to bind in the analogous M 2 R PAM LY2119620 binding site. The M 5 R NAM (ML-375) was predicted to bind in the PAM (ML-380) binding site with a unique induced-fit receptor conformation. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  17. Cholecystokinin-8 suppressed /sup 3/H-etorphine binding to rat brain opiate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Wang, X.J.; Fan, S.G.; Ren, M.F.; Han, J.S.

    1989-01-01

    Radioreceptor assay (RRA) was adopted to analyze the influence of CCK-8 on /sup 3/H-etorphine binding to opiate receptors in rat brain synaptosomal membranes (P2). In the competition experiment CCK-8 suppressed the binding of /sup 3/H-etorphine. This effect was completely reversed by proglumide at 1/mu/M. Rosenthal analysis for saturation revealed two populations of /sup 3/H-etorphine binding sites. CCK-8 inhibited /sup 3/H-etorphine binding to the high affinity sites by an increase in Kd and decrease in Bmax without significant changes in the Kd and Bmax of the low affinity sites. This effect of CCK-8 was also completely reversed by proglumide at 1/mu/M. Unsulfated CCK-8 produced only a slight increase in Kd of the high affinity sites without affecting Bmax. The results suggest that CCK-8 might be capable of suppressing the high affinity opioid binding sites via the activation of CCK receptor.

  18. A radioreceptor assay of luteinizing hormone-releasing hormone receptor and characterization of LHRH binding to pituitary receptors in Shao duck

    International Nuclear Information System (INIS)

    Yang Peixin; Wu Meiwen; Chen Ziyuan

    2000-01-01

    The properties of Shao duck pituitary luteinizing hormone-releasing hormone (LHRH) receptors were analyzed in pituitary membrane preparation and isolated pituitary cells prepared by enzymatic dispersion with collagenase and trypsin, by using a super-agonist analog of (D-Lys 6 ) LHRH. High binding of 125 I-(D-Lys 6 ) LHRH to 10 6 cultured cells of Shao duck was observed after a 90 minute incubation at 4 degree C, while binding was significantly reduced after a 24h incubation. Binding of the radioligand was a function of tissue concentration of Shao duck pituitary membrane preparation, with a positive correlation over the range of 1-2 pituitary per-tube. Specific binding for 125 I-(D-Lys 6 ) LHRH increased with the increase in the amount of 125 I-(D-Lys 6 ) LHRH. The Scatchard analysis of data revealed a linear relationship between the amount of specific binding and the ratio of specific binding to free 1 '2 5 I(D-Lys 6 )LHRH, indicating a single class of high affinity sites. Equilibrium dissociation constant (Kd) was 0.34 nM in pituitary membrane preparation and 0.43 nM in isolated pituitary cells. Both Kd values were near and the maximum binding capacity (B max ) was great in isolated cells, suggesting no significant loss of the LHRH receptor population caused by the enzymatic procedure employed for cell dispersion in the present study. Addition of 9D-Lys 6 ) LHRH displaced bound 125 I-(D-Lys 6 ) LHRH. These results demonstrated the presence and provided characterization of LHRH receptors in Shao duck pituitary

  19. The changes in drug binding activity of GABA receptor and animal neural-behavior after gamma irradiation

    International Nuclear Information System (INIS)

    Zheng Hui; Zhen Rong; Zhao Naikun; Xue Hong; Wang Zihui

    2004-01-01

    Objective: The purpose of this study was to investigate the effect of irradiation on gamma-aminobutyric-acid receptor (GABA-R) as well as behavioral changes after brain 60 Co γ-irradiation. Methods: The mice were irradiated with gamma rays (20 Gy; 10 Gy and 5 Gy) . The drug binding activity of GABA receptor in brain receptor was measured by fluorescence anisotropy (FA) and equilibrium dissociation constants. The behavioral changes were observed by the locomotor activity test, elevated plus-maze test and hole-board test at 1, 10, 24 and 48 hr after irradiation. Results: 1. The drug binding activity of the GABA receptor was decreased and the equilibrium dissociation constant (K d ) was significantly increased compared with the negative control group 2 hr after irradiation, and a spike value appeared at 24 hr. It showed that the irradiation might damage or decrease the binding activity and the bio-activity of GABA receptor. 2. The animal experiment confirmed that the irradiated animal model showed neural-behavioral changes of anxiety or depression. 3. The decreased binding activity of GABA receptor and changes in behavior of irradiated animal were dependent on radiation intensity. 4. The changes of behavior was similar to the blocked GABA receptor group. It suggests the relationship of radiation and GABA receptor. Conclusion: These results suggest that GABA receptor may be involved in radiation injury. The functional changes of GABA receptor may be an induction factor of behavioral disorder. The article also discussed the effect of anxiety and results obtained from the point of view of GABA receptor system involvement in the changes observed after irradiation. (authors)

  20. MAP kinase-independent signaling in angiotensin II regulation of neuromodulation in SHR neurons.

    Science.gov (United States)

    Yang, H; Raizada, M K

    1998-09-01

    Angiotensin II (Ang II), via its interaction with the angiotensin type 1 (AT1) receptor subtype, causes enhanced stimulation of norepinephrine (NE) neuromodulation. This involves increased transcription of NE transporter, tyrosine hydroxylase, and dopamine ss-hydroxylase genes in Wistar-Kyoto rat (WKY) brain neurons. AT1 receptor-mediated regulation of certain signaling events (such as activation of the Ras-Raf-1-mitogen activated protein (MAP) kinase signaling pathway, nuclear translocation of transcription factors such as Fos and Jun, and the interactions of these factors with AP-1 binding sites) is involved in this NE neuromodulation (Lu et al. J Cell Biol. 1996;135:1609-1617). The aim of this study was to compare the signal transduction mechanism of Ang II regulation of NE neuromodulation in WKY and spontaneously hypertensive rat (SHR) brain neurons, in view of the fact that AT1 receptor expression and Ang II stimulation of NE neuromodulation are higher in SHR neurons compared with WKY neurons. Despite this hyperactivity, Ang II stimulation of Ras, Raf-1, and MAP kinase activities was comparable between the neurons from WKY and SHR. Similarly, central injections of Ang II caused a comparable stimulation of MAP kinase in the hypothalamic and brain stem areas of adult WKY and SHR. Inhibition of MAP kinase by either an MAP kinase kinase inhibitor (PD98059) or an MAP kinase antisense oligonucleotide completely attenuated the stimulatory effects of Ang II on [3H]-NE uptake, NE transporter mRNA, and tyrosine hydroxylase mRNA levels in WKY neurons. These treatments resulted in only 43% to 50% inhibition of [3H]-NE uptake and NE transporter and tyrosine hydroxylase mRNAs in SHR neurons. Thus, Ang II stimulation of NE neuromodulation was completely blocked by MAP kinase inhibition in WKY neurons and only partially blocked in the SHR neurons. These observations suggest the presence of an additional signal transduction pathway involved in NE neuromodulation in SHR neurons

  1. Molecular Modeling of the M3 Acetylcholine Muscarinic Receptor and Its Binding Site

    Directory of Open Access Journals (Sweden)

    Marlet Martinez-Archundia

    2012-01-01

    Full Text Available The present study reports the results of a combined computational and site mutagenesis study designed to provide new insights into the orthosteric binding site of the human M3 muscarinic acetylcholine receptor. For this purpose a three-dimensional structure of the receptor at atomic resolution was built by homology modeling, using the crystallographic structure of bovine rhodopsin as a template. Then, the antagonist N-methylscopolamine was docked in the model and subsequently embedded in a lipid bilayer for its refinement using molecular dynamics simulations. Two different lipid bilayer compositions were studied: one component palmitoyl-oleyl phosphatidylcholine (POPC and two-component palmitoyl-oleyl phosphatidylcholine/palmitoyl-oleyl phosphatidylserine (POPC-POPS. Analysis of the results suggested that residues F222 and T235 may contribute to the ligand-receptor recognition. Accordingly, alanine mutants at positions 222 and 235 were constructed, expressed, and their binding properties determined. The results confirmed the role of these residues in modulating the binding affinity of the ligand.

