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Sample records for d3 receptors binding

  1. 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.

  2. Interactions of dopaminergic agonists and antagonists with dopaminergic D3 binding sites in rat striatum. Evidence that [3H]dopamine can label a high affinity agonist-binding state of the D1 dopamine receptor

    International Nuclear Information System (INIS)

    Leff, S.E.; Creese, I.

    1985-01-01

    The interactions of dopaminergic agonists and antagonists with 3 H-agonist labeled D3 dopaminergic binding sites of rat striatum have been characterized by radioligand-binding techniques. When the binding of [ 3 H]dopamine and [ 3 H]apomorphine to D2 dopamine receptors is blocked by the inclusion of D2 selective concentrations of unlabeled spiroperidol or domperidone, these ligands appear to label selectively the previously termed D3 binding site. Antagonist/[ 3 H]dopamine competition curves are of uniformly steep slope (nH . 1.0), suggesting the presence of a single D3 binding site. The relative potencies of antagonists to inhibit D3 specific [ 3 H]dopamine binding are significantly correlated with their potencies to block D1 dopamine receptors as measured by the inhibition of both dopamine-stimulated adenylate cyclase and [ 3 H]flupentixol-binding activities. The affinities of agonists to inhibit D3 specific [ 3 H]dopamine binding are also correlated with estimates of these agonists affinities for the high affinity binding component of agonist/[ 3 H]flupentixol competition curves. Both D3 specific [ 3 H] dopamine binding and the high affinity agonist-binding component of dopamine/[ 3 H]flupentixol competition curves show a similar sensitivity to guanine nucleotides. Taken together, these data strongly suggest that the D3 binding site is related to a high affinity agonist-binding state of the D1 dopamine receptor

  3. cis- and trans-2,3,3a,4,5,9b-Hexahydro-1H-benz[e]indoles: synthesis and evaluation of dopamine D2, and D3 receptor binding affinity

    DEFF Research Database (Denmark)

    Song, Xiaodong; Crider, Michael A.; Cruse, Sharon F.

    1999-01-01

    cis- and trans-2,3,3a,4,5,9b-hexahydro-1H-benz [e]indoles were synthesized as conformationally rigid analogues of 3-phenylpyrrolidine and evaluated for dopamine (DA) D2S and D3 receptor binding affinity. The tricyclic benz[e]indole nucleus was constructed by a previously reported reductive...... configuration. These novel ligands may be useful tools for gaining additional information about the DA D3 receptor. Copyright Elsevier, Paris.dopamine / D2S receptor / D3 receptor / cis- and trans-2,3,3a,4,5,9b-hexahydro-1H-benz[e]indoles / receptor binding affinity....... receptors was shown by compounds substituted with N-n-propyl or N-allyl groups. The cis-(+-)-N-allyl derivative 21e demonstrated a D2S/D3 selectivity of 290. Resolution of cis-(+-)-5 and trans-(+-)- 21c into individual enantiomers showed that in both series the more active isomer had 3aR absolute...

  4. Relationship of frontal D2/3 binding potentials to cognition

    DEFF Research Database (Denmark)

    Fagerlund, Birgitte; Pinborg, Lars H; Mortensen, Erik Lykke

    2013-01-01

    for set shifting. The main findings indicated a relation between D2/3 receptor binding in the frontal cortex and set shifting, planning and attention, but also support a differential involvement of cortical dopamine D2/3 receptor binding in at least some cognitive functions, perhaps particularly attention......Studies of in vivo dopamine receptors in schizophrenia have mostly focused on D2 receptors in striatal areas or on D1 receptors in cortex. No previous study has examined the correlation between cortical dopamine D2/3 receptor binding potentials and cognition in schizophrenia patients. The objective......, in schizophrenia patients compared to healthy people. The results suggest that cortical D2/3 receptor function may be more involved in some cognitive functions (i.e. attention, fluency and planning) in patients with schizophrenia than in healthy people, suggesting that information processing in schizophrenia may...

  5. [123I]epidepride binding to cerebellar dopamine D2/D3 receptors is displaceable: implications for the use of cerebellum as a reference region

    DEFF Research Database (Denmark)

    Pinborg, Lars H; Videbaek, Charlotte; Ziebell, Morten

    2007-01-01

    The low density of cerebellar dopamine D(2)/D(3) receptors provides the basis for using the cerebellum as a representation of free- and non-specifically bound radioligand in positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies. With the development...... of ultra high-affinity dopamine D(2)/D(3) ligands like [(123)I]epidepride, [(18)F]fallypride, and [(11)C]FLB-457, quantification of extrastriatal low density receptor populations including the cerebellum is possible with important implications for calculation of binding parameters. [(123)I...... [(123)I]epidepride binding to dopamine D(2)/D(3) receptors in the cerebellum. Using the cerebellum as a representation of free and non-specifically bound radioligand and neglecting the specifically bound component may lead to results that erroneously imply that antipsychotic drugs bind to extrastriatal...

  6. 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.

  7. Structure of the Human Dopamine D3 Receptor in Complex with a D2/D3 Selective Antagonist

    Energy Technology Data Exchange (ETDEWEB)

    Chien, Ellen Y.T.; Liu, Wei; Zhao, Qiang; Katritch, Vsevolod; Han, Gye Won; Hanson, Michael A.; Shi, Lei; Newman, Amy Hauck; Javitch, Jonathan A.; Cherezov, Vadim; Stevens, Raymond C. (Cornell); (Scripps); (NIDA); (Columbia); (UCSD); (Receptos)

    2010-11-30

    Dopamine modulates movement, cognition, and emotion through activation of dopamine G protein-coupled receptors in the brain. The crystal structure of the human dopamine D3 receptor (D3R) in complex with the small molecule D2R/D3R-specific antagonist eticlopride reveals important features of the ligand binding pocket and extracellular loops. On the intracellular side of the receptor, a locked conformation of the ionic lock and two distinctly different conformations of intracellular loop 2 are observed. Docking of R-22, a D3R-selective antagonist, reveals an extracellular extension of the eticlopride binding site that comprises a second binding pocket for the aryl amide of R-22, which differs between the highly homologous D2R and D3R. This difference provides direction to the design of D3R-selective agents for treating drug abuse and other neuropsychiatric indications.

  8. 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...

  9. Imaging dopamine D3 receptors in the human brain with positron emission tomography, [11C]PHNO, and a selective D3 receptor antagonist.

    Science.gov (United States)

    Searle, Graham; Beaver, John D; Comley, Robert A; Bani, Massimo; Tziortzi, Andri; Slifstein, Mark; Mugnaini, Manolo; Griffante, Cristiana; Wilson, Alan A; Merlo-Pich, Emilio; Houle, Sylvain; Gunn, Roger; Rabiner, Eugenii A; Laruelle, Marc

    2010-08-15

    Dopamine D(3) receptors are involved in the pathophysiology of several neuropsychiatric conditions. [(11)C]-(+)-PHNO is a radiolabeled D(2) and D(3) agonist, suitable for imaging the agonist binding sites (denoted D(2HIGH) and D(3)) of these receptors with positron emission tomography (PET). PET studies in nonhuman primates documented that, in vivo, [(11)C]-(+)-PHNO displays a relative selectivity for D(3) compared with D(2HIGH) receptor sites and that the [(11)C]-(+)-PHNO signal is enriched in D(3) contribution compared with conventional ligands such as [(11)C] raclopride. To define the D(3) contribution (f(PHNO)(D3)) to [(11)C]-(+)-PHNO binding potential (BP(ND)) in healthy humans, 52 PET scans were obtained in 19 healthy volunteers at baseline and following oral administration of various doses of the selective D(3) receptor antagonist, GSK598809. The impact of GSK598809 on [(11)C]-(+)-PHNO was regionally selective. In dorsal regions of the striatum, GSK598809 did not significantly affect [(11)C]-(+)-PHNO BP(ND) (f(PHNO)(D3) approximately 0%). Conversely, in the substantia nigra, GSK598809 dose-dependently reduced [(11)C]-(+)-PHNO binding to nonspecific level (f(PHNO)(D3) approximately 100%). In ventral striatum (VST), globus pallidus and thalamus (THA), [(11)C]-(+)-PHNO BP(ND) was attributable to a combination of D(2HIGH) and D(3) receptor sites, with f(PHNO)(D3) of 26%, 67% and 46%, respectively. D(3) receptor binding potential (BP(ND)(D3)) was highest in globus pallidus (1.90) and substantial nigra (1.39), with lower levels in VST (.77) and THA (.18) and negligible levels in dorsal striatum. This study elucidated the pharmacologic nature of the [(11)C]-(+)-PHNO signal in healthy subjects and provided the first quantification of D(3) receptor availability with PET in the living human brain. Copyright 2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  10. [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.

  11. Analysis of 3D models of octopus estrogen receptor with estradiol: evidence for steric clashes that prevent estrogen binding.

    Science.gov (United States)

    Baker, Michael E; Chandsawangbhuwana, Charlie

    2007-09-28

    Relatives of the vertebrate estrogen receptor (ER) are found in Aplysia californica, Octopus vulgaris, Thais clavigera, and Marisa cornuarietis. Unlike vertebrate ERs, invertebrate ERs are constitutively active and do not bind estradiol. To investigate the molecular basis of the absence of estrogen binding, we constructed a 3D model of the putative steroid-binding domain on octopus ER. Our 3D model indicates that binding of estradiol to octopus ER is prevented by steric clashes between estradiol and amino acids in the steroid-binding pocket. In this respect, octopus ER resembles vertebrate estrogen-related receptors (ERR), which have a ligand-binding pocket that cannot accommodate estradiol. Like ERR, octopus ER also may have the activation function 2 domain (AF2) in a configuration that can bind to coactivators in the absence of estrogens, which would explain constitutive activity of octopus ER.

  12. Crosstalk between the peroxisome proliferator-activated receptor γ (PPARγ) and the vitamin D receptor (VDR) in human breast cancer cells: PPARγ binds to VDR and inhibits 1α,25-dihydroxyvitamin D3 mediated transactivation

    International Nuclear Information System (INIS)

    Alimirah, Fatouma; Peng, Xinjian; Yuan, Liang; Mehta, Rajeshwari R.; Knethen, Andreas von; Choubey, Divaker; Mehta, Rajendra G.

    2012-01-01

    Heterodimerization and cross-talk between nuclear hormone receptors often occurs. For example, estrogen receptor alpha (ERα) physically binds to peroxisome proliferator-activated receptor gamma (PPARγ) and inhibits its transcriptional activity. The interaction between PPARγ and the vitamin D receptor (VDR) however, is unknown. Here, we elucidate the molecular mechanisms linking PPARγ and VDR signaling, and for the first time we show that PPARγ physically associates with VDR in human breast cancer cells. We found that overexpression of PPARγ decreased 1α,25-dihydroxyvitamin D 3 (1,25D 3 ) mediated transcriptional activity of the vitamin D target gene, CYP24A1, by 49% and the activity of VDRE-luc, a vitamin D responsive reporter, by 75% in T47D human breast cancer cells. Deletion mutation experiments illustrated that helices 1 and 4 of PPARγ's hinge and ligand binding domains, respectively, governed this suppressive function. Additionally, abrogation of PPARγ's AF2 domain attenuated its repressive action on 1,25D 3 transactivation, indicating that this domain is integral in inhibiting VDR signaling. PPARγ was also found to compete with VDR for their binding partner retinoid X receptor alpha (RXRα). Overexpression of RXRα blocked PPARγ's suppressive effect on 1,25D 3 action, enhancing VDR signaling. In conclusion, these observations uncover molecular mechanisms connecting the PPARγ and VDR pathways. -- Highlights: PPARγ's role on 1α,25-dihydroxyvitamin D 3 transcriptional activity is examined. ► PPARγ physically binds to VDR and inhibits 1α,25-dihydroxyvitamin D 3 action. ► PPARγ's hinge and ligand binding domains are important for this inhibitory effect. ► PPARγ competes with VDR for the availability of their binding partner, RXRα.

  13. ( sup 3 H)(D-PEN sup 2 , D-PEN sup 5 ) enkephalin binding to delta opioid receptors on intact neuroblastoma-glioma (NG 108-15) hybrid cells

    Energy Technology Data Exchange (ETDEWEB)

    Knapp, R.J.; Yamamura, H.I. (Univ. of Arizona College of Medicine, Tucson (USA))

    1990-01-01

    ({sup 3}H)(D-Pen{sup 2}, D-Pen{sup 5})enkephalin binding to intact NG 108-15 cells has been measured under physiological conditions of temperature and medium. The dissociation constant, receptor density, and Hill slope values measured under these conditions are consistent with values obtained by others using membranes prepared from these cells. Kinetic analysis of the radioligand binding to these cells show biphasic association and monophasic dissociation processes suggesting the presence of different receptor affinity states for the agonist. The data show that the binding affinity of ({sup 3}H)(D-Pen{sup 2}, D-Pen{sup 5})enkephalin under physiological conditions is not substantially different to that measured in 50 mM Tris buffer using cell membrane fractions. Unlike DPDPE, the {mu} opioid agonists morphine, normorphine, PL-17, and DAMGO, have much lower affinity for the {delta} receptor measured under these conditions than is observed by studies using 50 mM Tris buffer. The results described here suggest that this assay may serve as a useful model of {delta} opioid receptor binding in vivo.

  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. Biosynthesis, purification and receptor binding properties of high specific radioactivity 1α,24(R), 25-trihydroxy-[26,27-methyl-3H]-vitamin D3

    International Nuclear Information System (INIS)

    Chandler, J.S.; Pike, J.W.; Haussler, M.R.

    1982-01-01

    A procedure for the biosynthesis and purification of 1α,24(R),25-trihydroxy[26,27-methyl- 3 H]-vitamin D 3 (1,24,25-(OH) 3 [ 3 H]D 3 ) is reported. A kidney homogenate from chicks receiving a high calcium diet (3%) and oral supplements of 1α,25-dihydroxyvitamin D 3 (1,25-(OH) 2 D 3 ) was used for C-24-hydroxylation of 1α,25-dihydroxy[26,27-methyl- 3 H]-vitamin D 3 (1,25-(OH) 2 [ 3 H]D 3 ), in vitro. Extraction and purification of the homogenate lipid fraction by Sephadex LH-20 and high performance liquid chromatography yielded radiochemically pure 1,24,25-(OH) 3 [ 3 H]D 3 with a specific radioactivity equivalent to the initial substrate (166 Ci/mmol). The authenticity of the generated metabolite was assessed by co-migration with synthetic 1,24,25-(OH) 3 D 3 on high performance liquid chromatography and by equimolar competition with authentic radioinert 1,24,25-(OH) 3 D 3 for binding to a purified receptor protein from rat kidney. Binding studies indicate the trihydroxylated metabolite competes 40-50% as effectively as 1,25-(OH) 2 D 3 for hormone binding sites. Further analysis of 1,24,25-(OH) 3 D 3 -receptor interaction reveals a high-affinity, saturable binding with an apparent Ksub(d) of 2.2 x 10 -9 M. These studies demonstrate that although slightly less active than 1,25-(OH) 2 D 3 , 1,24,25-(OH) 3 D 3 is capable of hormone-like interactions, in vitro. The availability of this high specific radioactivity sterol should allow for clarification of its potential physiologic significance. (author)

  16. 3-D Image Analysis of Fluorescent Drug Binding

    Directory of Open Access Journals (Sweden)

    M. Raquel Miquel

    2005-01-01

    Full Text Available Fluorescent ligands provide the means of studying receptors in whole tissues using confocal laser scanning microscopy and have advantages over antibody- or non-fluorescence-based method. Confocal microscopy provides large volumes of images to be measured. Histogram analysis of 3-D image volumes is proposed as a method of graphically displaying large amounts of volumetric image data to be quickly analyzed and compared. The fluorescent ligand BODIPY FL-prazosin (QAPB was used in mouse aorta. Histogram analysis reports the amount of ligand-receptor binding under different conditions and the technique is sensitive enough to detect changes in receptor availability after antagonist incubation or genetic manipulations. QAPB binding was concentration dependent, causing concentration-related rightward shifts in the histogram. In the presence of 10 μM phenoxybenzamine (blocking agent, the QAPB (50 nM histogram overlaps the autofluorescence curve. The histogram obtained for the 1D knockout aorta lay to the left of that of control and 1B knockout aorta, indicating a reduction in 1D receptors. We have shown, for the first time, that it is possible to graphically display binding of a fluorescent drug to a biological tissue. Although our application is specific to adrenergic receptors, the general method could be applied to any volumetric, fluorescence-image-based assay.

  17. 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

  18. 3D model of amphioxus steroid receptor complexed with estradiol

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Michael E., E-mail: mbaker@ucsd.edu [Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0693 (United States); Chang, David J. [Department of Biology, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0693 (United States)

    2009-08-28

    The origins of signaling by vertebrate steroids are not fully understood. An important advance was the report that an estrogen-binding steroid receptor [SR] is present in amphioxus, a basal chordate with a similar body plan as vertebrates. To investigate the evolution of estrogen-binding to steroid receptors, we constructed a 3D model of amphioxus SR complexed with estradiol. This 3D model indicates that although the SR is activated by estradiol, some interactions between estradiol and human ER{alpha} are not conserved in the SR, which can explain the low affinity of estradiol for the SR. These differences between the SR and ER{alpha} in the steroid-binding domain are sufficient to suggest that another steroid is the physiological regulator of the SR. The 3D model predicts that mutation of Glu-346 to Gln will increase the affinity of testosterone for amphioxus SR and elucidate the evolution of steroid-binding to nuclear receptors.

  19. In vivo effects of olanzapine on striatal dopamine D[sub 2]/D[sub 3] receptor binding in schizophrenic patients: an iodine-123 iodobenzamide single-photon emission tomography study

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    Dresel, S.; Rossmueller, B.; Hahn, K.; Tatsch, K. (Department of Nuclear Medicine, University of Munich (Germany)); Mager, T.; Meisenzahl, E.; Moeller, H.J. (Department of Psychiatry, University of Munich (Germany))

    1999-08-01

    Olanzapine is a new atypical antipsychotic agent that belongs to the same chemical class as clozapine. The pharmacological efficacy of olanzapine (in contrast to that of risperidone) has been shown to be comparable to that of clozapine, but olanzapine has the advantage of producing a less pronounced bone marrow depressing effect than clozapine. The specific aims of this study were (a) to assess dopamine D[sub 2]/D[sub 3] receptor availability in patients treated with olanzapine by means of iodine-123 iodobenzamide [[sup 123]I]IBZM single-photon emission tomography (SPET), (b) to compare the results with findings of [[sup 123]I]IBZM SPET in patients under treatment with risperidone and (c) to correlate the results with the occurrance of extrapyramidal side-effects (EPMS). Brain SPET scans were performed in 20 schizophrenic patients (DSM III R) at 2 h after i.v. administration of 185 MBq [[sup 123]I]IBZM. Images were acquired using a triple-head gamma camera (Picker Prism 3000 XP). For semiquantitative evaluation of D[sub 2]/D[sub 3] receptor binding, transverse slices corrected for attenuation were used to calculate specific uptake values [STR-BKG]/BKG (STR=striatum; BKG=background). The mean daily dose of olanzapine ranged from 0.05 to 0.6 mg/kg body weight. The dopamine D[sub 2]/D[sub 3] receptor binding was reduced in all patients treated with olanzapine. Specific IBZM binding [STR-BKG]/BKG ranged from 0.13 to 0.61 (normal controls >0.95). The decreased D[sub 2]/D[sub 3] receptor availability revealed an exponential dose-response relationship (r=-0.85, P<0.001). The slope of the curve was similar to that of risperidone and considerably higher than that of clozapine as compared with the results of a previously published study. EPMS were observed in only one patient, presenting with the lowest D[sub 2]/D[sub 3] availability. The frequency of EPMS induced by olanzapine (5%) was considerably lower than the frequency under risperidone treatment (40%). Our findings

  20. In vivo effects of olanzapine on striatal dopamine D{sub 2}/D{sub 3} receptor binding in schizophrenic patients: an iodine-123 iodobenzamide single-photon emission tomography study

    Energy Technology Data Exchange (ETDEWEB)

    Dresel, S.; Rossmueller, B.; Hahn, K.; Tatsch, K. [Department of Nuclear Medicine, University of Munich (Germany); Mager, T.; Meisenzahl, E.; Moeller, H.J. [Department of Psychiatry, University of Munich (Germany)

    1999-08-01

    Olanzapine is a new atypical antipsychotic agent that belongs to the same chemical class as clozapine. The pharmacological efficacy of olanzapine (in contrast to that of risperidone) has been shown to be comparable to that of clozapine, but olanzapine has the advantage of producing a less pronounced bone marrow depressing effect than clozapine. The specific aims of this study were (a) to assess dopamine D{sub 2}/D{sub 3} receptor availability in patients treated with olanzapine by means of iodine-123 iodobenzamide [{sup 123}I]IBZM single-photon emission tomography (SPET), (b) to compare the results with findings of [{sup 123}I]IBZM SPET in patients under treatment with risperidone and (c) to correlate the results with the occurrance of extrapyramidal side-effects (EPMS). Brain SPET scans were performed in 20 schizophrenic patients (DSM III R) at 2 h after i.v. administration of 185 MBq [{sup 123}I]IBZM. Images were acquired using a triple-head gamma camera (Picker Prism 3000 XP). For semiquantitative evaluation of D{sub 2}/D{sub 3} receptor binding, transverse slices corrected for attenuation were used to calculate specific uptake values [STR-BKG]/BKG (STR=striatum; BKG=background). The mean daily dose of olanzapine ranged from 0.05 to 0.6 mg/kg body weight. The dopamine D{sub 2}/D{sub 3} receptor binding was reduced in all patients treated with olanzapine. Specific IBZM binding [STR-BKG]/BKG ranged from 0.13 to 0.61 (normal controls >0.95). The decreased D{sub 2}/D{sub 3} receptor availability revealed an exponential dose-response relationship (r=-0.85, P<0.001). The slope of the curve was similar to that of risperidone and considerably higher than that of clozapine as compared with the results of a previously published study. EPMS were observed in only one patient, presenting with the lowest D{sub 2}/D{sub 3} availability. The frequency of EPMS induced by olanzapine (5%) was considerably lower than the frequency under risperidone treatment (40%). Our findings

  1. 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

  2. Frontal dopamine D(2/3) receptor binding in drug-naive first-episode schizophrenic patients correlates with positive psychotic symptoms and gender

    DEFF Research Database (Denmark)

    Glenthoj, Birte Y; Mackeprang, Torben; Svarer, Claus

    2006-01-01

    BACKGROUND: The aim of the study was to examine extrastriatal dopamine D(2/3) receptor binding and psychopathology in schizophrenic patients, and to relate binding potential (BP) values to psychopathology. METHODS: Twenty-five drug-naive schizophrenic patients and 20 healthy controls were examined...

  3. Cocaine Disrupts Histamine H3 Receptor Modulation of Dopamine D1 Receptor Signaling: σ1-D1-H3 Receptor Complexes as Key Targets for Reducing Cocaine's Effects

    Science.gov (United States)

    Moreno, Estefanía; Moreno-Delgado, David; Navarro, Gemma; Hoffmann, Hanne M.; Fuentes, Silvia; Rosell-Vilar, Santi; Gasperini, Paola; Rodríguez-Ruiz, Mar; Medrano, Mireia; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carme; Ferré, Sergi; Ortiz, Jordi; Canela, Enric

    2014-01-01

    The general effects of cocaine are not well understood at the molecular level. What is known is that the dopamine D1 receptor plays an important role. Here we show that a key mechanism may be cocaine's blockade of the histamine H3 receptor-mediated inhibition of D1 receptor function. This blockade requires the σ1 receptor and occurs upon cocaine binding to σ1-D1-H3 receptor complexes. The cocaine-mediated disruption leaves an uninhibited D1 receptor that activates Gs, freely recruits β-arrestin, increases p-ERK 1/2 levels, and induces cell death when over activated. Using in vitro assays with transfected cells and in ex vivo experiments using both rats acutely treated or self-administered with cocaine along with mice depleted of σ1 receptor, we show that blockade of σ1 receptor by an antagonist restores the protective H3 receptor-mediated brake on D1 receptor signaling and prevents the cell death from elevated D1 receptor signaling. These findings suggest that a combination therapy of σ1R antagonists with H3 receptor agonists could serve to reduce some effects of cocaine. PMID:24599455

  4. 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)

  5. Incorporation of the purified epstein barr virus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions

    Energy Technology Data Exchange (ETDEWEB)

    Mold, C.; Cooper, N.R.; Nemerow, G.R.

    1986-06-01

    The 145-kDA molecule that has been identified as the C3d receptor CR2 was isolated from lysates of Raji cells by affinity chromatography by using the monoclonal antibody (MoAb)HB-5. The purified protein was incorporated into /sup 14/C-phosphatidylcholine liposomes by deoxycholate dialysis followed by flotation on discontinuous sucrose gradients. Incorporation of the receptor was verified by testing the gradient fractions for CR2 by an enzyme-linked immunosorbent assay. Liposomes were shown to be unilamellar vesicles ranging in diameter from 25 to 100 nm by electron microscopy. The external orientation of CR2 in the membranes was demonstrated by immunoelectron microscopy. The functional activities of liposomes containing CR2 and liposomes without protein were compared. CR2 liposomes bound to EC3d, but not to E, and this binding was inhibited by the anti-CR2 MoAb OKB7 and by a MoAb specific for C3d. Control liposomes failed to bind to either E or EC3D. The ability of CR2 to function as a receptor for Epstein Barr virus (EBV) was tested in two ways. First, CR2 liposomes bound to B95-8, a cell line expressing EBV membrane antigens, but not to B95-8 cells treated with the viral DNA polymerase inhibitor phosphonoformic acid. Second, liposomes containing CR2 were shown by ultracentrifugal analyses to bind directly to purified EBV, and this binding was also inhibited by OKB7. Control liposomes did not bind to B95-8 cells or to EBV. These findings show that CR2 purified from detergent extracts of Raji cells can be reconstituted into lipid membranes with maintenance of its dual functions as a receptor for C3d and EBV.

  6. Incorporation of the purified epstein barr virus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions

    International Nuclear Information System (INIS)

    Mold, C.; Cooper, N.R.; Nemerow, G.R.

    1986-01-01

    The 145-kDA molecule that has been identified as the C3d receptor CR2 was isolated from lysates of Raji cells by affinity chromatography by using the monoclonal antibody (MoAb)HB-5. The purified protein was incorporated into 14 C-phosphatidylcholine liposomes by deoxycholate dialysis followed by flotation on discontinuous sucrose gradients. Incorporation of the receptor was verified by testing the gradient fractions for CR2 by an enzyme-linked immunosorbent assay. Liposomes were shown to be unilamellar vesicles ranging in diameter from 25 to 100 nm by electron microscopy. The external orientation of CR2 in the membranes was demonstrated by immunoelectron microscopy. The functional activities of liposomes containing CR2 and liposomes without protein were compared. CR2 liposomes bound to EC3d, but not to E, and this binding was inhibited by the anti-CR2 MoAb OKB7 and by a MoAb specific for C3d. Control liposomes failed to bind to either E or EC3D. The ability of CR2 to function as a receptor for Epstein Barr virus (EBV) was tested in two ways. First, CR2 liposomes bound to B95-8, a cell line expressing EBV membrane antigens, but not to B95-8 cells treated with the viral DNA polymerase inhibitor phosphonoformic acid. Second, liposomes containing CR2 were shown by ultracentrifugal analyses to bind directly to purified EBV, and this binding was also inhibited by OKB7. Control liposomes did not bind to B95-8 cells or to EBV. These findings show that CR2 purified from detergent extracts of Raji cells can be reconstituted into lipid membranes with maintenance of its dual functions as a receptor for C3d and EBV

  7. 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.

  8. PET evaluation of the relationship between D2 receptor binding and glucose metabolism in patients with parkinsonism

    International Nuclear Information System (INIS)

    Nakagawa, Makoto; Kuwabara, Yasuo; Taniwaki, Takayuki; Koga, Hirofumi; Kaneko, Koichiro; Hayashi, Kazutaka; Kira, Jun-ichi; Honda, Hiroshi; Sasaki, Masayuki

    2005-01-01

    The objective of this study was to clarify the relationship between D 2 receptor binding and the cerebral metabolic rate for glucose (CMRGlu) in patients with parkinsonism, we simultaneously measured both of these factors, and then compared the results. The subjects consisted of 24 patients: 9 with Parkinson's disease (PD), 3 with Juvenile Parkinson's disease (JPD), 9 with multiple system atrophy (MSA), and 3 with progressive supranuclear palsy (PSP). The striatal D 2 receptor binding was measured by the C-11 raclopride transient equilibrium method. CMRGlu was investigated by the F-18 fluorodeoxyglucose autoradiographic method. The D 2 receptor binding in both the caudate nucleus and putamen showed a positive correlation with the CMRGlu in the PD-JPD group, but the two parameters demonstrated no correlation in the MSA-PSP group. The left/right (L/R) ratio of D 2 receptor binding in the putamen showed a positive correlation with that of CMRGlu in the MSA-PSP group, while the two demonstrated no correlation in the PD-JPD group. Our PET study showed striatal D 2 receptor binding and the CMRGlu to be closely related in patients with parkinsonism, even though the results obtained using the L/R ratios tended to differ substantially from those obtained using absolute values. The reason for this difference is not clear, but this finding may reflect the pathophysiology of these disease entities. (author)

  9. Adaptive increase in D3 dopamine receptors in the brain reward circuits of human cocaine fatalities.

    Science.gov (United States)

    Staley, J K; Mash, D C

    1996-10-01

    The mesolimbic dopaminergic system plays a primary role in mediating the euphoric and rewarding effects of most abused drugs. Chronic cocaine use is associated with an increase in dopamine neurotransmission resulting from the blockade of dopamine uptake and is mediated by the activation of dopamine receptors. Recent studies have suggested that the D3 receptor subtype plays a pivotal role in the reinforcing effects of cocaine. The D3 receptor-preferring agonist 7-hydroxy-N,N-di-n-propyl-2-aminotetralin (7-OH-DPAT) is a reinforcer in rhesus monkeys trained to self-administer cocaine, but not in cocainenaive monkeys. In vitro autoradiographic localization of [3H]-(+)-7-OH-DPAT binding in the human brain demonstrated that D3 receptors were prevalent and highly localized over the ventromedial sectors of the striatum. Pharmacological characterization of [3H]-(+)-7-OH-DPAT binding to the human nucleus accumbens demonstrated a rank order of potency similar to that observed for binding to the cloned D3 receptor expressed in transfected cell lines. Region-of-interest analysis of [3H]-(+)-7-OH-DPAT binding to the D3 receptor demonstrated a one- to threefold elevation in the number of binding sites over particular sectors of the striatum and substantia nigra in cocaine overdose victims as compared with age-matched and drug-free control subjects. The elevated number of [3H]-(+)-7-OH-DPAT binding sites demonstrates that adaptive changes in the D3 receptor in the reward circuitry of the brain are associated with chronic cocaine abuse. These results suggest that the D3 receptor may be a useful target for drug development of anticocaine medications.

  10. 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.

  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. Leptin Increases Striatal Dopamine D2 Receptor Binding in Leptin-Deficient Obese (ob/ob) Mice

    Energy Technology Data Exchange (ETDEWEB)

    Pfaffly, J.; Michaelides, M.; Wang, G-J.; Pessin, J.E.; Volkow, N.D.; Thanos, P.K.

    2010-06-01

    Peripheral and central leptin administration have been shown to mediate central dopamine (DA) signaling. Leptin-receptor deficient rodents show decreased DA D2 receptor (D2R) binding in striatum and unique DA profiles compared to controls. Leptin-deficient mice show increased DA activity in reward-related brain regions. The objective of this study was to examine whether basal D2R-binding differences contribute to the phenotypic behaviors of leptin-deficient ob/ob mice, and whether D2R binding is altered in response to peripheral leptin treatment in these mice. Leptin decreased body weight, food intake, and plasma insulin concentration in ob/ob mice but not in wild-type mice. Basal striatal D2R binding (measured with autoradiography [{sup 3}H] spiperone) did not differ between ob/ob and wild-type mice but the response to leptin did. In wild-type mice, leptin decreased striatal D2R binding, whereas, in ob/ob mice, leptin increased D2R binding. Our findings provide further evidence that leptin modulates D2R expression in striatum and that these effects are genotype/phenotype dependent.

  13. The Role of Endogenous D2 Receptor Levels in Morphine Addiction: A Correlative Study of Morphine Place Conditioning and In Vivo [3H]-Raclopride Binding

    Energy Technology Data Exchange (ETDEWEB)

    Khan, N.; Gatley, S.

    2004-01-01

    Dopamine is a neurotransmitter that has a wide array of effects on an individual’s mental state. It is vital in the regulation of motor skills and in generating the effects of substance abuse. This study examined the dopamine D2 receptors found in the striatum of the brain. The impetus for investigating this receptor lies in the perception that it plays an influential role in drug addiction. It has been conjectured on the basis of human PET studies that possession of low levels of D2 receptors will heighten an individual’s susceptibility to drug addiction. However, an alternative explanation of low D2 receptor levels in drug dependent individuals is that these levels are a consequence of drug abuse. To understand this phenomenon, the present study employed the paradigm of conditioned place preference (CPP). In CPP, individuals of an out-bred mouse strain are observed to spend time in environments where they had previously been exposed to a drug that is abused by humans. The drug chosen for our studies was morphine because it has been previously shown to generate a robust place preference in mice and is a prototypic abused drug in humans. D2 receptor levels were quantified using an in vivo binding study involving [3H]raclopride, a radioactive compound that binds to D2 receptors. The results showed a significant place preference for morphine following the conditioning procedure. Additionally, data from the binding analysis agreed with previous studies that the striatum contains high levels of D2 receptors. However, there was no consistent relationship between the extent of morphine CPP and D2 receptor levels as revealed by [3H]-RAC binding. This finding does not support the hypothesis that low levels of D2 receptors predispose a mouse to easy morphine conditioning. Further experiments are required to determine the ability to generalize our findings to other species and other drugs of abuse.

  14. Relationship of Structure and Function of DNA-Binding Domain in Vitamin D Receptor

    Directory of Open Access Journals (Sweden)

    Lin-Yan Wan

    2015-07-01

    Full Text Available While the structure of the DNA-binding domain (DBD of the vitamin D receptor (VDR has been determined in great detail, the roles of its domains and how to bind the motif of its target genes are still under debate. The VDR DBD consists of two zinc finger modules and a C-terminal extension (CTE, at the end of the C-terminal of each structure presenting α-helix. For the first zinc finger structure, N37 and S-box take part in forming a dimer with 9-cis retinoid X receptor (RXR, while V26, R50, P-box and S-box participate in binding with VDR response elements (VDRE. For the second zinc finger structure, P61, F62 and H75 are essential in the structure of the VDR homodimer with the residues N37, E92 and F93 of the downstream of partner VDR, which form the inter-DBD interface. T-box of the CTE, especially the F93 and I94, plays a critical role in heterodimerization and heterodimers–VDRE binding. Six essential residues (R102, K103, M106, I107, K109, and R110 of the CTE α-helix of VDR construct one interaction face, which packs against the DBD core of the adjacent symmetry mate. In 1,25(OH2D3-activated signaling, the VDR-RXR heterodimer may bind to DR3-type VDRE and ER9-type VDREs of its target gene directly resulting in transactivation and also bind to DR3-liked nVDRE of its target gene directly resulting in transrepression. Except for this, 1α,25(OH2D3 ligand VDR-RXR may bind to 1αnVDRE indirectly through VDIR, resulting in transrepression of the target gene. Upon binding of 1α,25(OH2D3, VDR can transactivate and transrepress its target genes depending on the DNA motif that DBD binds.