  2. Angiotensin II Regulation of Proliferation, Differentiation, and Engraftment of Hematopoietic Stem Cells.

    Science.gov (United States)

    Kim, Seungbum; Zingler, Michael; Harrison, Jeffrey K; Scott, Edward W; Cogle, Christopher R; Luo, Defang; Raizada, Mohan K

    2016-03-01

    Emerging evidence indicates that differentiation and mobilization of hematopoietic cell are critical in the development and establishment of hypertension and hypertension-linked vascular pathophysiology. This, coupled with the intimate involvement of the hyperactive renin-angiotensin system in hypertension, led us to investigate the hypothesis that chronic angiotensin II (Ang II) infusion affects hematopoietic stem cell (HSC) regulation at the level of the bone marrow. Ang II infusion resulted in increases in hematopoietic stem/progenitor cells (83%) and long-term HSC (207%) in the bone marrow. Interestingly, increases of HSCs and long-term HSCs were more pronounced in the spleen (228% and 1117%, respectively). Furthermore, we observed higher expression of C-C chemokine receptor type 2 in these HSCs, indicating there was increased myeloid differentiation in Ang II-infused mice. This was associated with accumulation of C-C chemokine receptor type 2(+) proinflammatory monocytes in the spleen. In contrast, decreased engraftment efficiency of GFP(+) HSC was observed after Ang II infusion. Time-lapse in vivo imaging and in vitro Ang II pretreatment demonstrated that Ang II induces untimely proliferation and differentiation of the donor HSC resulting in diminished HSC engraftment and bone marrow reconstitution. We conclude that (1) chronic Ang II infusion regulates HSC proliferation, mediated by angiotensin receptor type 1a, (2) Ang II accelerates HSC to myeloid differentiation resulting in accumulation of C-C chemokine receptor type 2(+) HSCs and inflammatory monocytes in the spleen, and (3) Ang II impairs homing and reconstitution potentials of the donor HSCs. These observations highlight the important regulatory roles of Ang II on HSC proliferation, differentiation, and engraftment. © 2016 American Heart Association, Inc.

  3. Dietary restriction but not angiotensin II type 1 receptor blockade improves DNA damage-related vasodilator dysfunction in rapidly aging Ercc1Δ/- mice.

    Science.gov (United States)

    Wu, Haiyan; van Thiel, Bibi S; Bautista-Niño, Paula K; Reiling, Erwin; Durik, Matej; Leijten, Frank P J; Ridwan, Yanto; Brandt, Renata M C; van Steeg, Harry; Dollé, Martijn E T; Vermeij, Wilbert P; Hoeijmakers, Jan H J; Essers, Jeroen; van der Pluijm, Ingrid; Danser, A H Jan; Roks, Anton J M

    2017-08-01

    DNA damage is an important contributor to endothelial dysfunction and age-related vascular disease. Recently, we demonstrated in a DNA repair-deficient, prematurely aging mouse model ( Ercc1 Δ/- mice) that dietary restriction (DR) strongly increases life- and health span, including ameliorating endothelial dysfunction, by preserving genomic integrity. In this mouse mutant displaying prominent accelerated, age-dependent endothelial dysfunction we investigated the signaling pathways involved in improved endothelium-mediated vasodilation by DR, and explore the potential role of the renin-angiotensin system (RAS). Ercc1 Δ/- mice showed increased blood pressure and decreased aortic relaxations to acetylcholine (ACh) in organ bath experiments. Nitric oxide (NO) signaling and phospho-Ser 1177 -eNOS were compromised in Ercc1 Δ / - DR improved relaxations by increasing prostaglandin-mediated responses. Increase of cyclo-oxygenase 2 and decrease of phosphodiesterase 4B were identified as potential mechanisms. DR also prevented loss of NO signaling in vascular smooth muscle cells and normalized angiotensin II (Ang II) vasoconstrictions, which were increased in Ercc1 Δ/- mice. Ercc1 Δ/ - mutants showed a loss of Ang II type 2 receptor-mediated counter-regulation of Ang II type 1 receptor-induced vasoconstrictions. Chronic losartan treatment effectively decreased blood pressure, but did not improve endothelium-dependent relaxations. This result might relate to the aging-associated loss of treatment efficacy of RAS blockade with respect to endothelial function improvement. In summary, DR effectively prevents endothelium-dependent vasodilator dysfunction by augmenting prostaglandin-mediated responses, whereas chronic Ang II type 1 receptor blockade is ineffective. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  4. Role of angiotensin II and vasopressin receptors within the supraoptic nucleus in water and sodium intake induced by the injection of angiotensin II into the medial septal area

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    Antunes V.R.

    1998-01-01

    Full Text Available In this study we investigated the effects of the injection into the supraoptic nucleus (SON of non-peptide AT1- and AT2-angiotensin II (ANG II receptor antagonists, DuP753 and PD123319, as well as of the arginine-vasopressin (AVP receptor antagonist d(CH25-Tyr(Me-AVP, on water and 3% NaCl intake induced by the injection of ANG II into the medial septal area (MSA. The effects on water or 3% NaCl intake were assessed in 30-h water-deprived or in 20-h water-deprived furosemide-treated adult male rats, respectively. The drugs were injected in 0.5 ml over 30-60 s. Controls were injected with a similar volume of 0.15 M NaCl. Antagonists were injected at doses of 20, 80 and 180 nmol. Water and sodium intake was measured over a 2-h period. Previous administration of the AT1 receptor antagonist DuP753 into the SON decreased water (65%, N = 10, P<0.01 and sodium intake (81%, N = 8, P<0.01 induced by the injection of ANG II (10 nmol into the MSA. Neither of these responses was significantly changed by injection of the AT2-receptor antagonist PD123319 into the SON. On the other hand, while there was a decrease in water intake (45%, N = 9, P<0.01, ANG II-induced sodium intake was significantly increased (70%, N = 8, P<0.01 following injection of the V1-type vasopressin antagonist d(CH25-Tyr(Me-AVP into the SON. These results suggest that both AT1 and V1 receptors within the SON may be involved in water and sodium intake induced by the activation of ANG II receptors within the MSA. Furthermore, they do not support the involvement of MSA AT2 receptors in the mediation of these responses.

  5. Evidence for modulation of pericryptal sheath myofibroblasts in rat descending colon by Transforming Growth Factor β and Angiotensin II.

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    Pedley Kevin C

    2002-02-01

    Full Text Available Abstract Background Absorption of water and Na+ in descending colonic crypts is dependent on the barrier function of the surrounding myofibroblastic pericryptal sheath. Here the effects of high and low Na+ diets and exposure to whole body ionising radiation on the growth and activation of the descending colonic pericryptal myofibroblasts are evaluated. In addition the effect of a post-irradiation treatment with the angiotensin converting enzyme inhibitor Captopril was investigated. Methods The levels of Angiotensin II type 1 receptor (AT1, ACE, collagen type IV, transforming growth factor-β type 1 receptor (TGF-βR1, OB cadherin and α-smooth muscle actin in both descending colon and caecum were evaluated, using immunocytochemistry and confocal microscopy, in rats fed on high and low Na+ diets (LS. These parameters were also determined during 3 months post-irradiation with 8Gy from a 60Co source in the presence and absence of the angiotensin converting enzyme inhibitor, Captopril. Results Increases in AT1 receptor (135.6% ± 18.3, P Conclusions These results demonstrate an activation of descending colonic myofibroblasts to trophic stimuli, or irradiation, which can be attenuated by Captopril, indicative of local trophic control by angiotensin II and TGF-β release.

  6. Genetic variation and activity of the renin-angiotensin system and severe hypoglycemia in type 1 diabetes

    DEFF Research Database (Denmark)

    Pedersen-Bjergaard, U.; Dhamrait, S.S.; Sethi, A.A.