  15. [123I]Epidepride neuroimaging of dopamine D2/D3 receptor in chronic MK-801-induced rat schizophrenia model

    International Nuclear Information System (INIS)

    Huang, Yuan-Ruei; Shih, Jun-Ming; Chang, Kang-Wei; Huang, Chieh; Wu, Yu-Lung; Chen, Chia-Chieh

    2012-01-01

    Purpose: [ 123 I]Epidepride is a radio-tracer with very high affinity for dopamine D 2 /D 3 receptors in brain. The importance of alteration in dopamine D 2 /D 3 receptor binding condition has been wildly verified in schizophrenia. In the present study we set up a rat schizophrenia model by chronic injection of a non-competitive NMDA receptor antagonist, MK-801, to examine if [ 123 I]epidepride could be used to evaluate the alterations of dopamine D 2 /D 3 receptor binding condition in specific brain regions. Method: Rats were given repeated injection of MK-801 (dissolved in saline, 0.3 mg/kg) or saline for 1 month. Afterwards, total distance traveled (cm) and social interaction changes were recorded. Radiochemical purity of [ 123 I]epidepride was analyzed by Radio-Thin-Layer Chromatography (chloroform: methanol, 9:1, v/v) and [ 123 I]epidepride neuroimages were obtained by ex vivo autoradiography and small animal SPECT/CT. Data obtained were then analyzed to determine the changes of specific binding ratio. Result: Chronic MK-801 treatment for a month caused significantly increased local motor activity and induced an inhibition of social interaction. As shown in [ 123 I]epidepride ex vivo autoradiographs, MK-801 induced a decrease of specific binding ratio in the striatum (24.01%), hypothalamus (35.43%), midbrain (41.73%) and substantia nigra (37.93%). In addition, [ 123 I]epidepride small animal SPECT/CT neuroimaging was performed in the striatum and midbrain. There were statistically significant decreases in specific binding ratio in both the striatum (P 123 I]epidepride is a useful radio-tracer to reveal the alterations of dopamine D 2 /D 3 receptor binding in a rat schizophrenia model and is also helpful to evaluate therapeutic effects of schizophrenia in the future.

  16. A radioligand immunoassay for 1,25-dihydroxyvitamin D3 receptors using monoclonal antibody: detection of a phenotypic receptor variant in vitamin D-dependency rickets (type II) which does not bind hormone

    International Nuclear Information System (INIS)

    Pike, J.W.; Dokoh, Shigeharu; Liberman, U.A.; Eil, C.; Haussler, M.R.; Marx, S.J.

    1984-01-01

    Vitamin D-dependency rickets, type II (VDDRII), is a well recognized heritable disorder characterized by peripheral target organ resistance to 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ), the hormonally active form of the vitamin. Recently, cultured skin fibroblasts obtained from a number of patients with VDDRII have been utilized to characterize the underlying molecular defects associated with this malady. Recently monoclonal antibodies to the vitamin D receptor have been generated, and a radioligand immunoassay (RLIA) for the detection of this molecule has been developed which is independent of its hormone-binding capacity. This report describes the application of the immunoassay in the detection of receptor-like molecules in fibroblasts derived from patients with VDDRII. The results indicate that the molecule is generally present in all patients, and provides a mechanism for individual responsiveness to pharmacologic treatment with vitamin D 3 metabolites. 8 refs.; 3 figs.; 1 table

  17. Dopamine D/sub 2/ and D/sub 1/ receptors: biochemical characterization

    Energy Technology Data Exchange (ETDEWEB)

    Niznik, H B

    1986-01-01

    In order to label dopamine D/sub 2/ receptors reversibly and selectively the potent substituted benzamide neuroleptic, YM-09151-2, was tritium labeled and its binding characteristics to striatal homogenates investigated. (/sup 3/H) YM-09151-2 bound to D/sub 2/ receptors with high affinity in a specific, saturable, reversible and sodium dependent fashion, displaying an appropriate pharmacological D/sub 2/ receptor profile. (/sup 3/H) YM-09151-2 appears to be the ligand of choice for labeling D/sub 2/ receptors since it displays approximately 20-fold lower affinity for serotonergic S/sub 2/ receptors than does (/sup 3/H) spiperone. As an initial step towards the molecular identification of the ligand binding subunit of the striatal D/sub 2/ receptor, photolabile analogues of the substituted benzamide clebopride were synthesized and their reversible and irreversible binding interactions to D/sub 2/ receptors characterized. D/sub 2/ receptor photoinactivation was prevented in a concentration and stereoselective manner by dopaminergic agonists and antagonists. In vivo biodistribution studies with (/sup 125/I) iodoazidoclebopride confirmed the ligand's ability to bind to D/sub 2/ receptor-rich regions and as such, may become a useful tool for the molecular characterization of D/sub 2/ receptor proteins. Digitonin solubilized striatal dopamine D/sub 2/ and D/sub 1/ receptors can be completely separated with full retention of biological activity by steric exclusion High Pressure Liquid Chromatography (HPLC) with corresponding Stokes radii of 7.1 and 5.6 nm.

  18. The E2.65A mutation disrupts dynamic binding poses of SB269652 at the dopamine D2 and D3 receptors.

    Directory of Open Access Journals (Sweden)

    Ravi Kumar Verma

    2018-01-01

    Full Text Available The dopamine D2 and D3 receptors (D2R and D3R are important targets for antipsychotics and for the treatment of drug abuse. SB269652, a bitopic ligand that simultaneously binds both the orthosteric binding site (OBS and a secondary binding pocket (SBP in both D2R and D3R, was found to be a negative allosteric modulator. Previous studies identified Glu2.65 in the SBP to be a key determinant of both the affinity of SB269652 and the magnitude of its cooperativity with orthosteric ligands, as the E2.65A mutation decreased both of these parameters. However, the proposed hydrogen bond (H-bond between Glu2.65 and the indole moiety of SB269652 is not a strong interaction, and a structure activity relationship study of SB269652 indicates that this H-bond may not be the only element that determines its allosteric properties. To understand the structural basis of the observed phenotype of E2.65A, we carried out molecular dynamics simulations with a cumulative length of ~77 μs of D2R and D3R wild-type and their E2.65A mutants bound to SB269652. In combination with Markov state model analysis and by characterizing the equilibria of ligand binding modes in different conditions, we found that in both D2R and D3R, whereas the tetrahydroisoquinoline moiety of SB269652 is stably bound in the OBS, the indole-2-carboxamide moiety is dynamic and only intermittently forms H-bonds with Glu2.65. Our results also indicate that the E2.65A mutation significantly affects the overall shape and size of the SBP, as well as the conformation of the N terminus. Thus, our findings suggest that the key role of Glu2.65 in mediating the allosteric properties of SB269652 extends beyond a direct interaction with SB269652, and provide structural insights for rational design of SB269652 derivatives that may retain its allosteric properties.

  19. Predicting treatment response in Schizophrenia: the role of stratal and frontal dopamine D2/D3 receptor binding potential

    DEFF Research Database (Denmark)

    Wulff, Sanne; Nørbak-Emig, Henrik; Nielsen, Mette Ødegaard

    2014-01-01

    Background One of the best validated findings in schizophrenia is an association between increased presynaptic striatal dopaminergic activity and psychotic symptoms. We have previously reported an association between positive symptoms and dopamine D2 receptor binding potentials (BPs) in frontal...... cortex in antipsychotic-naïve first-episode male schizophrenia patients(1). Preclinical studies suggest an inverse relationship between frontal and striatal dopamine activity. This activity can indirectly be expressed by the BP of dopamine receptors using Single Photon Emission Computed Tomography (SPECT......) where low striatal BP is believed to reflect high dopamine availability. We aim to assess the association between D2 receptor BPs in antipsychotic-naïve first-episode schizophrenia patients and their response to the first treatment with an antipsychotic compound. We hypothesise that patients with low...

  20. Involvement of 1,25D{sub 3}-MARRS (membrane associated, rapid response steroid-binding), a novel vitamin D receptor, in growth inhibition of breast cancer cells

    Energy Technology Data Exchange (ETDEWEB)

    Richard, Cynthia L. [Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada); Farach-Carson, Mary C.; Rohe, Ben [Department of Biological Sciences, University of Delaware, Newark, DE 19716 (United States); Nemere, Ilka [Department of Nutrition and Food Sciences, Center for Integrated BioSystems, Utah State University, Logan, UT 84322 8700 (United States); Meckling, Kelly A., E-mail: kmecklin@uoguelph.ca [Department of Human Health and Nutritional Sciences, University of Guelph, Guelph, Ontario, Canada N1G2W1 (Canada)

    2010-03-10

    In addition to classical roles in calcium homeostasis and bone development, 1,25 dihydroxyvitamin D{sub 3} [1,25(OH){sub 2}D{sub 3}] inhibits the growth of several cancer types, including breast cancer. Although cellular effects of 1,25(OH){sub 2}D{sub 3} traditionally have been attributed to activation of a nuclear vitamin D receptor (VDR), a novel receptor for 1,25(OH){sub 2}D{sub 3} called 1,25D{sub 3}-MARRS (membrane-associated, rapid response steroid-binding) protein was identified recently. The purpose of this study was to determine if the level of 1,25D{sub 3}-MARRS expression modulates 1,25(OH){sub 2}D{sub 3} activity in breast cancer cells. Relative levels of 1,25D{sub 3}-MARRS protein in MCF-7, MDA MB 231, and MCF-10A cells were estimated by real-time RT-PCR and Western blotting. To determine if 1,25D{sub 3}-MARRS receptor was involved in the growth inhibitory effects of 1,25(OH){sub 2}D{sub 3} in MCF-7 cells, a ribozyme construct designed to knock down 1,25D{sub 3}-MARRS mRNA was stably transfected into MCF-7 cells. MCF-7 clones in which 1,25D{sub 3}-MARRS receptor expression was reduced showed increased sensitivity to 1,25(OH){sub 2}D{sub 3} ( IC{sub 50} 56 {+-} 24 nM) compared to controls (319 {+-} 181 nM; P < 0.05). Reduction in 1,25D{sub 3}-MARRS receptor lengthened the doubling time in transfectants treated with 1,25(OH){sub 2}D{sub 3}. Knockdown of 1,25D{sub 3}-MARRS receptor also increased the sensitivity of MCF-7 cells to the vitamin D analogs KH1060 and MC903, but not to unrelated agents (all-trans retinoic acid, paclitaxel, serum/glucose starvation, or the isoflavone, pomiferin). These results suggest that 1,25D{sub 3}-MARRS receptor expression interferes with the growth inhibitory activity of 1,25(OH){sub 2}D{sub 3} in breast cancer cells, possibly through the nuclear VDR. Further research should examine the potential for pharmacological or natural agents that modify 1,25D{sub 3}-MARRS expression or activity as anticancer agents.

  1. Reduced striatal dopamine D2/3 receptor availability in Body Dysmorphic Disorder.

    Science.gov (United States)

    Vulink, Nienke C; Planting, Robin S; Figee, Martijn; Booij, Jan; Denys, Damiaan

    2016-02-01

    Though the dopaminergic system is implicated in Obsessive Compulsive and Related Disorders (OCRD), the dopaminergic system has never been investigated in-vivo in Body Dysmorphic Disorder (BDD). In line with consistent findings of reduced striatal dopamine D2/3 receptor availability in Obsessive Compulsive Disorder (OCD), we hypothesized that the dopamine D2/3 receptor availability in the striatum will be lower in patients with BDD in comparison to healthy subjects. Striatal dopamine D2/3 receptor Binding Potential (BPND) was examined in 12 drug-free BDD patients and 12 control subjects pairwise matched by age, sex, and handedness using [(123)I]iodobenzamide Single Photon Emission Computed Tomography (SPECT; bolus/constant infusion technique). Regions of interest were the caudate nucleus and the putamen. BPND was calculated as the ratio of specific striatal to binding in the occipital cortex (representing nonspecific binding). Compared to controls, dopamine D2/3 receptor BPND was significantly lower in BDD, both in the putamen (p=0.017) and caudate nucleus (p=0.022). This study provides the first evidence of a disturbed dopaminergic system in BDD patients. Although previously BDD was classified as a separate disorder (somatoform disorder), our findings give pathophysiological support for the recent reclassification of BDD to the OCRD in DSM-5. Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.

  2. Frontal D2/3 Receptor Availability in Schizophrenia Patients Before and After Their First Antipsychotic Treatment

    DEFF Research Database (Denmark)

    Nørbak-Emig, Henrik; Ebdrup, Bjørn H; Fagerlund, Birgitte

    2016-01-01

    the relation between frontal D2/3 receptor availability and treatment effect on positive symptoms. METHODS: Twenty-five antipsychotic-naïve first-episode schizophrenia patients were examined with the Positive and Negative Syndrome Scale, tested with the cognitive test battery Cambridge Neuropsychological Test......BACKGROUND: We have previously reported associations between frontal D2/3 receptor binding potential positive symptoms and cognitive deficits in antipsychotic-naïve schizophrenia patients. Here, we examined the effect of dopamine D2/3 receptor blockade on cognition. Additionally, we explored.......56, P=.003; D2/3 receptor binding potential right frontal cortex rho = 0.48, P=.016). CONCLUSIONS: Our data support the hypothesis of a negative influence of D2/3 receptor blockade on specific cognitive functions in schizophrenia. This is highly clinically relevant given the well-established association...

  3. Stronger Dopamine D1 Receptor-Mediated Neurotransmission in Dyskinesia.

    Science.gov (United States)

    Farré, Daniel; Muñoz, Ana; Moreno, Estefanía; Reyes-Resina, Irene; Canet-Pons, Júlia; Dopeso-Reyes, Iria G; Rico, Alberto J; Lluís, Carme; Mallol, Josefa; Navarro, Gemma; Canela, Enric I; Cortés, Antonio; Labandeira-García, José L; Casadó, Vicent; Lanciego, José L; Franco, Rafael

    2015-12-01

    Radioligand binding assays to rat striatal dopamine D1 receptors showed that brain lateralization of the dopaminergic system were not due to changes in expression but in agonist affinity. D1 receptor-mediated striatal imbalance resulted from a significantly higher agonist affinity in the left striatum. D1 receptors heteromerize with dopamine D3 receptors, which are considered therapeutic targets for dyskinesia in parkinsonian patients. Expression of both D3 and D1-D3 receptor heteromers were increased in samples from 6-hydroxy-dopamine-hemilesioned rats rendered dyskinetic by treatment with 3, 4-dihydroxyphenyl-L-alanine (L-DOPA). Similar findings were obtained using striatal samples from primates. Radioligand binding studies in the presence of a D3 agonist led in dyskinetic, but not in lesioned or L-DOPA-treated rats, to a higher dopamine sensitivity. Upon D3-receptor activation, the affinity of agonists for binding to the right striatal D1 receptor increased. Excess dopamine coming from L-DOPA medication likely activates D3 receptors thus making right and left striatal D1 receptors equally responsive to dopamine. These results show that dyskinesia occurs concurrently with a right/left striatal balance in D1 receptor-mediated neurotransmission.

  4. 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

  5. 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.

  6. [3H]AVP binding to rat renal tubular receptors during long-term treatment with an antagonist of arginine vasopressin

    International Nuclear Information System (INIS)

    Mah, S.C.; Whitebread, S.E.; De Gasparo, M.; Hofbauer, K.G.

    1988-01-01

    The interaction of an antagonist of arginine vasopressin (AVP), d(CH2)5-D-Tyr(Et)VAVP, with renal tubular V2 receptors were studied in medullary membrane preparations from kidneys of Sprague-Dawley and Brattleboro rats. In both rat strains, V2 receptors had comparable KD and Bmax values for binding of [3H]AVP. In vitro studies revealed that the V2-antagonist was more potent than cold AVP in displacing [3H]AVP. In vivo treatment of Sprague-Dawley rats with the antagonist over one week resulted only in a transient state of diabetes insipidus (DI). No specific [3H]AVP binding was detectable throughout the period of administration. Chronic treatment of Brattleboro rats resulted in a complete normalization of water intake. This agonistic effect was also associated with undetectable [3H]AVP binding. After stopping the infusion of d(CH2)5-D-Tyr(Et)VAVP, Bmax values tended to rise but had still not reached base line values after 6 days. In contrast, the chronic infusion of AVP in Brattleboro rats resulted in a reduction in water intake which was accompanied by a decreased Bmax. [3H]AVP binding remained detectable during the entire treatment period. Thereafter Bmax was restored to base line values within 2 days of stopping the infusion. These results suggest that d(CH2)5-D-Tyr(Et)VAVP has a high affinity for V2 receptors in both Sprague-Dawley and Brattleboro rats. Its rate of dissociation from the receptor appears to be much slower than that of AVP. In Brattleboro rats, the binding of d(CH2)5-D-Tyr(Et)VAVP leads to an antidiuretic response. In Sprague-Dawley rats, a transient diuretic response is followed by a progressive normalization in water intake. This occurs despite persistent and complete blockade of renal medullary V2 receptors

  7. Nicotinic cholinergic receptor in brain detected by binding of. cap alpha. -(/sup 3/H)bungarotoxin

    Energy Technology Data Exchange (ETDEWEB)

    Eterovic, V A; Bennett, E L

    1974-01-01

    ..cap alpha..-(/sup 3/H)bungarotoxin was prepared by catalytic reduction of iodinated ..cap alpha..-bungarotoxin with tritium gas. Crude mitochondrial fraction from rat cerebral cortex bound 40 x 10/sup -15/ to 60 x 10/sup -15/ moles of ..cap alpha..-(/sup 3/H)bungarotoxin per mg of protein. This binding was reduced by 50% in the presence of approx. 10/sup -6/ M d-tubocurarine or nicotine, 10/sup -5/ M acetylcholine, 10/sup -4/ M carbamylcholine or decamethonium or 10/sup -3/ M atropine. Hexamethonium and eserine were the least effective of the drugs tested. Crude mitochondrial fraction was separated into myelin, nerve endings, and mitochondria. The highest binding of toxin per mg of protein was found in nerve endings, as well as the greatest inhibition of toxin binding by d-tubocurarine. Binding of ..cap alpha..-(/sup 3/H)bungarotoxin to membranes obtained by osmotic shock of the crude mitochondrial fraction indicates that the receptor for the toxin is membrane bound. /sup 125/I-labeled ..cap alpha..-bungarotoxin, prepared with Na/sup 125/I and chloramine T, was highly specific for the acetylcholine receptor in diaphragm, however, it was less specific and less reliable than ..cap alpha..-(/sup 3/H)bungarotoxin in brain. It is concluded that a nicotinic cholinergic receptor exists in brain, and that ..cap alpha..-(/sup 3/H)bungarotoxin is a suitable probe for this receptor.