    2008-01-01

    BACKGROUND: The deletion-allele of the angiotensin-converting enzyme (ACE) gene and elevated ACE activity are associated with increased risk of severe hypoglycemia in type 1 diabetes. We explored whether genetic and phenotypic variations in other components of the renin-angiotensin system...... are similarly associated. METHODS: Episodes of severe hypoglycemia were recorded in 171 consecutive type 1 diabetic outpatients during a 1-year follow-up. Participants were characterized at baseline by gene polymorphisms in angiotensinogen, ACE, angiotensin-II receptor types 1 (AT1R) and 2 (AT2R), and by plasma...... associate with high risk of severe hypoglycemia in type 1 diabetes. A potential preventive effect of renin-angiotensin system blocking drugs in patients with recurrent severe hypoglycemia merits further investigation Udgivelsesdato: 2008/3...

  7. Targeting hepatic heparin-binding EGF-like growth factor (HB-EGF) induces anti-hyperlipidemia leading to reduction of angiotensin II-induced aneurysm development.

    Science.gov (United States)

    Kim, Seonwook; Yang, Lihua; Kim, Seongu; Lee, Richard G; Graham, Mark J; Berliner, Judith A; Lusis, Aldons J; Cai, Lei; Temel, Ryan E; Rateri, Debra L; Lee, Sangderk

    2017-01-01

    The upregulated expression of heparin binding EGF-like growth factor (HB-EGF) in the vessel and circulation is associated with risk of cardiovascular disease. In this study, we tested the effects of HB-EGF targeting using HB-EGF-specific antisense oligonucleotide (ASO) on the development of aortic aneurysm in a mouse aneurysm model. Low-density lipoprotein receptor (LDLR) deficient mice (male, 16 weeks of age) were injected with control and HB-EGF ASOs for 10 weeks. To induce aneurysm, the mice were fed a high fat diet (22% fat, 0.2% cholesterol; w/w) at 5 week point of ASO administration and infused with angiotensin II (AngII, 1,000ng/kg/min) for the last 4 weeks of ASO administration. We confirmed that the HB-EGF ASO administration significantly downregulated HB-EGF expression in multiple tissues including the liver. Importantly, the HB-EGF ASO administration significantly suppressed development of aortic aneurysms including thoracic and abdominal types. Interestingly, the HB-EGF ASO administration induced a remarkable anti-hyperlipidemic effect by suppressing very low density lipoprotein (VLDL) level in the blood. Mechanistically, the HB-EGF targeting suppressed hepatic VLDL secretion rate without changing heparin-releasable plasma triglyceride (TG) hydrolytic activity or fecal neutral cholesterol excretion rate. This result suggested that the HB-EGF targeting induced protection against aneurysm development through anti-hyperlipidemic effects. Suppression of hepatic VLDL production process appears to be a key mechanism for the anti-hyperlipidemic effects by the HB-EGF targeting.

  8. A camelid single-domain antibody neutralizes botulinum neurotoxin A by blocking host receptor binding

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Guorui; Lam, Kwok-ho; Weisemann, Jasmin; Peng, Lisheng; Krez, Nadja; Perry, Kay; Shoemaker, Charles B.; Dong, Min; Rummel, Andreas; Jin, Rongsheng (BCH); (Cornell); (Tufts CTSI); (UCI); (MHH)

    2017-08-07

    Antibody treatment is currently the only available countermeasure for botulism, a fatal illness caused by flaccid paralysis of muscles due to botulinum neurotoxin (BoNT) intoxication. Among the seven major serotypes of BoNT/A-G, BoNT/A poses the most serious threat to humans because of its high potency and long duration of action. Prior to entering neurons and blocking neurotransmitter release, BoNT/A recognizes motoneurons via a dual-receptor binding process in which it engages both the neuron surface polysialoganglioside (PSG) and synaptic vesicle glycoprotein 2 (SV2). Previously, we identified a potent neutralizing antitoxin against BoNT/A1 termed ciA-C2, derived from a camelid heavy-chain-only antibody (VHH). In this study, we demonstrate that ciA-C2 prevents BoNT/A1 intoxication by inhibiting its binding to neuronal receptor SV2. Furthermore, we determined the crystal structure of ciA-C2 in complex with the receptor-binding domain of BoNT/A1 (HCA1) at 1.68 Å resolution. The structure revealed that ciA-C2 partially occupies the SV2-binding site on HCA1, causing direct interference of HCA1 interaction with both the N-glycan and peptide-moiety of SV2. Interestingly, this neutralization mechanism is similar to that of a monoclonal antibody in clinical trials, despite that ciA-C2 is more than 10-times smaller. Taken together, these results enlighten our understanding of BoNT/A1 interactions with its neuronal receptor, and further demonstrate that inhibiting toxin binding to the host receptor is an efficient countermeasure strategy.

  9. Sequence similarity between the erythrocyte binding domain of the Plasmodium vivax Duffy binding protein and the V3 loop of HIV-1 strain MN reveals a functional heparin binding motif involved in binding to the Duffy antigen receptor for chemokines

    Directory of Open Access Journals (Sweden)

    Bolton Michael J

    2011-11-01

    Full Text Available Abstract Background The HIV surface glycoprotein gp120 (SU, gp120 and the Plasmodium vivax Duffy binding protein (PvDBP bind to chemokine receptors during infection and have a site of amino acid sequence similarity in their binding domains that often includes a heparin binding motif (HBM. Infection by either pathogen has been found to be inhibited by polyanions. Results Specific polyanions that inhibit HIV infection and bind to the V3 loop of X4 strains also inhibited DBP-mediated infection of erythrocytes and DBP binding to the Duffy Antigen Receptor for Chemokines (DARC. A peptide including the HBM of PvDBP had similar affinity for heparin as RANTES and V3 loop peptides, and could be specifically inhibited from heparin binding by the same polyanions that inhibit DBP binding to DARC. However, some V3 peptides can competitively inhibit RANTES binding to heparin, but not the PvDBP HBM peptide. Three other members of the DBP family have an HBM sequence that is necessary for erythrocyte binding, however only the protein which binds to DARC, the P. knowlesi alpha protein, is inhibited by heparin from binding to erythrocytes. Heparitinase digestion does not affect the binding of DBP to erythrocytes. Conclusion The HBMs of DBPs that bind to DARC have similar heparin binding affinities as some V3 loop peptides and chemokines, are responsible for specific sulfated polysaccharide inhibition of parasite binding and invasion of red blood cells, and are more likely to bind to negative charges on the receptor than cell surface glycosaminoglycans.

  10. Nanodiscs for immobilization of lipid bilayers and membrane receptors: kinetic analysis of cholera toxin binding to a glycolipid receptor

    DEFF Research Database (Denmark)

    Borch, Jonas; Torta, Federico; Sligar, Stephen G

    2008-01-01

    nanodiscs and their incorporated membrane receptors can be attached to surface plasmon resonance sensorchips and used to measure the kinetics of the interaction between soluble molecules and membrane receptors inserted in the bilayer of nanodiscs. Cholera toxin and its glycolipid receptor G(M1) constitute...... a system that can be considered a paradigm for interactions of soluble proteins with membrane receptors. In this work, we have investigated different technologies for capturing nanodiscs containing the glycolipid receptor G(M1) in lipid bilayers, enabling measurements of binding of its soluble interaction...

  11. Structure-based stabilization of HIV-1 gp120 enhances humoral immune responses to the induced co-receptor binding site.

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    Barna Dey

    2009-05-01

    Full Text Available The human immunodeficiency virus type 1 (HIV-1 exterior envelope glycoprotein, gp120, possesses conserved binding sites for interaction with the primary virus receptor, CD4, and also for the co-receptor, generally CCR5. Although gp120 is a major target for virus-specific neutralizing antibodies, the gp120 variable elements and its malleable nature contribute to evasion of effective host-neutralizing antibodies. To understand the conformational character and immunogenicity of the gp120 receptor binding sites as potential vaccine targets, we introduced structure-based modifications to stabilize gp120 core proteins (deleted of the gp120 major variable regions into the conformation recognized by both receptors. Thermodynamic analysis of the re-engineered core with selected ligands revealed significant stabilization of the receptor-binding regions. Stabilization of the co-receptor-binding region was associated with a marked increase in on-rate of ligand binding to this site as determined by surface plasmon resonance. Rabbit immunization studies showed that the conformational stabilization of core proteins, along with increased ligand affinity, was associated with strikingly enhanced humoral immune responses against the co-receptor-binding site. These results demonstrate that structure-based approaches can be exploited to stabilize a conformational site in a large functional protein to enhance immunogenic responses specific for that region.

  12. [Integration of pharmacokinetics and pharmacodynamics based on the in vivo analysis of drug-receptor binding].

    Science.gov (United States)

    Yamada, Shizuo

    2015-01-01

      As I was deeply interested in the effects of drugs on the human body, I chose pharmacology as the subject of special study when I became a 4th year student at Shizuoka College of Pharmacy. I studied abroad as a postdoctoral fellow for two years, from 1978, under the tutelage of Professor Henry I. Yamamura (pharmacology) in the College of Medicine at the University of Arizona, USA. He taught me a variety of valuable skills such as the radioreceptor binding assay, which represented the most advanced technology developed in the US at that time. After returning home, I engaged in clarifying receptor abnormalities in pathological conditions, as well as in drug action mechanisms, by making the best use of this radioreceptor binding assay. In 1989, following the founding of the University of Shizuoka, I was invited by Professor Ryohei Kimura to join the Department of Pharmacokinetics. This switch in discipline provided a good opportunity for me to broaden my perspectives in pharmaceutical sciences. I worked on evaluating drug-receptor binding in vivo as a combined index for pharmacokinetics and pharmacological effect manifestation, with the aim of bridging pharmacology and pharmacokinetics. In fact, by focusing on data from in vivo receptor binding, it became possible to clearly rationalize the important consideration of drug dose-concentration-action relationships, and to study quantitative and kinetic analyses of relationships among pharmacokinetics, receptor binding and pharmacological effects. Based on this concept, I was able to demonstrate the utility of dynamic analyses of drug-receptor binding in drug discovery, drug fostering, and the proper use of pharmacokinetics with regard to many drugs.

  13. Cloning, ligand-binding, and temporal expression of ecdysteroid receptors in the diamondback moth, Plutella xylostella

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    Tang Baozhen

    2012-10-01

    Full Text Available Abstract Background The diamondback moth, Plutella xylostella (L. (Lepidoptera: Plutellidae, is a devastating pest of cruciferous crops worldwide, and has developed resistance to a wide range of insecticides, including diacylhydrazine-based ecdysone agonists, a highly selective group of molt-accelerating biopesticides targeting the ecdysone receptors. Result In this study, we cloned and characterized the ecdysone receptors from P. xylostella, including the two isoforms of EcR and a USP. Sequence comparison and phylogenetic analysis showed striking conservations among insect ecdysone receptors, especially between P. xylostella and other lepidopterans. The binding affinity of ecdysteroids to in vitro-translated receptor proteins indicated that PxEcRB isoform bound specifically to ponasterone A, and the binding affinity was enhanced by co-incubation with PxUSP (Kd =3.0±1.7 nM. In contrast, PxEcRA did not bind to ponasterone A, even in the presence of PxUSP. The expression of PxEcRB were consistently higher than that of PxEcRA across each and every developmental stage, while the pattern of PxUSP expression is more or less ubiquitous. Conclusions Target site insensitivity, in which the altered binding of insecticides (ecdysone agonists to their targets (ecdysone receptors leads to an adaptive response (resistance, is one of the underlying mechanisms of diacylhydrazine resistance. Given the distinct differences at expression level and the ligand-binding capacity, we hypothesis that PxEcRB is the ecdysone receptor that controls the remodeling events during metamorphosis. More importantly, PxEcRB is the potential target site which is modified in the ecdysone agonist-resistant P. xylostella.

  14. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    Energy Technology Data Exchange (ETDEWEB)

    Ishizuka, Toshiaki, E-mail: tishizu@ndmc.ac.jp [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan); Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro [Department of Pharmacology, National Defense Medical College, Tokorozawa, Saitama 359-8513 (Japan)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the DNA synthesis via induction of superoxide. Black-Right-Pointing-Pointer Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. Black-Right-Pointing-Pointer Angiotensin II enhanced differentiation into mesodermal progenitor cells. Black-Right-Pointing-Pointer Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT{sub 1}R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression

  15. Effect of angiotensin II on proliferation and differentiation of mouse induced pluripotent stem cells into mesodermal progenitor cells

    International Nuclear Information System (INIS)

    Ishizuka, Toshiaki; Goshima, Hazuki; Ozawa, Ayako; Watanabe, Yasuhiro

    2012-01-01

    Highlights: ► Treatment with angiotensin II enhanced LIF-induced DNA synthesis of mouse iPS cells. ► Angiotensin II may enhance the DNA synthesis via induction of superoxide. ► Treatment with angiotensin II significantly increased JAK/STAT3 phosphorylation. ► Angiotensin II enhanced differentiation into mesodermal progenitor cells. ► Angiotensin II may enhance the differentiation via activation of p38 MAPK. -- Abstract: Previous studies suggest that angiotensin receptor stimulation may enhance not only proliferation but also differentiation of undifferentiated stem/progenitor cells. Therefore, in the present study, we determined the involvement of the angiotensin receptor in the proliferation and differentiation of mouse induced pluripotent stem (iPS) cells. Stimulation with angiotensin II (Ang II) significantly increased DNA synthesis in mouse iPS cells cultured in a medium with leukemia inhibitory factor (LIF). Pretreatment of the cells with either candesartan (a selective Ang II type 1 receptor [AT 1 R] antagonist) or Tempol (a cell-permeable superoxide scavenger) significantly inhibited Ang II-induced DNA synthesis. Treatment with Ang II significantly increased JAK/STAT3 phosphorylation. Pretreatment with candesartan significantly inhibited Ang II- induced JAK/STAT3 phosphorylation. In contrast, induction of mouse iPS cell differentiation into Flk-1-positive mesodermal progenitor cells was performed in type IV collagen (Col IV)- coated dishes in a differentiation medium without LIF. When Col IV-exposed iPS cells were treated with Ang II for 5 days, the expression of Flk-1 was significantly increased compared with that in the cells treated with the vehicle alone. Pretreatment of the cells with both candesartan and SB203580 (a p38 MAPK inhibitor) significantly inhibited the Ang II- induced increase in Flk-1 expression. Treatment with Ang II enhanced the phosphorylation of p38 MAPK in Col IV- exposed iPS cells. These results suggest that the stimulation

  16. Angiotensin II protects primary rat hepatocytes against bile salt-induced apoptosis.

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    Golnar Karimian

    Full Text Available UNLABELLED: Angiotensin II (AT-II is a pro-fibrotic compound that acts via membrane-bound receptors (AT-1R/AT-2R and thereby activates hepatic stellate cells (HSCs. AT-II receptor blockers (ARBs are thus important candidates in the treatment of liver fibrosis. However, multiple case reports suggest that AT-1R blockers may induce hepatocyte injury. Therefore, we investigated the effect of AT-II and its receptor blockers on cytokine-, oxidative stress- and bile salt-induced cell death in hepatocytes. Primary rat hepatocytes were exposed to TNF-α/Actinomycin D, the ROS-generating agent menadione or the bile salts: glycochenodeoxycholic acid (GCDCA and tauro-lithocholic acid-3 sulfate (TLCS, to induce apoptosis. AT-II (100 nmol/L was added 10 minutes prior to the cell death-inducing agent. AT-1R antagonists (Sartans and the AT-2R antagonist PD123319 were used at 1 µmol/L. Apoptosis (caspase-3 activity, acridine orange staining and necrosis (Sytox green staining were quantified. Expression of CHOP (marker for ER stress and AT-II receptor mRNAs were quantified by Q-PCR. AT-II dose-dependently reduced GCDCA-induced apoptosis of hepatocytes (-50%, p<0.05 without inducing necrosis. In addition, AT-II reduced TLCS-induced apoptosis of hepatocytes (-50%, p<0.05. However, AT-II did not suppress TNF/Act-D and menadione-induced apoptosis. Only the AT-1R antagonists abolished the protective effect of AT-II against GCDCA-induced apoptosis. AT-II increased phosphorylation of ERK and a significant reversal of the protective effect of AT-II was observed when signaling kinases, including ERK, were inhibited. Moreover, AT-II prevented the GCDCA-induced expression of CHOP (the marker of the ER-mediated apoptosis. CONCLUSION: Angiotensin II protects hepatocytes from bile salt-induced apoptosis through a combined activation of PI3-kinase, MAPKs, PKC pathways and inhibition of bile salt-induced ER stress. Our results suggest a mechanism for the observed hepatocyte