  8. Relative binding affinity prediction of farnesoid X receptor in the D3R Grand Challenge 2 using FEP+

    Science.gov (United States)

    Schindler, Christina; Rippmann, Friedrich; Kuhn, Daniel

    2018-01-01

    Physics-based free energy simulations have increasingly become an important tool for predicting binding affinity and the recent introduction of automated protocols has also paved the way towards a more widespread use in the pharmaceutical industry. The D3R 2016 Grand Challenge 2 provided an opportunity to blindly test the commercial free energy calculation protocol FEP+ and assess its performance relative to other affinity prediction methods. The present D3R free energy prediction challenge was built around two experimental data sets involving inhibitors of farnesoid X receptor (FXR) which is a promising anticancer drug target. The FXR binding site is predominantly hydrophobic with few conserved interaction motifs and strong induced fit effects making it a challenging target for molecular modeling and drug design. For both data sets, we achieved reasonable prediction accuracy (RMSD ≈ 1.4 kcal/mol, rank 3-4 according to RMSD out of 20 submissions) comparable to that of state-of-the-art methods in the field. Our D3R results boosted our confidence in the method and strengthen our desire to expand its applications in future in-house drug design projects.

  9. Relative binding affinity prediction of farnesoid X receptor in the D3R Grand Challenge 2 using FEP.

    Science.gov (United States)

    Schindler, Christina; Rippmann, Friedrich; Kuhn, Daniel

    2018-01-01

    Physics-based free energy simulations have increasingly become an important tool for predicting binding affinity and the recent introduction of automated protocols has also paved the way towards a more widespread use in the pharmaceutical industry. The D3R 2016 Grand Challenge 2 provided an opportunity to blindly test the commercial free energy calculation protocol FEP+ and assess its performance relative to other affinity prediction methods. The present D3R free energy prediction challenge was built around two experimental data sets involving inhibitors of farnesoid X receptor (FXR) which is a promising anticancer drug target. The FXR binding site is predominantly hydrophobic with few conserved interaction motifs and strong induced fit effects making it a challenging target for molecular modeling and drug design. For both data sets, we achieved reasonable prediction accuracy (RMSD ≈ 1.4 kcal/mol, rank 3-4 according to RMSD out of 20 submissions) comparable to that of state-of-the-art methods in the field. Our D3R results boosted our confidence in the method and strengthen our desire to expand its applications in future in-house drug design projects.

  10. Reduced striatal D2 receptor binding in myoclonus-dystonia

    International Nuclear Information System (INIS)

    Beukers, R.J.; Weisscher, N.; Tijssen, M.A.J.; Booij, J.; Zijlstra, F.; Amelsvoort, T.A.M.J. van

    2009-01-01

    To study striatal dopamine D 2 receptor availability in DYT11 mutation carriers of the autosomal dominantly inherited disorder myoclonus-dystonia (M-D). Fifteen DYT11 mutation carriers (11 clinically affected) and 15 age- and sex-matched controls were studied using 123 I-IBZM SPECT. Specific striatal binding ratios were calculated using standard templates for striatum and occipital areas. Multivariate analysis with corrections for ageing and smoking showed significantly lower specific striatal to occipital IBZM uptake ratios (SORs) both in the left and right striatum in clinically affected patients and also in all DYT11 mutation carriers compared to control subjects. Our findings are consistent with the theory of reduced dopamine D 2 receptor (D2R) availability in dystonia, although the possibility of increased endogenous dopamine, and consequently, competitive D2R occupancy cannot be ruled out. (orig.)

  11. 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

  12. 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

  13. Bitropic D3 Dopamine Receptor Selective Compounds as Potential Antipsychotics.

    Science.gov (United States)

    Luedtke, Robert R; Rangel-Barajas, Claudia; Malik, Mahinder; Reichert, David E; Mach, R H

    2015-01-01

    Neuropsychiatric disorders represent a substantial social and health care issue. The National Institutes of Health estimates that greater than 2 million adults suffer from neuropsychiatric disorders in the USA. These individuals experience symptoms that can include auditory hallucinations, delusions, unrealistic beliefs and cognitive dysfunction. Although antipsychotic medications are available, suboptimal therapeutic responses are observed for approximately one-third of patients. Therefore, there is still a need to explore new pharmacotherapeutic strategies for the treatment of neuropsychiatric disorders. Many of the medications that are used clinically to treat neuropsychiatric disorders have a pharmacological profile that includes being an antagonist at D2-like (D2, D3 and D4) dopamine receptor subtypes. However, dopamine receptor subtypes are involved in a variety of neuronal circuits that include movement coordination, cognition, emotion, affect, memory and the regulation of prolactin. Consequently, antagonism at D2-like receptors can also contribute to some of the adverse side effects associated with the long-term use of antipsychotics including the a) adverse extrapyramidal symptoms associated with the use of typical antipsychotics and b) metabolic side effects (weight gain, hyperglycemia, increased risk of diabetes mellitus, dyslipidemia and gynecomastia) associated with atypical antipsychotic use. Preclinical studies suggest that D3 versus D2 dopamine receptor selective compounds might represent an alternative strategy for the treatment of the symptoms of schizophrenia. In this review we discuss a) how bitropic Nphenylpiperazine D3 dopamine receptor selective compounds have been developed by modification of the primary (orthosteric) and secondary (allosteric or modulatory) pharmacophores to optimize D3 receptor affinity and D2/D3 binding selectivity ratios and b) the functional selectivity of these compounds. Examples of how these compounds might be

  14. Photoaffinity labeling of serum vitamin D binding protein by 3-deoxy-3-azido-25-hydroxyvitamin D3

    International Nuclear Information System (INIS)

    Link, R.P.; Kutner, A.; Schnoes, H.K.; DeLuca, H.F.

    1987-01-01

    3-Deoxy-3-azido-25-hydroxyvitamin D3 was covalently incorporated in the 25-hydroxyvitamin D3 binding site of purified human plasma vitamin D binding protein. Competition experiments showed that 3-deoxy-3-azido-25-hydroxyvitamin D3 and 25-hydroxyvitamin D3 bind at the same site on the protein. Tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was synthesized from tritiated 25-hydroxyvitamin D3, retaining the high specific activity of the parent compound. The tritiated azido label bound reversibly to human vitamin D binding protein in the dark and covalently to human vitamin D binding protein after exposure to ultraviolet light. Reversible binding of tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was compared to tritiated 25-hydroxyvitamin D3 binding to human vitamin D binding protein. Scatchard analysis of the data indicated equivalent maximum density binding sites with a KD,app of 0.21 nM for 25-hydroxyvitamin D3 and a KD,app of 1.3 nM for the azido derivative. Covalent binding was observed only after exposure to ultraviolet irradiation, with an average of 3% of the reversibly bound label becoming covalently bound to vitamin D binding protein. The covalent binding was reduced 70-80% when 25-hydroxyvitamin D3 was present, indicating strong covalent binding at the vitamin D binding site of the protein. When tritiated 3-deoxy-3-azido-25-hydroxyvitamin D3 was incubated with human plasma in the absence and presence of 25-hydroxyvitamin D3, 12% of the azido derivative was reversibly bound to vitamin D binding protein. After ultraviolet irradiation, four plasma proteins covalently bound the azido label, but vitamin D binding protein was the only protein of the four that was unlabeled in the presence of 25-hydroxyvitamin D3

  15. Cyclin D3 interacts with vitamin D receptor and regulates its transcription activity

    International Nuclear Information System (INIS)

    Jian Yongzhi; Yan Jun; Wang Hanzhou; Chen Chen; Sun Maoyun; Jiang Jianhai; Lu Jieqiong; Yang Yanzhong; Gu Jianxin

    2005-01-01

    D-type cyclins are essential for the progression through the G1 phase of the cell cycle. Besides serving as cell cycle regulators, D-type cyclins were recently reported to have transcription regulation functions. Here, we report that cyclin D3 is a new interacting partner of vitamin D receptor (VDR), a member of the superfamily of nuclear receptors for steroid hormones, thyroid hormone, and the fat-soluble vitamins A and D. The interaction was confirmed with methods of yeast two-hybrid system, in vitro binding analysis and in vivo co-immunoprecipitation. Cyclin D3 interacted with VDR in a ligand-independent manner, but treatment of the ligand, 1,25-dihydroxyvitamin D3, strengthened the interaction. Confocal microscopy analysis showed that ligand-activated VDR led to an accumulation of cyclin D3 in the nuclear region. Cyclin D3 up-regulated transcriptional activity of VDR and this effect was counteracted by overexpression of CDK4 and CDK6. These findings provide us a new clue to understand the transcription regulation functions of D-type cyclins

  16. The role of receptor topology in the vitamin D3 uptake and Ca"2"+ response systems

    International Nuclear Information System (INIS)

    Morrill, Gene A.; Kostellow, Adele B.; Gupta, Raj K.

    2016-01-01

    The steroid hormone, vitamin D_3, regulates gene transcription via at least two receptors and initiates putative rapid response systems at the plasma membrane. The vitamin D receptor (VDR) binds vitamin D_3 and a second receptor, importin-4, imports the VDR-vitamin D_3 complex into the nucleus via nuclear pores. Here we present evidence that the Homo sapiens VDR homodimer contains two transmembrane (TM) helices ("3"2"7E – D"3"4"2), two TM “half-helix” ("2"6"4K − N"2"7"6), one or more large channels, and 16 cholesterol binding (CRAC/CARC) domains. The importin-4 monomer exhibits 3 pore-lining regions ("2"2"6E – L"2"5"1; "7"6"8V – G"7"8"3; "8"7"6S – A"8"9"1) and 16 CRAC/CARC domains. The MEMSAT algorithm indicates that VDR and importin-4 may not be restricted to cytoplasm and nucleus. VDR homodimer TM helix-topology predicts insertion into the plasma membrane, with two 84 residue C-terminal regions being extracellular. Similarly, MEMSAT predicts importin-4 insertion into the plasma membrane with 226 residue extracellular N-terminal regions and 96 residue C-terminal extracellular loops; with the pore-lining regions contributing gated Ca"2"+ channels. The PoreWalker algorithm indicates that, of the 427 residues in each VDR monomer, 91 line the largest channel, including two vitamin D_3 binding sites and residues from both the TM helix and “half-helix”. Cholesterol-binding domains also extend into the channel within the ligand binding region. Programmed changes in bound cholesterol may regulate both membrane Ca"2"+ response systems and vitamin D_3 uptake as well as receptor internalization by the endomembrane system culminating in uptake of the vitamin D_3-VDR-importin-4 complex into the nucleus.

  17. Neurovascular coupling to D2/D3 dopamine receptor occupancy using simultaneous PET/functional MRI

    DEFF Research Database (Denmark)

    Sander, Christin Y; Hooker, Jacob M; Catana, Ciprian

    2013-01-01

    This study employed simultaneous neuroimaging with positron emission tomography (PET) and functional magnetic resonance imaging (fMRI) to demonstrate the relationship between changes in receptor occupancy measured by PET and changes in brain activity inferred by fMRI. By administering the D2/D3...... dopamine receptor antagonist [(11)C]raclopride at varying specific activities to anesthetized nonhuman primates, we mapped associations between changes in receptor occupancy and hemodynamics [cerebral blood volume (CBV)] in the domains of space, time, and dose. Mass doses of raclopride above tracer levels...... caused increases in CBV and reductions in binding potential that were localized to the dopamine-rich striatum. Moreover, similar temporal profiles were observed for specific binding estimates and changes in CBV. Injection of graded raclopride mass doses revealed a monotonic coupling between neurovascular...

  18. Frontal D2/3 Receptor Availability in Schizophrenia Patients Before and After Their First Antipsychotic Treatment: Relation to Cognitive Functions and Psychopathology.

    Science.gov (United States)

    Nørbak-Emig, Henrik; Ebdrup, Bjørn H; Fagerlund, Birgitte; Svarer, Claus; Rasmussen, Hans; Friberg, Lars; Allerup, Peter N; Rostrup, Egill; Pinborg, Lars H; Glenthøj, Birte Y

    2016-05-01

    We have previously reported associations between frontal D2/3 receptor binding potential positive symptoms and cognitive deficits in antipsychotic-naïve schizophrenia patients. Here, we examined the effect of dopamine D2/3 receptor blockade on cognition. Additionally, we explored the relation between frontal D2/3 receptor availability and treatment effect on positive symptoms. Twenty-five antipsychotic-naïve first-episode schizophrenia patients were examined with the Positive and Negative Syndrome Scale, tested with the cognitive test battery Cambridge Neuropsychological Test Automated Battery, scanned with single-photon emission computerized tomography using the dopamine D2/3 receptor ligand [(123)I]epidepride, and scanned with MRI. After 3 months of treatment with either risperidone (n=13) or zuclopenthixol (n=9), 22 patients were reexamined. Blockade of extrastriatal dopamine D2/3 receptors was correlated with decreased attentional focus (r = -0.615, P=.003) and planning time (r = -0.436, P=.048). Moreover, baseline frontal dopamine D2/3 binding potential and positive symptom reduction correlated positively (D2/3 receptor binding potential left frontal cortex rho = 0.56, P=.003; D2/3 receptor binding potential right frontal cortex rho = 0.48, P=.016). Our data support the hypothesis of a negative influence of D2/3 receptor blockade on specific cognitive functions in schizophrenia. This is highly clinically relevant given the well-established association between severity of cognitive disturbances and a poor functional outcome in schizophrenia. Additionally, the findings support associations between frontal D2/3 receptor binding potential at baseline and the effect of antipsychotic treatment on positive symptoms. © The Author 2016. Published by Oxford University Press on behalf of CINP.

  19. 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)

  20. GPR17: Molecular modeling and dynamics studies of the 3-D structure and purinergic ligand binding features in comparison with P2Y receptors

    Directory of Open Access Journals (Sweden)

    Ranghino Graziella

    2008-06-01

    Full Text Available Abstract Background GPR17 is a G-protein-coupled receptor located at intermediate phylogenetic position between two distinct receptor families: the P2Y and CysLT receptors for extracellular nucleotides and cysteinyl-LTs, respectively. We previously showed that GPR17 can indeed respond to both classes of endogenous ligands and to synthetic compounds active at the above receptor families, thus representing the first fully characterized non-peptide "hybrid" GPCR. In a rat brain focal ischemia model, the selective in vivo knock down of GPR17 by anti-sense technology or P2Y/CysLT antagonists reduced progression of ischemic damage, thus highlighting GPR17 as a novel therapeutic target for stroke. Elucidation of the structure of GPR17 and of ligand binding mechanisms are the necessary steps to obtain selective and potent drugs for this new potential target. On this basis, a 3-D molecular model of GPR17 embedded in a solvated phospholipid bilayer and refined by molecular dynamics simulations has been the first aim of this study. To explore the binding mode of the "purinergic" component of the receptor, the endogenous agonist UDP and two P2Y receptor antagonists demonstrated to be active on GPR17 (MRS2179 and cangrelor were then modeled on the receptor. Results Molecular dynamics simulations suggest that GPR17 nucleotide binding pocket is similar to that described for the other P2Y receptors, although only one of the three basic residues that have been typically involved in ligand recognition is conserved (Arg255. The binding pocket is enclosed between the helical bundle and covered at the top by EL2. Driving interactions are H-bonds and salt bridges between the 6.55 and 6.52 residues and the phosphate moieties of the ligands. An "accessory" binding site in a region formed by the EL2, EL3 and the Nt was also found. Conclusion Nucleotide binding to GPR17 occurs on the same receptor regions identified for already known P2Y receptors. Agonist

  1. Vitamin D receptor displays DNA binding and transactivation as a heterodimer with the retinoid X receptor, but not with the thyroid hormone receptor.

    Science.gov (United States)

    Thompson, P D; Hsieh, J C; Whitfield, G K; Haussler, C A; Jurutka, P W; Galligan, M A; Tillman, J B; Spindler, S R; Haussler, M R

    1999-12-01

    The vitamin D receptor (VDR) is a transcription factor believed to function as a heterodimer with the retinoid X receptor (RXR). However, it was reported [Schräder et al., 1994] that, on putative vitamin D response elements (VDREs) within the rat 9k and mouse 28k calcium binding protein genes (rCaBP 9k and mCaBP 28k), VDR and thyroid hormone receptor (TR) form heterodimers that transactivate in response to both 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) and triiodothyronine (T(3)). We, therefore, examined associations of these receptors on the putative rCaBP 9k and mCaBP 28k VDREs, as well as on established VDREs from the rat osteocalcin (rOC) and mouse osteopontin (mOP) genes, plus the thyroid hormone response element (TRE) from the rat myosin heavy chain (rMHC) gene. In gel mobility shift assays, we found no evidence for VDR-TR heterodimer interaction with any tested element. Further, employing these hormone response elements linked to reporter genes in transfected cells, VDR and TR mediated responses to their cognate ligands only from the rOC/mOP and rMHC elements, respectively, while the CaBP elements were unresponsive to any combination of ligand(s). Utilizing the rOC and mOP VDREs, two distinct repressive actions of TR on VDR-mediated signaling were demonstrated: a T(3)-independent action, presumably via direct TR-RXR competition for DNA binding, and a T(3)-dependent repression, likely by diversion of limiting RXR from VDR-RXR toward the formation of TR-RXR heterodimers. The relative importance of these two mechanisms differed in a response element-specific manner. These results may provide a partial explanation for the observed association between hyperthyroidism and bone demineralization/osteoporosis. Copyright 1999 Wiley-Liss, Inc.