  17. Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

    Science.gov (United States)

    Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

    2017-08-01

    While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

  18. Mood stabilizer treatment increases serotonin type 1A receptor binding in bipolar depression

    Science.gov (United States)

    Nugent, Allison C; Carlson, Paul J; Bain, Earle E; Eckelman, William; Herscovitch, Peter; Manji, Husseini; Zarate, Carlos A; Drevets, Wayne C

    2013-01-01

    Abnormal serotonin type 1A (5-HT1A) receptor function and binding have been implicated in the pathophysiology of mood disorders. Preclinical studies have consistently shown that stress decreases the gene expression of 5-HT1A receptors in experimental animals, and that the associated increase in hormone secretion plays a crucial role in mediating this effect. Chronic administration of the mood stabilizers lithium and divalproex (valproate semisodium) reduces glucocorticoid signaling and function in the hippocampus. Lithium has further been shown to enhance 5-HT1A receptor function. To assess whether these effects translate to human subject with bipolar disorder (BD), positron emission tomography (PET) and [18F]trans-4-fluoro-N-(2-[4-(2-methoxyphenyl) piperazino]-ethyl)-N-(2-pyridyl) cyclohexanecarboxamide ([18F]FCWAY) were used to acquire PET images of 5-HT1A receptor binding in 10 subjects with BD, before and after treatment with lithium or divalproex. Mean 5-HT1A binding potential (BPP) significantly increased following mood stabilizer treatment, most prominently in the mesiotemporal cortex (hippocampus plus amygdala). When mood state was also controlled for, treatment was associated with increases in BPP in widespread cortical areas. These preliminary findings are consistent with the hypothesis that these mood stabilizers enhance 5-HT1A receptor expression in BD, which may underscore an important component of these agents' mechanism of action. PMID:23926239

  19. In vitro receptor autoradiography reveals angiotensin IL (ANG II) binding associated with sensory and motor components of the vagus

    International Nuclear Information System (INIS)

    Diz, D.I.; Barnes, K.L.; Ferrario, C.M.

    1986-01-01

    Specific, high affinity Ang II binding in the dog's dorsal medulla is concentrated in the area postrema, nucleus tractus solitarii (nTS) and dorsal motor nucleus of the vagus (dmnX). More recently Ang II binding sites were observed where bundles of vagal afferent fibers enter the dorsal medulla 6 mm rostral to obex and in the nodose ganglia and peripheral vagal nerves. Since Ang II binding in the nTS and dmnX overlies the distribution of vagal afferent fibers and efferent neurons, the effects of nodose ganglionectomy and cervical vagotomy on Ang II binding in the dorsal medulla were studied in rats and dogs using autoradiography after incubation of 14 μm coronal sections with 0.4 nM 125 I-Ang II. Nonspecific binding was determined in the presence of 1 μm unlabeled Ang II. Two weeks after unilateral nodose ganglionectomy Ang II binding sites were absent ipsilaterally in the region where vagal afferent fibers enter the dorsal medulla. In the nTS and dmnX, binding near obex was reduced, while more rostrally these nuclei were almost completely devoid of Ang II binding on the denervated side. After cervical vagotomy, the loss of binding was restricted to the ipsilateral dmnX. These data are the first to reveal that Ang II binding in the dorsal medulla requires an intact vagal system

  20. Overexpression of ß-Arrestin1 in the Rostral Ventrolateral Medulla Downregulates Angiotensin Receptor and Lowers Blood Pressure in Hypertension

    Directory of Open Access Journals (Sweden)

    Jia-Cen Sun

    2018-03-01

    Full Text Available Background: Hypertension is characterized by sympathetic overactivity, which is associated with an enhancement in angiotensin receptor type I (AT1R in the rostral ventrolateral medulla (RVLM. β-arrestin1, a canonical scaffold protein, has been suggested to show a negative effect on G protein-coupled receptors via its internalization and desensitization and/or the biased signaling pathway. The major objectives of the present study were to observe the effect of β-arrestin1 overexpression in the RVLM on cardiovascular regulation in spontaneously hypertensive rats (SHR, and further determine the effect of β-arrestin1 on AT1R expression in the RVLM.Methods: The animal model of β-arrestin1 overexpression was induced by bilateral injection of adeno-associated virus containing Arrb1 gene (AAV-Arrb1 into the RVLM of WKY and SHR.Results: β-arrestin1 was expressed on the pre-sympathetic neurons in the RVLM, and its expression in the RVLM was significantly (P < 0.05 downregulated by an average of 64% in SHR than WKY. Overexpression of β-arrestin1 in SHR significantly decreased baseline levels of blood pressure and renal sympathetic nerve activity, and attenuated cardiovascular effects induced by RVLM injection of angiotensin II (100 pmol. Furthermore, β-arrestin1 overexpression in the RVLM significantly reduced the expression of AT1R by 65% and NF-κB p65 phosphorylation by 66% in SHR. It was confirmed that β-arrestin1 overexpression in the RVLM led to an enhancement of interaction between β-arrestin1 and IκB-α.Conclusion: Overexpression of β-arrestin1 in the RVLM reduces BP and sympathetic outflow in hypertension, which may be associated with NFκB-mediated AT1R downregulation.

  1. Interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway in renal carcinogenesis of uninephrectomized rats.

    Science.gov (United States)

    Yang, Ke-Ke; Sui, Yi; Zhou, Hui-Rong; Zhao, Hai-Lu

    2017-05-01

    Renin-angiotensin system and adenosine monophosphate-activated protein kinase signaling pathway both play important roles in carcinogenesis, but the interplay of renin-angiotensin system and adenosine monophosphate-activated protein kinase in carcinogenesis is not clear. In this study, we researched the interaction of renin-angiotensin system and adenosine monophosphate-activated protein kinase in renal carcinogenesis of uninephrectomized rats. A total of 96 rats were stratified into four groups: sham, uninephrectomized, and uninephrectomized treated with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker. Renal adenosine monophosphate-activated protein kinase and its downstream molecule acetyl coenzyme A carboxylase were detected by immunohistochemistry and western blot at 10 months after uninephrectomy. Meanwhile, we examined renal carcinogenesis by histological transformation and expressions of Ki67 and mutant p53. During the study, fasting lipid profiles were detected dynamically at 3, 6, 8, and 10 months. The results indicated that adenosine monophosphate-activated protein kinase expression in uninephrectomized rats showed 36.8% reduction by immunohistochemistry and 89.73% reduction by western blot. Inversely, acetyl coenzyme A carboxylase expression increased 83.3% and 19.07% in parallel to hyperlipidemia at 6, 8, and 10 months. The histopathology of carcinogenesis in remnant kidneys was manifested by atypical proliferation and carcinoma in situ, as well as increased expressions of Ki67 and mutant p53. Intervention with angiotensin-converting enzyme inhibitor or angiotensin receptor blocker significantly prevented the inhibition of adenosine monophosphate-activated protein kinase signaling pathway and renal carcinogenesis in uninephrectomized rats. In conclusion, the novel findings suggest that uninephrectomy-induced disturbance in adenosine monophosphate-activated protein kinase signaling pathway resulted in hyperlipidemia and

  2. Intracerebroventricular Infusion of the (Pro)renin Receptor Antagonist PRO20 Attenuates Deoxycorticosterone Acetate-Salt–Induced Hypertension

    Science.gov (United States)

    Li, Wencheng; Sullivan, Michelle N.; Zhang, Sheng; Worker, Caleb J.; Xiong, Zhenggang; Speth, Robert C.; Feng, Yumei

    2016-01-01

    We previously reported that binding of prorenin to the (pro)renin receptor (PRR) plays a major role in brain angiotensin II formation and the development of deoxycorticosterone acetate (DOCA)-salt hypertension. Here, we designed and developed an antagonistic peptide, PRO20, to block prorenin binding to the PRR. Fluorescently labeled PRO20 bound to both mouse and human brain tissues with dissociation constants of 4.4 and 1.8 nmol/L, respectively. This binding was blocked by coincubation with prorenin and was diminished in brains of neuron-specific PRR-knockout mice, indicating specificity of PRO20 for PRR. In cultured human neuroblastoma cells, PRO20 blocked prorenin-induced calcium influx in a concentration- and AT1 receptor–dependent manner. Intracerebroventricular infusion of PRO20 dose-dependently inhibited prorenin-induced hypertension in C57Bl6/J mice. Furthermore, acute intracerebroventricular infusion of PRO20 reduced blood pressure in both DOCA-salt and genetically hypertensive mice. Chronic intracerebroventricular infusion of PRO20 attenuated the development of hypertension and the increase in brain hypothalamic angiotensin II levels induced by DOCA-salt. In addition, chronic intracerebroventricular infusion of PRO20 improved autonomic function and spontaneous baroreflex sensitivity in mice treated with DOCA-salt. In summary, PRO20 binds to both mouse and human PRRs and decreases angiotensin II formation and hypertension induced by either prorenin or DOCA-salt. Our findings highlight the value of the novel PRR antagonist, PRO20, as a lead compound for a novel class of antihypertensive agents and as a research tool to establish the validity of brain PRR antagonism as a strategy for treating hypertension. PMID:25421983

  3. Single-molecule photobleaching reveals increased MET receptor dimerization upon ligand binding in intact cells

    International Nuclear Information System (INIS)

    Dietz, Marina S; Haße, Daniel; Ferraris, Davide M; Göhler, Antonia; Niemann, Hartmut H; Heilemann, Mike

    2013-01-01

    The human receptor tyrosine kinase MET and its ligand hepatocyte growth factor/scatter factor are essential during embryonic development and play an important role during cancer metastasis and tissue regeneration. In addition, it was found that MET is also relevant for infectious diseases and is the target of different bacteria, amongst them Listeria monocytogenes that induces bacterial uptake through the surface protein internalin B. Binding of ligand to the MET receptor is proposed to lead to receptor dimerization. However, it is also discussed whether preformed MET dimers exist on the cell membrane. To address these issues we used single-molecule fluorescence microscopy techniques. Our photobleaching experiments show that MET exists in dimers on the membrane of cells in the absence of ligand and that the proportion of MET dimers increases significantly upon ligand binding. Our results indicate that partially preformed MET dimers may play a role in ligand binding or MET signaling. The addition of the bacterial ligand internalin B leads to an increase of MET dimers which is in agreement with the model of ligand-induced dimerization of receptor tyrosine kinases.

  4. Histamine type I (H1) receptor radioligand binding studies on normal T cell subsets, B cells, and monocytes

    International Nuclear Information System (INIS)

    Cameron, W.; Doyle, K.; Rocklin, R.E.

    1986-01-01

    A single, specific binding site for [ 3 H]pyrilamine on normal human T helper, T suppressor, B cells, and monocytes was documented. The binding of the radioligand to its receptor is reversible with cold H 1 antagonist, saturates at 40 to 60 nM, and binding equilibrium is achieved in 2 to 4 min. Using a computer program (Ligand), the authors calculated the dissociation constants, binding capacities, and numbers of receptors per cell for each of the different cell types. Monocytes were found to have the highest affinity for [ 3 H]pyrilamine, followed by T helper cells, B cells and T suppressor cells (K/sub D/ = 44.6 +/- 49.4 nM). T suppressor cells were found to express the higher number of H 1 receptors per cell followed by B cells, T helper cells, and monocytes. The binding affinity for [ 3 H]pyrilamine increased over a 48-hr period, whereas the number of receptors per T cell was essentially unchanged. In contrast, T cells stimulated with Con A or PHA were shown to have a greater than fourfold increase in the number of receptors per cell, whereas the binding affinity for [ 3 H]pyrilamine decreased over the 48-hr period. Although the function of H 1 receptors on T cells, B cells, and monocytes has not been completely defined, this receptor has the potential of playing an important role in the modulating the immune response

  5. Estrogen receptor determination in endometrial carcinoma: ligand binding assay versus enzyme immunoassay

    DEFF Research Database (Denmark)

    Nyholm, H C; Nielsen, Anette Lynge; Lyndrup, J

    1995-01-01

    We compared concentrations of cytosolic estrogen receptors (ERc) measured in 35 postmenopausal endometrial carcinomas by ligand binding method (LBA) (dextran-coated charcoal assay) and enzyme immunoassay (EIA). Correlations between ERc, nuclear estrogen receptors (ERn) determined by EIA......, and cytosolic progesterone receptors (PR) measured by LBA were also studied. While ERc concentrations determined by LBA and EIA were highly correlated (r: 0.94), ERc values detected by LBA were approximately twice those found by EIA (median values of ERc: 155 vs. 64 fmol/mg cytosol protein, DCC vs. EIA......). The percentages of ERc positive tumors were 89% by LBA and 77% by EIA. The median fraction of total ER present as ERn was 63%. PR levels correlated positively with ERn concentrations (r: 0.73). We explore possible reasons why greater concentrations of ERc are determined by estradiol binding than by the ER-EIA kit...

  6. Label-Free, LC-MS-Based Assays to Quantitate Small-Molecule Antagonist Binding to the Mammalian BLT1 Receptor.

    Science.gov (United States)

    Chen, Xun; Stout, Steven; Mueller, Uwe; Boykow, George; Visconti, Richard; Siliphaivanh, Phieng; Spencer, Kerrie; Presland, Jeremy; Kavana, Michael; Basso, Andrea D; McLaren, David G; Myers, Robert W

    2017-08-01

    We have developed and validated label-free, liquid chromatography-mass spectrometry (LC-MS)-based equilibrium direct and competition binding assays to quantitate small-molecule antagonist binding to recombinant human and mouse BLT1 receptors expressed in HEK 293 cell membranes. Procedurally, these binding assays involve (1) equilibration of the BLT1 receptor and probe ligand, with or without a competitor; (2) vacuum filtration through cationic glass fiber filters to separate receptor-bound from free probe ligand; and (3) LC-MS analysis in selected reaction monitoring mode for bound probe ligand quantitation. Two novel, optimized probe ligands, compounds 1 and 2, were identified by screening 20 unlabeled BLT1 antagonists for direct binding. Saturation direct binding studies confirmed the high affinity, and dissociation studies established the rapid binding kinetics of probe ligands 1 and 2. Competition binding assays were established using both probe ligands, and the affinities of structurally diverse BLT1 antagonists were measured. Both binding assay formats can be executed with high specificity and sensitivity and moderate throughput (96-well plate format) using these approaches. This highly versatile, label-free method for studying ligand binding to membrane-associated receptors should find broad application as an alternative to traditional methods using labeled ligands.

  7. Analysis of fluorescently labeled substance P analogs: binding, imaging and receptor activation

    Directory of Open Access Journals (Sweden)

    Simmons Mark A

    2001-06-01

    Full Text Available Abstract Background Substance P (SP is a peptide neurotransmitter found in central and peripheral nerves. SP is involved in the control of smooth muscle, inflammation and nociception. The amino acid sequence of SP is Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2. Five different forms of fluorescently labeled SP have recently been synthesized, in which Alexa 488, BODIPY Fl, fluorescein, Oregon Green 488 or tetramethylrhodamine has been covalently linked to SP at Lys3. Here, these novel analogs are characterized as to their ligand binding, receptor activation and fluorescence labeling properties. Results Competition binding studies, using radiolabeled [125I] SP, revealed that all of the labeled forms of SP, except for Alexa 488-SP, effectively competed with radiolabeled SP for binding at the rat SP receptor. With the exception of Alexa 488-SP, all of the SP analogs produced Ca++ elevations and fluorescence labeling of the SP receptor expressed in Chinese hamster ovary cells. In SP-responsive neurons, BODIPY Fl-SP and Oregon Green 488-SP were as effective as unlabeled SP in producing a reduction of the M-type K+ current. Fluorescein-SP produced variable results, while tetramethylrhodamine-SP was less potent and Alexa 488-SP was less effective on intact neurons. Conclusions The above results show that fluorescent labeling of SP altered the biological activity and the binding properties of the parent peptide. Oregon Green 488 and BODIPY FL-SP are the most useful fluorophores for labeling SP without affecting its biological activity. Given these results, these probes can now be utilized in further investigations of the mechanisms of SPR function, including receptor localization, internalization and recycling.