  2. Linearized method: A new approach for kinetic analysis of central dopamine D2 receptor specific binding

    International Nuclear Information System (INIS)

    Watabe, Hiroshi; Hatazawa, Jun; Ishiwata, Kiichi; Ido, Tatsuo; Itoh, Masatoshi; Iwata, Ren; Nakamura, Takashi; Takahashi, Toshihiro; Hatano, Kentaro

    1995-01-01

    The authors proposed a new method (Linearized method) to analyze neuroleptic ligand-receptor specific binding in a human brain using positron emission tomography (PET). They derived the linear equation to solve four rate constants, k 3 , k 4 , k 5 , k 6 from PET data. This method does not demand radioactivity curve in plasma as an input function to brain, and can do fast calculations in order to determine rate constants. They also tested Nonlinearized method including nonlinear equations which is conventional analysis using plasma radioactivity corrected for ligand metabolites as an input function. The authors applied these methods to evaluate dopamine D 2 receptor specific binding of [ 11 C] YM-09151-2. The value of B max /K d = k 3 k 4 obtained by Linearized method was 5.72 ± 3.1 which was consistent with the value of 5.78 ± 3.4 obtained by Nonlinearized method

  3. 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

  4. 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.

  5. [¹¹C]-(+)-PHNO PET imaging of dopamine D(2/3) receptors in Parkinson's disease with impulse control disorders.

    Science.gov (United States)

    Payer, Doris E; Guttman, Mark; Kish, Stephen J; Tong, Junchao; Strafella, Antonio; Zack, Martin; Adams, John R; Rusjan, Pablo; Houle, Sylvain; Furukawa, Yoshiaki; Wilson, Alan A; Boileau, Isabelle

    2015-02-01

    Dopamine agonist medications with high affinity for the D3 dopamine receptor are commonly used to treat Parkinson's disease, and have been associated with pathological behaviors categorized under the umbrella of impulse control disorders (ICD). The aim of this study was to investigate whether ICD in Parkinson's patients are associated with greater D3 dopamine receptor availability. We used positron emission tomography (PET) radioligand imaging with the D3 dopamine receptor preferring agonist [¹¹C]-(+)-propyl-hexahydro-naphtho-oxazin (PHNO) in Parkinson's patients with (n = 11) and without (n = 21) ICD, and age-, sex-, and education-matched healthy control subjects (n = 18). Contrary to hypotheses, [¹¹C]-(+)-PHNO binding in D3 -rich brain areas was not elevated in Parkinson's patients with ICD compared with those without; instead, [¹¹C]-(+)-PHNO binding in ventral striatum was 20% lower (P = 0.011), correlating with two measures of ICD severity (r = -0.8 and -0.9), which may reflect higher dopamine tone in ventral striatum. In dorsal striatum, where [¹¹C]-(+)-PHNO binding is associated with D2 receptor levels, [¹¹C]-(+)-PHNO binding was elevated across patients compared with controls. We conclude that although D3 dopamine receptors have been linked to the occurrence of ICD in Parkinson's patients. Our findings do not support the hypothesis that D3 receptor levels are elevated in Parkinson's patients with ICD. We also did not find ICD-related abnormalities in D2 receptor levels. Our findings argue against the possibility that differences in D2/3 receptor levels can account for the development of ICD in PD; however, we cannot rule out that differences in dopamine levels (particularly in ventral striatum) may be involved. © 2015 International Parkinson and Movement Disorder Society.

  6. Binding of the ligand [3H]MK-801 to the MK-801 binding site of the N-methyl-D-aspartate receptor during experimental encephalopathy from acute liver failure and from acute hyperammonemia in the rabbit

    NARCIS (Netherlands)

    R.J. de Knegt (Robert); J. Kornhuber (Johannes); S.W. Schalm (Solko); K. Rusche (K.); P.F. Riederer (Peter); J. Tan (J.)

    1993-01-01

    textabstractBinding of the ligand [3H]MK-801 to the MK-801 binding site of the N-methyl-D-aspartate (NMDA) receptor population on brain homogenates in rabbits was studied during experimental encephalopathy from acute liver failure and from acute hyperammonemia in the rabbit. Homogenates were

  7. [18F]fallypride characterization of striatal and extrastriatal D2/3 receptors in Parkinson's disease.

    Science.gov (United States)

    Stark, Adam J; Smith, Christopher T; Petersen, Kalen J; Trujillo, Paula; van Wouwe, Nelleke C; Donahue, Manus J; Kessler, Robert M; Deutch, Ariel Y; Zald, David H; Claassen, Daniel O

    2018-01-01

    Parkinson's disease (PD) is characterized by widespread degeneration of monoaminergic (especially dopaminergic) networks, manifesting with a number of both motor and non-motor symptoms. Regional alterations to dopamine D 2/3 receptors in PD patients are documented in striatal and some extrastriatal areas, and medications that target D 2/3 receptors can improve motor and non-motor symptoms. However, data regarding the combined pattern of D 2/3 receptor binding in both striatal and extrastriatal regions in PD are limited. We studied 35 PD patients off-medication and 31 age- and sex-matched healthy controls (HCs) using PET imaging with [ 18 F]fallypride, a high affinity D 2/3 receptor ligand, to measure striatal and extrastriatal D 2/3 nondisplaceable binding potential (BP ND ). PD patients completed PET imaging in the off medication state, and motor severity was concurrently assessed. Voxel-wise evaluation between groups revealed significant BP ND reductions in PD patients in striatal and several extrastriatal regions, including the locus coeruleus and mesotemporal cortex. A region-of-interest (ROI) based approach quantified differences in dopamine D 2/3 receptors, where reduced BP ND was noted in the globus pallidus, caudate, amygdala, hippocampus, ventral midbrain, and thalamus of PD patients relative to HC subjects. Motor severity positively correlated with D 2/3 receptor density in the putamen and globus pallidus. These findings support the hypothesis that abnormal D 2/3 expression occurs in regions related to both the motor and non-motor symptoms of PD, including areas richly invested with noradrenergic neurons.

  8. Targeting the dopamine D3 receptor: an overview of drug design strategies.

    Science.gov (United States)

    Cortés, Antoni; Moreno, Estefanía; Rodríguez-Ruiz, Mar; Canela, Enric I; Casadó, Vicent

    2016-07-01

    Dopamine is a neurotransmitter widely distributed in both the periphery and the central nervous system (CNS). Its physiological effects are mediated by five closely related G protein-coupled receptors (GPCRs) that are divided into two major subclasses: the D1-like (D1, D5) and the D2-like (D2, D3, D4) receptors. D3 receptors (D3Rs) have the highest density in the limbic areas of the brain, which are associated with cognitive and emotional functions. These receptors are therefore attractive targets for therapeutic management. This review summarizes the functional and pharmacological characteristics of D3Rs, including the design and clinical relevance of full agonists, partial agonists and antagonists, as well as the capacity of these receptors to form active homodimers, heterodimers or higher order receptor complexes as pharmacological targets in several neurological and neurodegenerative disorders. The high sequence homology between D3R and the D2-type challenges the development of D3R-selective compounds. The design of new D3R-preferential ligands with improved physicochemical properties should provide a better pharmacokinetic/bioavailability profile and lesser toxicity than is found with existing D3R ligands. It is also essential to optimize D3R affinity and, especially, D3R vs. D2-type binding and functional selectivity ratios. Developing allosteric and bitopic ligands should help to improve the D3R selectivity of these drugs. As most evidence points to the ability of GPCRs to form homomers and heteromers, the most promising therapeutic strategy in the future is likely to involve the application of heteromer-selective drugs. These selective ligands would display different affinities for a given receptor depending on the receptor partners within the heteromer. Therefore, designing novel compounds that specifically target and modulate D1R-D3R heteromers would be an interesting approach for the treatment of levodopa (L-DOPA)-induced dyskinesias.

  9. 124I-Epidepride: A PET radiotracer for extended imaging of dopamine D2/D3 receptors

    International Nuclear Information System (INIS)

    Pandey, Suresh; Venugopal, Archana; Kant, Ritu; Coleman, Robert; Mukherjee, Jogeshwar

    2014-01-01

    Objectives: A new radiotracer, 124 I-epidepride, has been developed for the imaging of dopamine D2/3 receptors (D2/3Rs). 124 I-Epidepride (half-life of 124 I = 4.2 days) allows imaging over extended periods compared to 18 F-fallypride (half-life of 18 F = 0.076 days) and may maximize visualization of D2/3Rs in the brain and pancreas (allowing clearance from adjacent organs). D2/3Rs are also present in pancreatic islets where they co-localize with insulin to produce granules and may serve as a surrogate marker for imaging diabetes. Methods: 124 I-Epidepride was synthesized using N-[[(2S)-1-ethylpyrrolidin-2-yl]methyl]-5-tributyltin-2, 3-dimethoxybenzamide and 124 I-iodide under no carrier added condition. Rats were used for in vitro and in vivo imaging. Brain slices were incubated with 124 I-epidepride (0.75 μCi/cc) and nonspecific binding measured with 10 μM haloperidol. Autoradiograms were analyzed by OptiQuant. 124 I-Epidepride (0.2 to 0.3 mCi, iv) was administered to rats and brain uptake at 3 hours, 24 hours, and 48 hours post injection was evaluated. Results: 124 I-Epidepride was obtained with 50% radiochemical yield and high radiochemical purity (> 95%). 124 I-Epidepride localized in the striatum with a striatum to cerebellum ratio of 10. Binding was displaced by dopamine and haloperidol. Brain slices demonstrated localization of 124 I-epidepride up until 48 hours in the striatum. However, the extent of binding was reduced significantly. Conclusions: 124 I-Epidepride is a new radiotracer suitable for extended imaging of dopamine D2/3 receptors and may have applications in imaging of receptors in the brain and monitoring pancreatic islet cell grafting

  10. The role of receptor topology in the vitamin D3 uptake and Ca{sup 2+} response systems

    Energy Technology Data Exchange (ETDEWEB)

    Morrill, Gene A., E-mail: gene.morrill@einstein.yu.edu; Kostellow, Adele B.; Gupta, Raj K.

    2016-09-02

    The steroid hormone, vitamin D{sub 3}, regulates gene transcription via at least two receptors and initiates putative rapid response systems at the plasma membrane. The vitamin D receptor (VDR) binds vitamin D{sub 3} and a second receptor, importin-4, imports the VDR-vitamin D{sub 3} complex into the nucleus via nuclear pores. Here we present evidence that the Homo sapiens VDR homodimer contains two transmembrane (TM) helices ({sup 327}E – D{sup 342}), two TM “half-helix” ({sup 264}K − N{sup 276}), one or more large channels, and 16 cholesterol binding (CRAC/CARC) domains. The importin-4 monomer exhibits 3 pore-lining regions ({sup 226}E – L{sup 251}; {sup 768}V – G{sup 783}; {sup 876}S – A{sup 891}) and 16 CRAC/CARC domains. The MEMSAT algorithm indicates that VDR and importin-4 may not be restricted to cytoplasm and nucleus. VDR homodimer TM helix-topology predicts insertion into the plasma membrane, with two 84 residue C-terminal regions being extracellular. Similarly, MEMSAT predicts importin-4 insertion into the plasma membrane with 226 residue extracellular N-terminal regions and 96 residue C-terminal extracellular loops; with the pore-lining regions contributing gated Ca{sup 2+} channels. The PoreWalker algorithm indicates that, of the 427 residues in each VDR monomer, 91 line the largest channel, including two vitamin D{sub 3} binding sites and residues from both the TM helix and “half-helix”. Cholesterol-binding domains also extend into the channel within the ligand binding region. Programmed changes in bound cholesterol may regulate both membrane Ca{sup 2+} response systems and vitamin D{sub 3} uptake as well as receptor internalization by the endomembrane system culminating in uptake of the vitamin D{sub 3}-VDR-importin-4 complex into the nucleus.

  11. Effects of single and repeated administration of 1,2,3,4-tetrahydroisoquinoline analogs on the binding of [11C]raclopride to dopamine D2 receptors in the mouse brain

    International Nuclear Information System (INIS)

    Ishiwata, K.; Senda, M.; Saitoh, T.; Taguchi, K.; Toda, J.; Sano, T.; Koyanagi, Y.

    2001-01-01

    We investigated the effects of intraperitoneal injection of 1,2,3,4-tetrahydroisoquinoline (TIQ) analogs and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on the binding of [ 11 C]raclopride to striatal dopamine D 2 receptors in mice. The binding of [ 11 C]raclopride, but not of [ 11 C]N-methylspiperone or [ 11 C]nemonapride with higher affinity, to the receptors was significantly decreased immediately after TIQ injection. Neither a dopamine transporter blocker induced such effect nor TIQ affected the dopamine transporter-radioligand binding. Among the compounds investigated, including parkinsonism-inducing TIQ and (R/S)-1-benzyl-TIQ, parkinsonism-preventing (R)- and (S)-1-methyl-TIQ, and probable N-methylated metabolites of TIQ and 1-methyl-TIQ, TIQ and (S)-1-methyl-TIQ had the strongest effect on the binding of [ 11 C]raclopride, and N-methylated derivatives showed less of an effect than the respective parent compounds. The decrease in the binding of [ 11 C]raclopride continued for 7 hours and was followed by an increase until 10 days after the single and subchronic administration of TIQ. These findings suggest that TIQ analogs profoundly stimulated dopamine release which resulted in the competitive inhibition of the binding of [ 11 C]raclopride to dopamine D 2 receptors, but did not induce degeneration of the receptors. (author)

  12. Recent Methods for Measuring Dopamine D3 receptor Occupancy In Vivo: Importance for Drug Development

    Directory of Open Access Journals (Sweden)

    Bernard eLe Foll

    2014-07-01

    Full Text Available There is considerable interest in developing highly selective dopamine D3 receptor ligands for a variety of mental health disorders. Dopamine D3 receptors have been implicated in Parkinson’s Disease, schizophrenia, anxiety, depression, and substance use disorders. The most concrete evidence suggests a role for the D3 receptor in drug-seeking behaviors. D3 receptors are a subtype of D2 receptors, and traditionally the functional role of these two receptors has been difficult to differentiate. Over the past 10-15 years a number of compounds selective for D3 over D2 receptors have been developed. However, translating these findings into clinical research has been difficult as many of these compounds cannot be used in humans. Therefore, the functional data involving the D3 receptor in drug addiction mostly comes from preclinical studies. Recently, with the advent of [11C]-(+-PHNO, it has become possible to image D3 receptors in the human brain with increased selectivity and sensitivity. This is a significant innovation over traditional methods such as [11C]-raclopride that cannot differentiate between D2 and D3 receptors. The use of [11C]-(+-PHNO will allow for further delineation of the role of D3 receptors. Here, we review recent evidence that the role of the D3 receptor has functional importance and is distinct from the role of the D2 receptor. We then introduce the utility of analyzing [11C]-(+-PHNO binding by region of interest. This novel methodology can be used in preclinical and clinical approaches for the measurement of occupancy of both D3 and D2 receptors. Evidence that [11C]-(+-PHNO can provide insights into the function of D3 receptors in addiction is also presented.

  13. PET measurement of D2 and S2 receptor binding of 3-N-([2'-18F]fluoroethyl)-spiperone in baboon brain

    International Nuclear Information System (INIS)

    Coenen, H.H.; Stoecklin, G.; Laufer, P.

    1988-01-01

    The regional pharmacokinetic behavior in baboon brain of 18 F-fluoro-ethyl and 18 F-fluoropropylspiperone ( 18 FESP, 18 FPSP) at specific activities ≥1000 Ci/mmol was studied with PET. Four hours after injection of 5-10 mCi 18 FESP, uptake in striatum was 0.048%±0.005% of injected dose per cm 3 , which is almost the same as with 18 F- and 11 C-methylspiperone. While 18 FPSP was taken up in much smaller amounts than 18 FESP, striatum to cerebellum activity ratios were quite similar for both ligands (about 9 to 10 at 4 h p.i.). Because of its higher striatal uptake, 18 FESP seems to be better suited for PET. Furthermore, relative binding to S 2 receptors was much smaller for FESP: Competing cold S 2 antagonists (ritanserin, ketanserin) did not alter 18 8FESP binding to striatum, concurrently reducing uptake in frontal cortex by only 15%-20%. With coinjection of increasing amounts of cold FESP, saturation of 18 FESP binding to striatum occurred at doses exceeding 10 μg per kg. Quantitative analysis of radiolabelled ligand in arterial plasma (decrease to 8% at 4 h p.i.) demonstrated identical metabolic turnover for both ligands. Direct use of binding fractions from the saturation curve resulted in overestimation of the receptor density in striatum. Using the 18 FESP plasma concentration time curve and the dynamic uptake data, k 3 of a three compartment model could be determined by non linear regression. However, dramatic changes of the dependence of k 3 on the specifically bound ligand concentration were observed even at small loading doses of FESP. Estimation of B max yielded a D 2 receptor density of only 6 pmol per cm 3 in baboon striatum. (orig.)

  14. Study on dopamine D2 binding capacity in vascular parkinsonism

    International Nuclear Information System (INIS)

    Terashi, Hiroo; Nagata, Ken; Hirata, Yutaka; Hatazawa, Jun; Utsumi, Hiroya

    2001-01-01

    To investigate whether the striatal dopamine receptor function is involved in the development of vascular parkinsonism (VP), a positron emission tomography (PET) study was conducted on 9 patients with VP by using [ 11 C] N-methylspiperone as the tracer. The rate of binding availability in the striatal dopamine D 2 receptor (k 3 ) was determined semiquantitatively, and the values were compared to the predicted normal values based on the results from 7 normal volunteers. Of 9 patients with VP, the normalized D 2 receptor binding [%k 3 ] was more than 90% in 5 patients, 89 to 87% in 3, and 75% in one. These values showed no evident correlation with the Hoehn and Yahr stage. The laterality of the striatal %k 3 did not correspond to that of the parkinsonism. Thus, the striatal dopamine D 2 receptor binding was not severely impaired and did not correlate with the neurological status in patients with VP. This may indicate that striatal dopamine D 2 receptor function is not primarily associated with the development of the parkinsonism in VP. (author)

  15. Link between D1 and D2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain

    International Nuclear Information System (INIS)

    Seeman, P.; Niznik, H.B.; Guan, H.C.; Booth, G.; Ulpian, C.

    1989-01-01

    Dopamine receptor types D 1 and D 2 can oppose enhance each other's actions for electrical, biochemical, and psychomotor effects. The authors report a D 1 -D 2 interaction in homogenized tissue as revealed by ligand binding. D 2 agonists lowered the binding of [ 3 H]raclopride to D 2 receptors in striatal and anterior pituitary tissues. Pretreating the tissue with the D 1 -selective antagonist SCH 23390 prevented the agonist-induced decrease in [ 3 H]raclopride binding to D 2 sites in the striatum but not in the anterior pituitary, which has no D 1 receptors. Conversely, a dopamine-induced reduction in the binding of [ 3 H]SCH 23390 to D 1 receptors could be prevented by the D 2 -selective antagonist eticlopride. Receptor photolabeling experiments confirmed both these D 1 -D 2 interactions. The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D 2 receptors in the high-affinity state. Thus, the D 1 -D 2 link may be mediated by guanine nucleotide-binding protein components. The link may underlie D 1 -D 2 interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata

  16. [123I]Iodobenzamide binding to the rat dopamine D2 receptor in competition with haloperidol and endogenous dopamine - an in vivo imaging study with a dedicated small animal SPECT

    International Nuclear Information System (INIS)

    Nikolaus, Susanne; Larisch, Rolf; Wirrwar, Andreas; Jamdjeu-Noune, Marlyse; Antke, Christina; Beu, Markus; Mueller, Hans-Wilhelm; Schramm, Nils

    2005-01-01

    This study assessed [ 123 I]iodobenzamide binding to the rat dopamine D 2 receptor in competition with haloperidol and endogenous dopamine using a high-resolution small animal SPECT. Subsequent to baseline quantifications of D 2 receptor binding, imaging studies were performed on the same animals after pre-treatment with haloperidol and methylphenidate, which block D 2 receptors and dopamine transporters, respectively. Striatal baseline equilibrium ratios (V 3 '' ) of [ 123 I]iodobenzamide binding were 1.42±0.31 (mean±SD). After pre-treatment with haloperidol and methylphenidate, V 3 '' values decreased to 0.54±0.46 (p 123 I]iodobenzamide binding induced by pre-treatment with haloperidol reflects D 2 receptor blockade, whereas the decrease in receptor binding induced by pre-treatment with methylphenidate can be interpreted in terms of competition between [ 123 I]IBZM and endogenous dopamine. Findings show that multiple in vivo measurements of [ 123 I]iodobenzamide binding to D 2 receptors in competition with exogenous and endogenous ligands are feasible in the same animal. This may be of future relevance for the in vivo evaluation of novel radioligands as well as for studying the interrelations between pre- and/or postsynaptic radioligand binding and different levels of endogenous dopamine. (orig.)

  17. Effects of acute nicotine on hemodynamics and binding of [11C]raclopride to dopamine D2,3 receptors in pig brain.

    Science.gov (United States)

    Cumming, Paul; Rosa-Neto, Pedro; Watanabe, Hideaki; Smith, Donald; Bender, Dirk; Clarke, Paul B S; Gjedde, Albert

    2003-07-01

    Positive reinforcing properties of nicotine and the psychostimulants have been attributed to elevated dopamine release in the basal ganglia. It is well known that the specific binding of [(11)C]raclopride to dopamine D(2,3) receptors in living striatum is reduced by cocaine and amphetamines, revealing increased competition between endogenous dopamine and [(11)C]raclopride for dopamine D(2,3) receptors. However, the sensitivity of [(11)C]raclopride binding to nicotine-induced dopamine release is less well documented. In order to provide the basis for mapping effects of nicotine, we first optimized reference tissue methods for quantifying [(11)C]raclopride binding sites in striatum of living pigs (n = 16). In the same animals, the rate of cerebral blood flow (CBF) was mapped using [(15)O]water. Neither a low dose of nicotine (50 mu kg(-1), iv) nor a high dose of nicotine (500 microg kg(-1), iv) altered CBF in the pig brain, an important condition for calculating the binding of radioligands when using a reference tissue to estimate the free ligand concentration. The methods of Logan and of Lammertsma were compared using the cerebellum or the occipital cortex as reference tissues for calculating the binding potential (pB) of [(11)C]raclolpride in brain. Irrespective of the method used, the mean undrugged baseline pB in striatum (ca. 2.0) was significantly asymmetric, with highest binding in the left caudate and right putamen. Test-retest estimates of pB were stable. Subtraction of Logan pB maps revealed that the low dose of nicotine reduced the pB of [(11)C]raclopride by 10% in a cluster of voxels in the left anteroventral striatum, but this effect did not persist after correction for multiple comparisons. The high dose of nicotine (n = 9) acutely reduced pB by 10% bilaterally in the ventral striatum; 3 h after the high nicotine dose, the reductions had shifted dorsally and caudally into the caudate and putamen. Evidently, nicotine challenge enhances the competition

  18. Identification of D3 and sigma receptors in the rat striatum and nucleus accumbens using (+/-)-7-hydroxy-N,N-di-n-[3H]propyl-2-aminotetralin and carbetapentane.

    Science.gov (United States)

    Wallace, D R; Booze, R M

    1995-02-01

    Cross-reactions between dopamine D3 and sigma receptor ligands were investigated using (+/-)-7-hydroxy-N,N-di-n-[3H]propyl-2-aminotetralin [(+/-)-7-OH-[3H]-DPAT], a putative D3-selective radioligand, in conjunction with the unlabeled sigma ligands 1,3-di(2-tolyl)guanidine (DTG), carbetapentane, and R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane [R(-)-PPAP]. In transfected CCL1.3 mouse fibroblasts expressing the human D3 receptor, neither DTG nor carbetapentane (0.1 microM) displaced (+/-)-7-OH-[3H]DPAT binding. R(-)-PPAP (0.1 microM) displaced 39.6 +/- 1.0% of total (+/-)-7-OH-[3H]DPAT binding. In striatal and nucleus accumbens homogenates, (+/-)-7-OH-[3H]DPAT labeled a single site (15-20 fmol/mg of protein) with high (1 nM) affinity. Competition analysis with carbetapentane defined both high- and low-affinity sites in striatal (35 and 65%, respectively) and nucleus accumbens (59 and 41%, respectively) tissue, yet R(-)-PPAP identified two sites in equal proportion. Carbetapentane and R(-)-PPAP (0.1 microM) displaced approximately 20-50% of total (+/-)-7-OH-[3H]DPAT binding in striatum, nucleus accumbens, and olfactory tubercle in autoradiographic studies, with the nucleus accumbens shell subregion exhibiting the greatest displacement. To determine directly (+)-7-OH-[3H]DPAT binding to sigma receptors, saturation analysis was performed in the cerebellum while masking D3 receptors with 1 microM dopamine. Under these conditions (+)-7-OH-[3H]DPAT labeled sigma receptors with an affinity of 24 nM. These results suggest that (a) (+/-)-7-OH-[3H]DPAT binds D3 receptors with high affinity in rat brain and (b) a significant proportion of (+/-)-7-OH-[3H]DPAT binding consists of sigma 1 sites and the percentages of these sites differ among the subregions of the striatum and nucleus accumbens.

  19. Receptor-based 3D-QSAR in Drug Design: Methods and Applications in Kinase Studies.

    Science.gov (United States)

    Fang, Cheng; Xiao, Zhiyan

    2016-01-01

    Receptor-based 3D-QSAR strategy represents a superior integration of structure-based drug design (SBDD) and three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis. It combines the accurate prediction of ligand poses by the SBDD approach with the good predictability and interpretability of statistical models derived from the 3D-QSAR approach. Extensive efforts have been devoted to the development of receptor-based 3D-QSAR methods and two alternative approaches have been exploited. One associates with computing the binding interactions between a receptor and a ligand to generate structure-based descriptors for QSAR analyses. The other concerns the application of various docking protocols to generate optimal ligand poses so as to provide reliable molecular alignments for the conventional 3D-QSAR operations. This review highlights new concepts and methodologies recently developed in the field of receptorbased 3D-QSAR, and in particular, covers its application in kinase studies.

  20. Yttrium-90 and indium-111 labelling, receptor binding and biodistribution of [DOTA0,d-Phe1,Tyr3[octreotide, a promising somatostatin analogue for radionuclide therapy

    International Nuclear Information System (INIS)

    Jong, M. de; Bakker, W.H.; Krenning, E.P.; Breeman, W.A.P.; Pluijm, M.E. van der; Bernard, B.F.; Visser, T.J.; Jermann, E.; Behe, M.; Powell, P.; Maecke, H.R.

    1997-01-01

    In vitro octreotide receptor binding of [ 111 In-DOTA 0 ,d-Phe 1 ,Tyr 3 [octreotide ( 111 In-DOTATOC) and the in vivo metabolism of 90 Y- or 111 In-labelled DOTATOC were investigated in rats in comparison with [ 111 In-DTPA 0 [octreotide [ 111 In-DTPAOC). 111 In-DOTATOC was found to have an affinity similar to octreotide itself for the octreotide receptor in rat cerebral cortex microsomes. Twenty-four hours after injection of 90 Y- or 111 In-labelled DOTATOC, uptake of radioactivity in the octreotide receptor-expressing tissues pancreas, pituitary, adrenals and tumour was a factor of 2-6 that after injection of 111 In-DTPAOC. Uptake of labelled DOTATOC in pituitary, pancreas, adrenals and tumour was almost completely blocked by pretreatment with 0.5 mg unlabelled octreotide, indicating specific binding to the octreotide receptors. These findings strongly indicate that 90 Y-DOTATOC is a promising radiopharmaceutical for radiotherapy and that 111 In-DOTATOC is of potential value for diagnosis of patients with octreotide receptor-positive lesions, such as most neuroendocrine tumours. (orig.). With 3 figs., 2 tabs

  1. A new point mutation in the deoxyribonuclic acid-binding domain of the vitamine D receptor in a kindred with hereditary 1,25-dihydroxyvitamin d-resistant rickets

    Energy Technology Data Exchange (ETDEWEB)

    Yagi, Hideki; Miyake, Hiroshi; Nagashima, Kanji; Kuroume, Takayoshi (Gunma Univ. School of Medicine (Japan)); Ozone, K.; Pike, J.W. (Baylor College of Medicine, Houston, TX (United States))

    1993-02-01

    Hereditary 1,25-dihydroxyvitamin D [1,25-(OH)[sub 2]D]-resistant rickets (HVDRR) is a rare disorder characterized by rickets, alopecia, hypocalcemia, secondary hyperparathyroidism, and normal or elevated serum 1,25-dihydroxyvitamin D levels. The authors describe a patient with typical clinical characteristics of HVDRR, except that elevated levels of serum phosphorus were present coincident with increased levels of serum intact PTH. The patient was treated with high dose calcium infusion after an ineffective treatment with 1[alpha]-hydroxyvitamin D[sub 3]; serum calcium and phosphorus as well as intact PTH and alkaline phosphatase levels were normalized. Evaluation of phytohemagglutinin-activated lymphocytes derived from this patient revealed that 1,25-(OH)[sub 2]D[sub 3] was unable to inhibit thymidine incooperation, a result that contrast with the capacity of 1,25-(OH)[sub 2]D[sub 3] to inhibit uptake into normal activated lymphocytes. 1,25-(OH)[sub 2]D[sub 3] did not induce human osteocalcin promoter activity after transfection of this DNA linked to a reporter gene into patient cells. Cointroduction of a human vitamin D receptor (VDR) cDNA expression vector with the reporter plasmid, however, restored the hormone response. Evaluation of extracts from the patient cells for VDR DNA binding revealed a defect in DNA binding. Analysis of genomic DNA from the patient's cells by PCR confirmed the presence of a point mutation in exon 2 of the VDR. This exon directs synthesis of a portion of the DNA-binding domain of the receptor. We conclude that the genetic basis for 1,25-(OH)[sub 2]D[sub 3] resistance in this kindred with VDR-positive HVDRR is due to a single base mutation in the VDR that leads to production of a receptor unable to interact appropriately with DNA. 20 refs., 3 figs., 1 tab.

  2. Ventral striatal D2/3 receptor availability is associated with impulsive choice behavior as well aslimbic corticostriatal connectivity.

    Science.gov (United States)

    Barlow, Rebecca L; Gorges, Martin; Wearn, Alfie; Niessen, Heiko G; Kassubek, Jan; Dalley, Jeffrey W; Pekcec, Anton

    2018-03-15

    Low dopamine D2/3 receptor availability in the nucleus accumbens (NAcb) shell is associated with highly-impulsive behavior in rats, as measured by premature responses in a cued attentional task. However, it is unclear whether dopamine D2/3 receptor availability in the NAcb is equally linked to intolerance for delayed rewards, a related form of impulsivity. We investigated the relationship between D2/3 receptor availability in the NAcb and impulsivity in a delay-discounting task (DDT) where animals must choose between immediate small-magnitude rewards and delayed larger-magnitude rewards. Corticostriatal D2/3 receptor availability was measured in rats stratified for high-, and low-impulsivity using in-vivo [18F]fallypride positron emission tomography (PET) and ex-vivo [3H]raclopride autoradiography. Resting-state functional connectivity in limbic corticostriatal networks was also assessed using fMRI. DDT impulsivity was inversely related to D2/3 receptor availability in the NAcb core but not the dorsal striatum with higher D2/3 binding in the NAcb shell of high-impulsive rats compared with low-impulsive rats. D2/3 receptor availability was associated with stronger connectivity between the cingulate cortex and hippocampus of high versus low impulsive rats. We conclude that DDT impulsivity is associated with low D2/3 receptor binding in the NAcb core. Thus two related forms of waiting impulsivity - premature responding and delay intolerance in a delay-of-reward task - implicate an involvement of D2/3 receptor availability in the NAcb shell and core, respectively. This dissociation may be causal or consequential to enhanced functional connectivity of limbic brain circuitry and hold relevance for attention-deficit/hyperactivity disorder, drug addiction and other psychiatric disorders.

  3. An ochre mutation in the vitamin D receptor gene causes hereditary 1,25-dihydroxyvitamin D3-resistant rickets in three families

    International Nuclear Information System (INIS)

    Ritchie, H.H.; Hughes, M.R.; Thompson, E.T.; Pike, J.W.; O'Malley, B.W.; Malloy, P.J.; Feldman, D.; Hochberg, Z.

    1989-01-01

    Hereditary 1,25-dihydroxyvitamin D 3 -resistant rickets is a rare autosomal-recessive disease resulting from target-organ resistance to the action of the active hormonal form of vitamin D. Four affected children from three related families with the classical syndrome of hereditary 1,25-dihydroxyvitamin D 3 -resistant rickets and the absence of detectable binding to the vitamin D receptor (VDR) in cultured fibroblasts or lymphoblasts were examined for genetic abnormalities in the VDR gene. Genomic DNA from Epstein-Barr virus-transformed lymphoblasts of eight family members was isolated and amplified by polymerase chain reaction techniques. Amplified fragments containing the eight structural exons encoding the VDR protein were sequenced. The DNA from all affected children exhibited a single C → A base substitution within exon 7 at nucleotide 970. Although the affected children were all homozygotic for the mutation, the four parents tested all exhibited both wild-type and mutant alleles, indicating a heterozygous state. Recreated mutant receptor exhibited no specific 1,25-[ 3 H]dihydroxyvitamin D 3 binding and failed to activate a cotransfected VDR promoter-reporter gene construct. Thus these findings identify an ochre mutation in a human steroid hormone receptor in patients with hereditary 1,25-dihydroxyvitamin D 3 -resistant rickets

  4. [(3)H]-YM-09151-2 binding sites in human brain postmortem.

    Science.gov (United States)

    Marazziti, Donatella; Baroni, Stefano; Masala, Irene; Giannaccini, Gino; Betti, Laura; Palego, Lionella; Catena Dell'Osso, Mario; Consoli, Giorgio; Castagna, Maura; Lucacchini, Antonio

    2009-12-01

    The controversial and limited data on the distribution of dopamine (DA) receptors of type 4 (D(4)) in the human brain prompted us to explore their density and pharmacological characteristics in the prefrontal cortex, striatum and hippocampus, through a series of binding assays. Brain samples were taken during autopsy from seven subjects. Tissue homogenates were incubated with increasing concentration of [(3)H]-YM-09151-2, a D(2)-like receptor antagonist, and L-745,870 and/or sulpiride to define the non-specific binding, while PPAP was used to block sigma receptors. The results showed a low density of D(4) receptors in the hippocampus only, with a preponderance of D(2)/D(3) and sigma receptors in the prefrontal cortex and striatum. In conclusion, these findings underline that it is possible to label D(4) receptors by means of [(3)H]-YM-09151-2, provided that D(2), D(3) and sigma receptors are blocked.

  5. Diphtheria toxin can simultaneously bind to its receptor and adenylyl-(3',5')-uridine 3'-monophosphate

    International Nuclear Information System (INIS)

    Barbieri, J.T.; Collins, C.M.; Collier, R.J.