  8. Reduction of GABA/sub B/ receptor binding induced by climbing fiber degeneration in the rat cerebellum

    International Nuclear Information System (INIS)

    Kato, K.; Fukuda, H.

    1985-01-01

    When the rat cerebellar climbing fibers degenerated, as induced by lesioning the inferior olive with 3-acetylpyridine (3-AP), GABA/sub B/ receptor binding determined with 3 H-(+/-)baclofen was reduced in the cerebellum but not in the cerebral cortex of rats. Computer analysis of saturation data revealed two components of the binding sites, and indicated that decrease of the binding in the cerebellum was due to reduction in receptor density, mainly of the high-affinity sites, the B/sub max/ of which was reduced to one-third that in the control animals. In vitro treatment with 3-AP, of the membranes prepared from either the cerebellum or the cerebral cortex, induced no alteration in the binding sites, thereby indicating that the alteration of GABA/sub B/ sites induced by in vivo treatment with 3-AP is not due to a direct action of 3-AP on the receptor. GABA/sub A/ and benzodiazepine receptor binding labelled with 3 H-muscimol and 3 H-diazepam, respectively, in both of brain regions was not affected by destruction of the inferior olive. These results provide evidence that some of the GABA/sub B/ sites but neither GABA/sub A/ nor benzodiazepine receptors in the cerebellum are located at the climbing fiber terminals. 28 references, 4 figures, 2 tables

  9. Diversifying selection and functional analysis of interleukin-4 suggests antagonism-driven evolution at receptor-binding interfaces

    Directory of Open Access Journals (Sweden)

    Brown Scott

    2010-07-01

    Full Text Available Abstract Background Interleukin-4 (IL4 is a secreted immunoregulatory cytokine critically involved in host protection from parasitic helminths 1. Reasoning that helminths may have evolved mechanisms to antagonize IL4 to maximize their dispersal, we explored mammalian IL4 evolution. Results This analysis revealed evidence of diversifying selection at 15 residues, clustered in epitopes responsible for IL4 binding to its Type I and Type II receptors. Such a striking signature of selective pressure suggested either recurrent episodes of pathogen antagonism or ligand/receptor co-evolution. To test the latter possibility, we performed detailed functional analysis of IL4 allotypes expressed by Mus musculus musculus and Mus musculus castaneus, which happen to differ at 5 residues (including three at positively selected sites in and adjacent to the site 1 epitope that binds the IL4Rα subunit shared by the Type I and Type II IL4 receptors. We show that this intra-species variation affects the ability of IL4 neither to bind IL4 receptor alpha (IL4Rα nor to signal biological responses through its Type I receptor. Conclusions Our results -- reminiscent of clustered positively selected sites revealing functionally important residues at host-virus interaction interfaces -- are consistent with IL4 having evolved to avoid recurrent pathogen antagonism, while maintaining the capacity to bind and signal through its cognate receptor. This work exposes what may be a general feature of evolutionary conflicts fought by pathogen antagonists at host protein-protein interaction interfaces involved in immune signaling: the emergence of receptor-binding ligand epitopes capable of buffering amino acid variation.

  10. Competitive receptor binding radioassay for β-1 and β-2 adrenergic agents

    International Nuclear Information System (INIS)

    Hussain, M.N.; Culbreth, W.; Dalrymple, R.; Fung, C.; Ricks, C.

    1987-01-01

    A rapid and sensitive competitive receptor bonding assay for β-1 and β-2 adrenergic binding for adrenergic agents has been developed. The steps that are critical for the success of the assay are given in detail so that the assay can be set up in any routine laboratory with relative ease. The rationale behind the use of specific reagents is discussed. The assay requires microgram quantities of test compound, a radiolabeled specific β adrenergic antagonist [ 3 H]dihydroalprenolol (DHA), and turkey erythrocyte β-1 and rat erythrocyte β-2 receptor membranes. Serial dilutions of sample are incubated with appropriate receptor membranes and DHA for 1 hr at room temperature. After equilibrium is attained, the bound radioligand is separated by rapid filtration under vacuum through Whatman GF/B filters. The amount of bound DHA trapped on the filter is inversely proportional to the degree of β-1 and β-2 adrenergic binding of the sample. Separation of bound from free radioligand by filtration permits rapid determination of a large number of samples. This assay quantitates and differentiates β-1 and β-2 adrenergic binding of synthetic adrenergic agents

  11. Regulation of the renin–angiotensin system in coronary atherosclerosis: A review of the literature

    Directory of Open Access Journals (Sweden)

    Ramadan A Hammoud

    2008-01-01

    Full Text Available Ramadan A Hammoud, Christopher S Vaccari, Sameer H Nagamia, Bobby V KhanEmory University School of Medicine, Division of Cardiology, Grady Memorial Hospital Vascular Research Laboratory, Atlanta, Georgia, USAAbstract: Activation of the renin–angiotensin system (RAS is significant in the pathogenesis of cardiovascular disease and specifically coronary atherosclerosis. There is strong evidence that the RAS has effects on the mechanisms of action of atherosclerosis, including fibrinolytic balance, endothelial function, and plaque stability. Pharmacological inhibition of the renin angiotensin system includes angiotensin converting enzyme (ACE inhibitors, angiotensin receptor blockers (ARBs, and renin inhibitors. These agents have clinical benefits in reducing morbidity and mortality in the management of hypertension. In addition, ACE inhibitors and ARBs have shown to be effective in the management of congestive heart failure and acute myocardial infarction. This review article discusses the biochemical and molecular mechanisms involving the RAS in coronary atherosclerosis as well as the effects of RAS inhibition in clinical studies involving coronary atherosclerosis.Keywords: angiotensin II, atherosclerosis, endothelium, inflammation, vasculature

  12. Testin, a novel binding partner of the calcium-sensing receptor, enhances receptor-mediated Rho-kinase signalling

    International Nuclear Information System (INIS)

    Magno, Aaron L.; Ingley, Evan; Brown, Suzanne J.; Conigrave, Arthur D.; Ratajczak, Thomas; Ward, Bryan K.

    2011-01-01

    Highlights: → A yeast two-hybrid screen revealed testin bound to the calcium-sensing receptor. → The second zinc finger of LIM domain 1 of testin is critical for interaction. → Testin bound to a region of the receptor tail important for cell signalling. → Testin and receptor interaction was confirmed in mammalian (HEK293) cells. → Overexpression of testin enhanced receptor-mediated Rho signalling in HEK293 cells. -- Abstract: The calcium-sensing receptor (CaR) plays an integral role in calcium homeostasis and the regulation of other cellular functions including cell proliferation and cytoskeletal organisation. The multifunctional nature of the CaR is manifested through ligand-dependent stimulation of different signalling pathways that are also regulated by partner binding proteins. Following a yeast two-hybrid library screen using the intracellular tail of the CaR as bait, we identified several novel binding partners including the focal adhesion protein, testin. Testin has not previously been shown to interact with cell surface receptors. The sites of interaction between the CaR and testin were mapped to the membrane proximal region of the receptor tail and the second zinc-finger of LIM domain 1 of testin, the integrity of which was found to be critical for the CaR-testin interaction. The CaR-testin association was confirmed in HEK293 cells by coimmunoprecipitation and confocal microscopy studies. Ectopic expression of testin in HEK293 cells stably expressing the CaR enhanced CaR-stimulated Rho activity but had no effect on CaR-stimulated ERK signalling. These results suggest an interplay between the CaR and testin in the regulation of CaR-mediated Rho signalling with possible effects on the cytoskeleton.