    1986-01-01

    Diphtheria toxin (DT) that was bound to receptors on BS-C-1 cells was able to bind approximately 1 molar equiv of adenylyl-(3',5')-uridine 3'-monophosphate (ApUp). In contrast, receptor-bound CRM197, a mutant form of toxin with greatly diminished affinity for dinucleotides, did not bind ApUp. Affinity of the dinucleotide for receptor-bound toxin differed from that for free toxin by less than an order of magnitude. These results indicate that the receptor site and the ApUp site on the toxin do not significantly overlap. BS-C-1 cells were incubated with or without 125 I-DT or CRM 197. They were then incubated with [ 32 P]ApUp, and assayed

  6. Destabilization of strigolactone receptor DWARF14 by binding of ligand and E3-ligase signaling effector DWARF3

    Science.gov (United States)

    Zhao, Li-Hua; Zhou, X Edward; Yi, Wei; Wu, Zhongshan; Liu, Yue; Kang, Yanyong; Hou, Li; de Waal, Parker W; Li, Suling; Jiang, Yi; Scaffidi, Adrian; Flematti, Gavin R; Smith, Steven M; Lam, Vinh Q; Griffin, Patrick R; Wang, Yonghong; Li, Jiayang; Melcher, Karsten; Xu, H Eric

    2015-01-01

    Strigolactones (SLs) are endogenous hormones and exuded signaling molecules in plant responses to low levels of mineral nutrients. Key mediators of the SL signaling pathway in rice include the α/β-fold hydrolase DWARF 14 (D14) and the F-box component DWARF 3 (D3) of the ubiquitin ligase SCFD3 that mediate ligand-dependent degradation of downstream signaling repressors. One perplexing feature is that D14 not only functions as the SL receptor but is also an active enzyme that slowly hydrolyzes diverse natural and synthetic SLs including GR24, preventing the crystallization of a binary complex of D14 with an intact SL as well as the ternary D14/SL/D3 complex. Here we overcome these barriers to derive a structural model of D14 bound to intact GR24 and identify the interface that is required for GR24-mediated D14-D3 interaction. The mode of GR24-mediated signaling, including ligand recognition, hydrolysis by D14, and ligand-mediated D14-D3 interaction, is conserved in structurally diverse SLs. More importantly, D14 is destabilized upon the binding of ligands and D3, thus revealing an unusual mechanism of SL recognition and signaling, in which the hormone, the receptor, and the downstream effectors are systematically destabilized during the signal transduction process. PMID:26470846

  7. Study on dopamine D{sub 2} binding capacity in vascular parkinsonism

    Energy Technology Data Exchange (ETDEWEB)

    Terashi, Hiroo; Nagata, Ken; Hirata, Yutaka; Hatazawa, Jun [Research Inst. for Brain and Blood Vessels, Akita (Japan); Utsumi, Hiroya [Tokyo Medical Coll. (Japan)

    2001-10-01

    To investigate whether the striatal dopamine receptor function is involved in the development of vascular parkinsonism (VP), a positron emission tomography (PET) study was conducted on 9 patients with VP by using [{sup 11}C] N-methylspiperone as the tracer. The rate of binding availability in the striatal dopamine D{sub 2} receptor (k{sub 3}) was determined semiquantitatively, and the values were compared to the predicted normal values based on the results from 7 normal volunteers. Of 9 patients with VP, the normalized D{sub 2} receptor binding [%k{sub 3}] was more than 90% in 5 patients, 89 to 87% in 3, and 75% in one. These values showed no evident correlation with the Hoehn and Yahr stage. The laterality of the striatal %k{sub 3} did not correspond to that of the parkinsonism. Thus, the striatal dopamine D{sub 2} receptor binding was not severely impaired and did not correlate with the neurological status in patients with VP. This may indicate that striatal dopamine D{sub 2} receptor function is not primarily associated with the development of the parkinsonism in VP. (author)

  8. /sup 3/H)-(H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) ((/sup 3/H)CTOP), a potent and highly selective peptide for mu opioid receptors in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Hawkins, K.N.; Knapp, R.J.; Lui, G.K.; Gulya, K.; Kazmierski, W.; Wan, Y.P.; Pelton, J.T.; Hruby, V.J.; Yamamura, H.I.

    1989-01-01

    The cyclic, conformationally restricted octapeptide (3H)-(H-D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2) ((3H)CTOP) was synthesized and its binding to mu opioid receptors was characterized in rat brain membrane preparations. Association rates (k+1) of 1.25 x 10(8) M-1 min-1 and 2.49 x 10(8) M-1 min-1 at 25 and 37 degrees C, respectively, were obtained, whereas dissociation rates (k-1) at the same temperatures were 1.93 x 10(-2) min-1 and 1.03 x 10(-1) min-1 at 25 and 37 degrees C, respectively. Saturation isotherms of (3H)CTOP binding to rat brain membranes gave apparent Kd values of 0.16 and 0.41 nM at 25 and 37 degrees C, respectively. Maximal number of binding sites in rat brain membranes were found to be 94 and 81 fmol/mg of protein at 25 and 37 degrees C, respectively. (3H)CTOP binding over a concentration range of 0.1 to 10 nM was best fit by a one site model consistent with binding to a single site. The general effect of different metal ions and guanyl-5'-yl-imidodiphosphate on (3H)CTOP binding was to reduce its affinity. High concentrations (100 mM) of sodium also produced a reduction of the apparent mu receptor density. Utilizing the delta opioid receptor specific peptide (3H)-(D-Pen2,D-Pen5)enkephalin, CTOP appeared to be about 2000-fold more specific for mu vs. delta opioid receptor than naloxone. Specific (3H)CTOP binding was inhibited by a large number of opioid or opiate ligands.

  9. 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.

  10. 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

  11. Characterization of dFOXO binding sites upstream of the Insulin Receptor P2 promoter across the Drosophila phylogeny.

    Directory of Open Access Journals (Sweden)

    Dorcas J Orengo

    Full Text Available The insulin/TOR signal transduction pathway plays a critical role in determining such important traits as body and organ size, metabolic homeostasis and life span. Although this pathway is highly conserved across the animal kingdom, the affected traits can exhibit important differences even between closely related species. Evolutionary studies of regulatory regions require the reliable identification of transcription factor binding sites. Here we have focused on the Insulin Receptor (InR expression from its P2 promoter in the Drosophila genus, which in D. melanogaster is up-regulated by hypophosphorylated Drosophila FOXO (dFOXO. We have finely characterized this transcription factor binding sites in vitro along the 1.3 kb region upstream of the InR P2 promoter in five Drosophila species. Moreover, we have tested the effect of mutations in the characterized dFOXO sites of D. melanogaster in transgenic flies. The number of experimentally established binding sites varies across the 1.3 kb region of any particular species, and their distribution also differs among species. In D. melanogaster, InR expression from P2 is differentially affected by dFOXO binding sites at the proximal and distal halves of the species 1.3 kb fragment. The observed uneven distribution of binding sites across this fragment might underlie their differential contribution to regulate InR transcription.

  12. Regulation of 1,25-dihydroxyvitamin D, receptors by [3H]-1,25-dihydroxyvitamin D3 in cultured cells (T-47D): evidence for receptor upregulation

    International Nuclear Information System (INIS)

    Reinhardt, T.A.; Horst, R.L.

    1986-01-01

    The authors examined the effect of 1,25-(OH) 2 D 3 on receptor concentration in cultured cells (T-47D). Two days prior to experiment, cells were fed with RPMI 1640 + 10% serum and 24-32 hours prior to experiment the media was replaced with RPMI 1640 + 25 mM Hepes + 1% serum. [ 3 H]-1,25-(OH) 2 D 3 +/- 100-fold molar excess cold hormone was used to treat the cells. Occupied receptors were measured in freshly prepared cytosols. Total receptors were measured following a 16-hour incubation of cytosols in the presence of 0.6 nM [ 3 H]-1,25-(OH) 2 D 3 +/- 100-fold molar excess of cold hormone at 4 0 C. Treatment of cell cultures for 16-18 hours with 0.5-1.0 nM [ 3 H]-1,25-(OH) 2 D 3 resulted in a 30-40% receptor occupancy by the hormone and a 2- to 3-fold increase in total cell receptor as compared to vehicle-treated controls. Time course studies showed a rapid increase in total receptors up to 16 hours post-treatment in the face of declining receptor occupancy. Actinomycin D blocked the [ 3 H]-1,25-(OH) 2 D 3 -dependent rise in cell receptor. The physiological significance of this receptor upregulation is not known nor is it known whether upregulation results from synthesis of new receptors and/or is the result of the activation of preformed receptors by a inducible activator protein

  13. An ochre mutation in the vitamin D receptor gene causes hereditary 1,25-dihydroxyvitamin D sub 3 -resistant rickets in three families

    Energy Technology Data Exchange (ETDEWEB)

    Ritchie, H.H.; Hughes, M.R.; Thompson, E.T.; Pike, J.W.; O' Malley, B.W. (Baylor College of Medicine, Houston, TX (USA)); Malloy, P.J.; Feldman, D. (Stanford Univ. School of Medicine, CA (USA)); Hochberg, Z. (Rambam Medical Center, Haifa (Israel))

    1989-12-01

    Hereditary 1,25-dihydroxyvitamin D{sub 3}-resistant rickets is a rare autosomal-recessive disease resulting from target-organ resistance to the action of the active hormonal form of vitamin D. Four affected children from three related families with the classical syndrome of hereditary 1,25-dihydroxyvitamin D{sub 3}-resistant rickets and the absence of detectable binding to the vitamin D receptor (VDR) in cultured fibroblasts or lymphoblasts were examined for genetic abnormalities in the VDR gene. Genomic DNA from Epstein-Barr virus-transformed lymphoblasts of eight family members was isolated and amplified by polymerase chain reaction techniques. Amplified fragments containing the eight structural exons encoding the VDR protein were sequenced. The DNA from all affected children exhibited a single C {yields} A base substitution within exon 7 at nucleotide 970. Although the affected children were all homozygotic for the mutation, the four parents tested all exhibited both wild-type and mutant alleles, indicating a heterozygous state. Recreated mutant receptor exhibited no specific 1,25-({sup 3}H)dihydroxyvitamin D{sub 3} binding and failed to activate a cotransfected VDR promoter-reporter gene construct. Thus these findings identify an ochre mutation in a human steroid hormone receptor in patients with hereditary 1,25-dihydroxyvitamin D{sub 3}-resistant rickets.

  14. Neuronal Calcium Sensor-1 Binds the D2 Dopamine Receptor and G-protein-coupled Receptor Kinase 1 (GRK1) Peptides Using Different Modes of Interactions.

    Science.gov (United States)

    Pandalaneni, Sravan; Karuppiah, Vijaykumar; Saleem, Muhammad; Haynes, Lee P; Burgoyne, Robert D; Mayans, Olga; Derrick, Jeremy P; Lian, Lu-Yun

    2015-07-24

    Neuronal calcium sensor-1 (NCS-1) is the primordial member of the neuronal calcium sensor family of EF-hand Ca(2+)-binding proteins. It interacts with both the G-protein-coupled receptor (GPCR) dopamine D2 receptor (D2R), regulating its internalization and surface expression, and the cognate kinases GRK1 and GRK2. Determination of the crystal structures of Ca(2+)/NCS-1 alone and in complex with peptides derived from D2R and GRK1 reveals that the differential recognition is facilitated by the conformational flexibility of the C-lobe-binding site. We find that two copies of the D2R peptide bind within the hydrophobic crevice on Ca(2+)/NCS-1, but only one copy of the GRK1 peptide binds. The different binding modes are made possible by the C-lobe-binding site of NCS-1, which adopts alternative conformations in each complex. C-terminal residues Ser-178-Val-190 act in concert with the flexible EF3/EF4 loop region to effectively form different peptide-binding sites. In the Ca(2+)/NCS-1·D2R peptide complex, the C-terminal region adopts a 310 helix-turn-310 helix, whereas in the GRK1 peptide complex it forms an α-helix. Removal of Ser-178-Val-190 generated a C-terminal truncation mutant that formed a dimer, indicating that the NCS-1 C-terminal region prevents NCS-1 oligomerization. We propose that the flexible nature of the C-terminal region is essential to allow it to modulate its protein-binding sites and adapt its conformation to accommodate both ligands. This appears to be driven by the variability of the conformation of the C-lobe-binding site, which has ramifications for the target specificity and diversity of NCS-1. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. Link between D sub 1 and D sub 2 dopamine receptors is reduced in schizophrenia and Huntington diseased brain

    Energy Technology Data Exchange (ETDEWEB)

    Seeman, P.; Niznik, H.B.; Guan, H.C.; Booth, G.; Ulpian, C. (Univ. of Toronto (Canada))

    1989-12-01

    Dopamine receptor types D{sub 1} and D{sub 2} can oppose enhance each other's actions for electrical, biochemical, and psychomotor effects. The authors report a D{sub 1}-D{sub 2} interaction in homogenized tissue as revealed by ligand binding. D{sub 2} agonists lowered the binding of ({sup 3}H)raclopride to D{sub 2} receptors in striatal and anterior pituitary tissues. Pretreating the tissue with the D{sub 1}-selective antagonist SCH 23390 prevented the agonist-induced decrease in ({sup 3}H)raclopride binding to D{sub 2} sites in the striatum but not in the anterior pituitary, which has no D{sub 1} receptors. Conversely, a dopamine-induced reduction in the binding of ({sup 3}H)SCH 23390 to D{sub 1} receptors could be prevented by the D{sub 2}-selective antagonist eticlopride. Receptor photolabeling experiments confirmed both these D{sub 1}-D{sub 2} interactions. The blocking effect by SCH 23390 was similar to that produced by a nonhydrolyzable guanine nucleotide analogue, and SCH 23390 reduced the number of agonist-labeled D{sub 2} receptors in the high-affinity state. Thus, the D{sub 1}-D{sub 2} link may be mediated by guanine nucleotide-binding protein components. The link may underlie D{sub 1}-D{sub 2} interactions influencing behavior, since the link was missing in over half the postmortem striata from patients with schizophrenia and Huntington disease (both diseases that show some hyperdopamine signs) but was present in human control, Alzheimer, and Parkinson striata.

  16. Striatal dopamine D2/3 receptor availability in treatment resistant depression.

    Directory of Open Access Journals (Sweden)

    Bart P de Kwaasteniet

    Full Text Available Several studies demonstrated improvement of depressive symptoms in treatment resistant depression (TRD after administering dopamine agonists which suggest abnormal dopaminergic neurotransmission in TRD. However, the role of dopaminergic signaling through measurement of striatal dopamine D(2/3 receptor (D2/3R binding has not been investigated in TRD subjects. We used [(123I]IBZM single photon emission computed tomography (SPECT to investigate striatal D2/3R binding in TRD. We included 6 severe TRD patients, 11 severe TRD patients on antipsychotics (TRD AP group and 15 matched healthy controls. Results showed no significant difference (p = 0.75 in striatal D2/3R availability was found between TRD patients and healthy controls. In the TRD AP group D2/3R availability was significantly decreased (reflecting occupancy of D2/3Rs by antipsychotics relative to TRD patients and healthy controls (p<0.001 but there were no differences in clinical symptoms between TRD AP and TRD patients. This preliminary study therefore does not provide evidence for large differences in D2/3 availability in severe TRD patients and suggests this TRD subgroup is not characterized by altered dopaminergic transmission. Atypical antipsychotics appear to have no clinical benefit in severe TRD patients who remain depressed, despite their strong occupancy of D2/3Rs.

  17. A novel thromboxane A2 receptor D304N variant that abrogates ligand binding in a patient with a bleeding diathesis.

    Science.gov (United States)

    Mumford, Andrew D; Dawood, Ban B; Daly, Martina E; Murden, Sherina L; Williams, Michael D; Protty, Majd B; Spalton, Jennifer C; Wheatley, Mark; Mundell, Stuart J; Watson, Steve P

    2010-01-14

    We investigated the cause of mild mucocutaneous bleeding in a 14-year-old male patient (P1). Platelet aggregation and ATP secretion induced by arachidonic acid and the thromboxane A(2) receptor (TxA(2)R) agonist U46619 were reduced in P1 compared with controls, whereas the responses to other platelet agonists were retained. P1 was heterozygous for a transversion within the TBXA2R gene predictive of a D304N substitution in the TxA(2)R. In Chinese hamster ovary-K1 cells expressing the variant D304N TxA(2)R, U46619 did not increase cytosolic free Ca(2+) concentration, indicating loss of receptor function. The TxA(2)R antagonist [(3)H]-SQ29548 showed an approximate 50% decrease in binding to platelets from P1 but absent binding to Chinese hamster ovary-K1 cells expressing variant D304N TxA(2)R. This is the second naturally occurring TxA(2)R variant to be associated with platelet dysfunction and the first in which loss of receptor function is associated with reduced ligand binding. D304 lies within a conserved NPXXY motif in transmembrane domain 7 of the TxA(2)R that is a key structural element in family A G protein-coupled receptors. Our demonstration that the D304N substitution causes clinically significant platelet dysfunction by reducing ligand binding establishes the importance of the NPXXY motif for TxA(2)R function in vivo.

  18. 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.

  19. The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

    DEFF Research Database (Denmark)

    Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus

    2017-01-01

    Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D...

  20. N-methyl-D-aspartic acid receptor agonists

    DEFF Research Database (Denmark)

    Madsen, U; Frydenvang, Karla Andrea; Ebert, B

    1996-01-01

    (R,S)-2-Amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid [(R,S)-AMAA, 4] is a potent and selective agonist at the N-methyl-D-aspartic acid (NMDA) subtype of excitatory amino acid receptors. Using the Ugi "four-component condensation" method, the two diastereomers (2R)- and (2S)-2-[3-(benzyloxy......) showed peak affinity for [3H]AMPA receptor sites (IC50 = 72 +/- 13 microM) and was shown to be a more potent inhibitor of [3H]CPP binding (IC50 = 3.7 +/- 1.5 microM) than (S)-AMAA (9) (IC50 = 61 +/- 6.4 microM). Neither enantiomer of AMAA affected [3H]kainic acid receptor binding significantly...

  1. 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

  2. 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

  3. Gliadin induces an increase in intestinal permeability and zonulin release by binding to the chemokine receptor CXCR3.