  13. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Directory of Open Access Journals (Sweden)

    Person Alexandra M

    2011-11-01

    Full Text Available Abstract Background Along with high affinity binding of epibatidine (Kd1≈10 pM to α4β2 nicotinic acetylcholine receptor (nAChR, low affinity binding of epibatidine (Kd2≈1-10 nM to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after

  14. Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

    Science.gov (United States)

    2011-01-01

    Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of

  15. Insulin receptor binding and protein kinase activity in muscles of trained rats

    International Nuclear Information System (INIS)

    Dohm, G.L.; Sinha, M.K.; Caro, J.F.

    1987-01-01

    Exercise has been shown to increase insulin sensitivity, and muscle is quantitatively the most important tissue of insulin action. Since the first step in insulin action is the binding to a membrane receptor, the authors postulated that exercise training would change insulin receptors in muscle and in this study they have investigated this hypothesis. Female rats initially weighing ∼ 100 g were trained by treadmill running for 2 h/day, 6 days/wk for 4 wk at 25 m/min (0 grade). Insulin receptors from vastus intermedius muscles were solubilized by homogenizing in a buffer containing 1% Triton X-100 and then partially purified by passing the soluble extract over a wheat germ agglutinin column. The 4 wk training regimen resulted in a 65% increase in citrate synthase activity in red vastus lateralis muscle, indicating an adaptation to exercise [ 125 I]. Insulin binding by the partially purified receptor preparations was approximately doubled in muscle of trained rats at all insulin concentrations, suggesting an increase in the number of receptors. Training did not alter insulin receptor structure as evidenced by electrophoretic mobility under reducing and nonreducing conditions. Basal insulin receptor protein kinase activity was higher in trained than untrained animals and this was likely due to the greater number of receptors. However, insulin stimulation of the protein kinase activity was depressed by training. These results demonstrate that endurance training does alter receptor number and function in muscle and these changes may be important in increasing insulin sensitivity after exercise training

  16. A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

    Science.gov (United States)

    Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

    2011-05-01

    A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

  17. Circadian rhythm of urinary potassium excretion during treatment with an angiotensin receptor blocker.

    Science.gov (United States)

    Ogiyama, Yoshiaki; Miura, Toshiyuki; Watanabe, Shuichi; Fuwa, Daisuke; Tomonari, Tatsuya; Ota, Keisuke; Kato, Yoko; Ichikawa, Tadashi; Shirasawa, Yuichi; Ito, Akinori; Yoshida, Atsuhiro; Fukuda, Michio; Kimura, Genjiro

    2014-12-01

    We have reported that the circadian rhythm of urinary potassium excretion (U(K)V) is determined by the rhythm of urinary sodium excretion (U(Na)V) in patients with chronic kidney disease (CKD). We also reported that treatment with an angiotensin receptor blocker (ARB) increased the U(Na)V during the daytime, and restored the non-dipper blood pressure (BP) rhythm into a dipper pattern. However, the circadian rhythm of U(K)V during ARB treatment has not been reported. Circadian rhythms of U(Na)V and U(K)V were examined in 44 patients with CKD undergoing treatment with ARB. Whole-day U(Na)V was not altered by ARB whereas whole-day U(K)V decreased. Even during the ARB treatment, the significant relationship persisted between the night/day ratios of U(Na)V and U(K)V (r=0.56, pcircadian rhythm of U(K)V was determined by the rhythm of UNaV even during ARB treatment. Changes in the circadian U(K)V rhythm were not determined by aldosterone but by U(Na)V. © The Author(s) 2013.

  18. The minor binding pocket: a major player in 7TM receptor activation

    DEFF Research Database (Denmark)

    Rosenkilde, Mette Marie; Benned-Jensen, Tau; Frimurer, Thomas M.

    2010-01-01

    residue located in one of two adjacent positions. Here we argue that this minor binding pocket is important for receptor activation. Functional coupling of the receptors seems to be mediated through the hydrogen bond network located between the intracellular segments of these TMs, with the allosteric...... targeted in the development of functionally biased drugs....

  19. Harmaline competitively inhibits [3H]MK-801 binding to the NMDA receptor in rabbit brain.

    Science.gov (United States)

    Du, W; Aloyo, V J; Harvey, J A

    1997-10-03

    Harmaline, a beta-carboline derivative, is known to produce tremor through a direct activation of cells in the inferior olive. However, the receptor(s) through which harmaline acts remains unknown. It was recently reported that the tremorogenic actions of harmaline could be blocked by the noncompetitive NMDA channel blocker, MK-801. This study examined whether the blockade of harmaline's action, in the rabbit, by MK-801 was due to a pharmacological antagonism at the MK-801 binding site. This was accomplished by measurement of [3H]MK-801 binding in membrane fractions derived from tissue containing the inferior olivary nucleus and from cerebral cortex. Harmaline completely displaced saturable [3H]MK-801 binding in both the inferior olive and cortex with apparent IC50 values of 60 and 170 microM, respectively. These IC50 values are consistent with the high doses of harmaline required to produce tremor, e.g., 10-30 mg/kg. Non-linear curve fitting analysis of [3H]MK-801 saturation experiments indicated that [3H]MK-801 bound to a single site and that harmaline's displacement of [3H]MK-801 binding to the NMDA receptor was competitive as indicated by a shift in Kd but not in Bmax. In addition, a Schild plot gave a slope that was not significantly different from 1 indicating that harmaline was producing a displacement of [3H]MK-801 from its binding site within the NMDA cation channel and not through an action at the glutamate or other allosteric sites on the NMDA receptor. These findings provide in vitro evidence that the competitive blockade of harmaline-induced tremor by MK-801 occurs within the calcium channel coupled to the NMDA receptor. Our hypothesis is that harmaline produces tremor by acting as an inverse agonist at the MK-801 binding site and thus opening the cation channel.

  20. Serum creatinine elevation after renin-angiotensin system blockade and long term cardiorenal risks: cohort study.

    Science.gov (United States)

    Schmidt, Morten; Mansfield, Kathryn E; Bhaskaran, Krishnan; Nitsch, Dorothea; Sørensen, Henrik Toft; Smeeth, Liam; Tomlinson, Laurie A

    2017-03-09

    Objective  To examine long term cardiorenal outcomes associated with increased concentrations of creatinine after the start of angiotensin converting enzyme inhibitor/angiotensin receptor blocker treatment. Design  Population based cohort study using electronic health records from the Clinical Practice Research Datalink and Hospital Episode Statistics. Setting  UK primary care, 1997-2014. Participants  Patients starting treatment with angiotensin converting enzyme inhibitors or angiotensin receptor blockers (n=122 363). Main outcome measures  Poisson regression was used to compare rates of end stage renal disease, myocardial infarction, heart failure, and death among patients with creatinine increases of 30% or more after starting treatment against those without such increases, and for each 10% increase in creatinine. Analyses were adjusted for age, sex, calendar period, socioeconomic status, lifestyle factors, chronic kidney disease, diabetes, cardiovascular comorbidities, and use of other antihypertensive drugs and non-steroidal anti-inflammatory drugs. Results  Among the 2078 (1.7%) patients with creatinine increases of 30% or more, a higher proportion were female, were elderly, had cardiorenal comorbidity, and used non-steroidal anti-inflammatory drugs, loop diuretics, or potassium sparing diuretics. Creatinine increases of 30% or more were associated with an increased adjusted incidence rate ratio for all outcomes, compared with increases of less than 30%: 3.43 (95% confidence interval 2.40 to 4.91) for end stage renal disease, 1.46 (1.16 to 1.84) for myocardial infarction, 1.37 (1.14 to 1.65) for heart failure, and 1.84 (1.65 to 2.05) for death. The detailed categorisation of increases in creatinine concentrations (creatinine increases of less than 30% were also associated with increased incidence rate ratios for all outcomes, including death (1.15 (1.09 to 1.22) for increases of 10-19% and 1.35 (1.23 to 1.49) for increases of 20-29%, using creatinine