    Science.gov (United States)

    Lammers, Karen M; Lu, Ruliang; Brownley, Julie; Lu, Bao; Gerard, Craig; Thomas, Karen; Rallabhandi, Prasad; Shea-Donohue, Terez; Tamiz, Amir; Alkan, Sefik; Netzel-Arnett, Sarah; Antalis, Toni; Vogel, Stefanie N; Fasano, Alessio

    2008-07-01

    Celiac disease is an immune-mediated enteropathy triggered by gliadin, a component of the grain protein gluten. Gliadin induces an MyD88-dependent zonulin release that leads to increased intestinal permeability, a postulated early element in the pathogenesis of celiac disease. We aimed to establish the molecular basis of gliadin interaction with intestinal mucosa leading to intestinal barrier impairment. Alpha-gliadin affinity column was loaded with intestinal mucosal membrane lysates to identify the putative gliadin-binding moiety. In vitro experiments with chemokine receptor CXCR3 transfectants were performed to confirm binding of gliadin and/or 26 overlapping 20mer alpha-gliadin synthetic peptides to the receptor. CXCR3 protein and gene expression were studied in intestinal epithelial cell lines and human biopsy specimens. Gliadin-CXCR3 interaction was further analyzed by immunofluorescence microscopy, laser capture microscopy, real-time reverse-transcription polymerase chain reaction, and immunoprecipitation/Western blot analysis. Ex vivo experiments were performed using C57BL/6 wild-type and CXCR3(-/-) mouse small intestines to measure intestinal permeability and zonulin release. Affinity column and colocalization experiments showed that gliadin binds to CXCR3 and that at least 2 alpha-gliadin 20mer synthetic peptides are involved in this binding. CXCR3 is expressed in mouse and human intestinal epithelia and lamina propria. Mucosal CXCR3 expression was elevated in active celiac disease but returned to baseline levels following implementation of a gluten-free diet. Gliadin induced physical association between CXCR3 and MyD88 in enterocytes. Gliadin increased zonulin release and intestinal permeability in wild-type but not CXCR3(-/-) mouse small intestine. Gliadin binds to CXCR3 and leads to MyD88-dependent zonulin release and increased intestinal permeability.

  4. 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.

  5. 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.

  6. The binding characteristics and orientation of a novel radioligand with distinct properties at 5-HT3A and 5-HT3AB receptors

    NARCIS (Netherlands)

    Thompson, Andrew J; Verheij, Mark H P; Verbeek, Joost; Windhorst, Albert D; de Esch, Iwan J P; Lummis, Sarah C R

    2014-01-01

    VUF10166 (2-chloro-3-(4-methyl piperazin-1-yl)quinoxaline) is a ligand that binds with high affinity to 5-HT3 receptors. Here we synthesise [(3)H]VUF10166 and characterise its binding properties at 5-HT3A and 5-HT3AB receptors. At 5-HT3A receptors [(3)H]VUF10166 displayed saturable binding with a Kd

  7. Effect of Exercise Training on Striatal Dopamine D2/D3 Receptors in Methamphetamine Users during Behavioral Treatment.

    Science.gov (United States)

    Robertson, Chelsea L; Ishibashi, Kenji; Chudzynski, Joy; Mooney, Larissa J; Rawson, Richard A; Dolezal, Brett A; Cooper, Christopher B; Brown, Amira K; Mandelkern, Mark A; London, Edythe D

    2016-05-01

    Methamphetamine use disorder is associated with striatal dopaminergic deficits that have been linked to poor treatment outcomes, identifying these deficits as an important therapeutic target. Exercise attenuates methamphetamine-induced neurochemical damage in the rat brain, and a preliminary observation suggests that exercise increases striatal D2/D3 receptor availability (measured as nondisplaceable binding potential (BPND)) in patients with Parkinson's disease. The goal of this study was to evaluate whether adding an exercise training program to an inpatient behavioral intervention for methamphetamine use disorder reverses deficits in striatal D2/D3 receptors. Participants were adult men and women who met DSM-IV criteria for methamphetamine dependence and were enrolled in a residential facility, where they maintained abstinence from illicit drugs of abuse and received behavioral therapy for their addiction. They were randomized to a group that received 1 h supervised exercise training (n=10) or one that received equal-time health education training (n=9), 3 days/week for 8 weeks. They came to an academic research center for positron emission tomography (PET) using [(18)F]fallypride to determine the effects of the 8-week interventions on striatal D2/D3 receptor BPND. At baseline, striatal D2/D3 BPND did not differ between groups. However, after 8 weeks, participants in the exercise group displayed a significant increase in striatal D2/D3 BPND, whereas those in the education group did not. There were no changes in D2/D3 BPND in extrastriatal regions in either group. These findings suggest that structured exercise training can ameliorate striatal D2/D3 receptor deficits in methamphetamine users, and warrants further evaluation as an adjunctive treatment for stimulant dependence.

  8. 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.

  9. Synthesis and anion binding studies of tris(3-aminopropyl)amine-based tripodal urea and thiourea receptors: Proton transfer-induced selectivity for hydrogen sulfate over sulfate.

    Science.gov (United States)

    Khansari, Maryam Emami; Johnson, Corey R; Basaran, Ismet; Nafis, Aemal; Wang, Jing; Leszczynski, Jerzy; Hossain, Md Alamgir

    2015-01-01

    Tris(3-aminopropyl)amine-based tripodal urea and thiourea receptors, tris([(4-cyanophenyl)amino]propyl)urea ( L1 ) and tris([(4-cyanophenyl)amino]propyl)thiourea ( L2 ), have been synthesized and their anion binding properties have been investigated for halides and oxoanions. As investigated by 1 H NMR titrations, each receptor binds an anion with a 1:1 stoichiometry via hydrogen-bonding interactions (NH⋯anion), showing the binding trend in the order of F - > H 2 PO 4 - > HCO 3 - > HSO 4 - > CH 3 COO - > SO 4 2- > Cl - > Br - > I in DMSO- d 6 . The interactions of the receptors were further studied by 2D NOESY, showing the loss of NOESY contacts of two NH resonances for the complexes of F - , H 2 PO 4 - , HCO 3 - , HSO 4 - or CH 3 COO - due to the strong NH⋯anion interactions. The observed higher binding affinity for HSO 4 - than SO 4 2- is attributed to the proton transfer from HSO 4 - to the central nitrogen of L1 or L2 which was also supported by the DFT calculations, leading to the secondary acid-base interactions. The thiourea receptor L2 has a general trend to show a higher affinity for an anion as compared to the urea receptor L1 for the corresponding anion in DMSO- d 6 . In addition, the compound L2 has been exploited for its extraction properties for fluoride in water using a liquid-liquid extraction technique, and the results indicate that the receptor effectively extracts fluoride from water showing ca. 99% efficiency (based on L2 ).

  10. Cocaine Inhibits Dopamine D2 Receptor Signaling via Sigma-1-D2 Receptor Heteromers

    Science.gov (United States)

    Navarro, Gemma; Moreno, Estefania; Bonaventura, Jordi; Brugarolas, Marc; Farré, Daniel; Aguinaga, David; Mallol, Josefa; Cortés, Antoni; Casadó, Vicent; Lluís, Carmen; Ferre, Sergi

    2013-01-01

    Under normal conditions the brain maintains a delicate balance between inputs of reward seeking controlled by neurons containing the D1-like family of dopamine receptors and inputs of aversion coming from neurons containing the D2-like family of dopamine receptors. Cocaine is able to subvert these balanced inputs by altering the cell signaling of these two pathways such that D1 reward seeking pathway dominates. Here, we provide an explanation at the cellular and biochemical level how cocaine may achieve this. Exploring the effect of cocaine on dopamine D2 receptors function, we present evidence of σ1 receptor molecular and functional interaction with dopamine D2 receptors. Using biophysical, biochemical, and cell biology approaches, we discovered that D2 receptors (the long isoform of the D2 receptor) can complex with σ1 receptors, a result that is specific to D2 receptors, as D3 and D4 receptors did not form heteromers. We demonstrate that the σ1-D2 receptor heteromers consist of higher order oligomers, are found in mouse striatum and that cocaine, by binding to σ1 -D2 receptor heteromers, inhibits downstream signaling in both cultured cells and in mouse striatum. In contrast, in striatum from σ1 knockout animals these complexes are not found and this inhibition is not seen. Taken together, these data illuminate the mechanism by which the initial exposure to cocaine can inhibit signaling via D2 receptor containing neurons, destabilizing the delicate signaling balance influencing drug seeking that emanates from the D1 and D2 receptor containing neurons in the brain. PMID:23637801

  11. How membrane lipids control the 3D structure and function of receptors

    Directory of Open Access Journals (Sweden)

    Jacques Fantini

    2018-02-01

    Full Text Available The cohabitation of lipids and proteins in the plasma membrane of mammalian cells is controlled by specific biochemical and biophysical rules. Lipids may be either constitutively tightly bound to cell-surface receptors (non-annular lipids or less tightly attached to the external surface of the protein (annular lipids. The latter are exchangeable with surrounding bulk membrane lipids on a faster time scale than that of non-annular lipids. Not only do non-annular lipids bind to membrane proteins through stereoselective mechanisms, they can also help membrane receptors acquire (or maintain a functional 3D structure. Cholesterol is the prototype of membrane lipids that finely controls the 3D structure and function of receptors. However, several other lipids such as sphingolipids may also modulate the function of membrane proteins though conformational adjustments. All these concepts are discussed in this review in the light of representative examples taken from the literature.

  12. 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

  13. Interaction between LSD and dopamine D2/3 binding sites in pig brain.

    Science.gov (United States)

    Minuzzi, Luciano; Nomikos, George G; Wade, Mark R; Jensen, Svend B; Olsen, Aage K; Cumming, Paul

    2005-06-15

    The psychoactive properties of the hallucinogen LSD have frequently been attributed to high affinity interactions with serotonin 5HT2 receptors in brain. Possible effects of LSD on dopamine D2/3 receptor availability have not previously been investigated in living brain. Therefore, we used PET to map the binding potential (pB) of [11C]raclopride in brain of three pigs, first in a baseline condition, and again at 1 and 4 h after administration of LSD (2.5 microg/kg, i.v.). There was a progressive treatment effect in striatum, where the pB was significantly reduced by 19% at 4 h after LSD administration. Concomitant maps of cerebral blood flow did not reveal significant changes in perfusion during this interval. Subsequent in vitro studies showed that LSD displaced [3H]raclopride (2 nM) from pig brain cryostat sections with an IC50 of 275 nM according to a one-site model. Fitting of a two-site model to the data suggested the presence of a component of the displacement curves with a subnanomolar IC50, comprising 20% of the total [3H]raclopride binding. In microdialysis experiments, LSD at similar and higher doses did not evoke changes in the interstitial concentration of dopamine or its acidic metabolites in rat striatum. Together, these results are consistent with a direct interaction between LSD and a portion of dopamine D2/3 receptors in pig brain, possibly contributing to the psychopharmacology of LSD. (c) 2005 Wiley-Liss, Inc.

  14. G protein-coupled receptor transmembrane binding pockets and their applications in GPCR research and drug discovery: a survey.

    Science.gov (United States)

    Kratochwil, Nicole A; Gatti-McArthur, Silvia; Hoener, Marius C; Lindemann, Lothar; Christ, Andreas D; Green, Luke G; Guba, Wolfgang; Martin, Rainer E; Malherbe, Pari; Porter, Richard H P; Slack, Jay P; Winnig, Marcel; Dehmlow, Henrietta; Grether, Uwe; Hertel, Cornelia; Narquizian, Robert; Panousis, Constantinos G; Kolczewski, Sabine; Steward, Lucinda

    2011-01-01

    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. Hence, an automated method was developed that allows a fast analysis and comparison of these generic ligand binding pockets across the entire GPCR family by providing the relevant information for all GPCRs in the same format. This methodology compiles amino acids lining the TM binding pocket including parts of the ECL2 loop in a so-called 1D ligand binding pocket vector and translates these 1D vectors in a second step into 3D receptor pharmacophore models. It aims to support various aspects of GPCR drug discovery in the pharmaceutical industry. Applications of pharmacophore similarity analysis of these 1D LPVs include definition of receptor subfamilies, prediction of species differences within subfamilies in regard to in vitro pharmacology and identification of nearest neighbors for GPCRs of interest to generate starting points for GPCR lead identification programs. These aspects of GPCR research are exemplified in the field of melanopsins, trace amine-associated receptors and somatostatin receptor subtype 5. In addition, it is demonstrated how 3D pharmacophore models of the LPVs can support the prediction of amino acids involved in ligand recognition, the understanding of mutational data in a 3D context and the elucidation of binding modes for GPCR ligands and their evaluation. Furthermore, guidance through 3D receptor pharmacophore modeling for the synthesis of subtype-specific GPCR ligands will be reported. Illustrative examples are taken from the GPCR family class C, metabotropic glutamate receptors 1 and 5 and sweet taste receptors, and from the GPCR class A, e.g. nicotinic acid and 5-hydroxytryptamine 5A receptor. © 2011 Bentham Science Publishers

  15. Synthesis of 11C-SCH 23390, a dopamine D-1 receptor antagonist, for use in in vivo receptor binding studies with PET

    International Nuclear Information System (INIS)

    Halldin, Christer; Stone-Elander, Sharon; Farde, Lars; Ehrin, Erling; Fasth, Karl-Johan; Langstroem, Bengt; Sedvall, Goeran; Karolinska Hospital, Stockholm; Uppsala Univ.

    1986-01-01

    Central dopamine receptors are generally accepted to exist in at least two distinct subtypes: D-1 and D-2. Recently a benzazepine, SCH 23390, was reported to be a selective D-1 dopaminergic antagonist. PET studies of the radio-brominated 76 Br-SCH 23390 reported by Friedman, et al. indicated that the analog exhibits specific binding in the striatum of the monkey brain. Here we report the synthesis of 11 C-SCH 23390 suitable for the in vivo study of dopamine D-1 receptors in the human brain. (author)

  16. 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.)

  17. 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.

  18. 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

  19. In vivo measurement of haloperidol affinity to dopamine D2/D3 receptors by [123I]IBZM and single photon emission computed tomography

    DEFF Research Database (Denmark)

    Videbaek, C; Toska, K; Friberg, L

    2001-01-01

    This study examines the feasibility of a steady-state bolus-integration method with the dopamine D2/D3 receptor single photon emission computer tomography (SPECT) tracer, [123I]IBZM, for determination of in vivo affinity of haloperidol. The nonspecific binding of [123I]IBZM was examined in the rat...... brain by infusion of haloperidol to plasma levels approximately 100 times the Kd level in man. In humans, Kd for haloperidol binding was measured in four healthy volunteers that were examined twice: once with partial dopamine D2/D3 receptor blockade obtained by a scheduled infusion of unlabeled...... haloperidol (0.7 mg total dosage), and once in an unblocked state. Blood sampling and SPECT were performed intermittently during 6 hours after intravenous [123I]IBZM bolus injection. Plasma [123I]IBZM was determined by octane extraction. Plasma haloperidol was determined by a radioimmunoassay, and plasma...

  20. Functional modulation of cerebral gamma-aminobutyric acidA receptor/benzodiazepine receptor/chloride ion channel complex with ethyl beta-carboline-3-carboxylate: Presence of independent binding site for ethyl beta-carboline-3-carboxylate

    Energy Technology Data Exchange (ETDEWEB)

    Taguchi, J.; Kuriyama, K. (Kyoto Prefectural Univ. of Medicine (Japan))

    1990-05-01

    Effect of ethyl beta-carboline-3-carboxylate (beta-CCE) on the function of gamma-aminobutyric acid (GABA)A receptor/benzodiazepine receptor/chloride ion channel complex was studied. Beta-CCE noncompetitively and competitively inhibited (3H)flunitrazepam binding to benzodiazepine receptor, but not (3H)muscimol binding to GABAA receptor as well as t-(3H)butylbicycloorthobenzoate (( 3H) TBOB) binding to chloride ion channel, in particulate fraction of the mouse brain. Ro15-1788 also inhibited competitively (3H) flunitrazepam binding. On the other hand, the binding of beta-(3H)CCE was inhibited noncompetitively and competitively by clonazepam and competitively by Ro15-1788. In agreement with these results, benzodiazepines-stimulated (3H)muscimol binding was antagonized by beta-CCE and Ro15-1788. Gel column chromatography for the solubilized fraction from cerebral particulate fraction by 0.2% sodium deoxycholate (DOC-Na) in the presence of 1 M KCl indicated that beta-(3H)CCE binding site was eluted in the same fraction (molecular weight, 250,000) as the binding sites for (3H)flunitrazepam, (3H)muscimol and (3H)TBOB. GABA-stimulated 36Cl- influx into membrane vesicles prepared from the bovine cerebral cortex was stimulated and attenuated by flunitrazepam and beta-CCE, respectively. These effects of flunitrazepam and beta-CCE on the GABA-stimulated 36Cl- influx were antagonized by Ro15-1788. The present results suggest that the binding site for beta-CCE, which resides on GABAA receptor/benzodiazepine receptor/chloride ion channel complex, may be different from that for benzodiazepine. Possible roles of beta-CCE binding site in the allosteric inhibitions on benzodiazepine binding site as well as on the functional coupling between chloride ion channel and GABAA receptor are also suggested.

  1. [99mTc]MAG3-mannosyl-dextran: a receptor-binding radiopharmaceutical for sentinel node detection

    International Nuclear Information System (INIS)

    Vera, David R.; Wallace, Anne M.; Hoh, Carl K.

    2001-01-01

    Technetium-99m-labeled benzoyl-mercaptoacetylglycylglycyl-glycine-mannosyl-dextran ([ 99m Tc]MAG 3 -mannosyl-dextran) is a receptor-binding radiotracer that binds to mannose-binding protein, a receptor expressed by reticuloendothelial tissue. This agent is composed of a 10.5-kilodalton molecule of dextran and multiple units of mannose, and benzoyl-mercaptoacetylglycylglycyl-glycine (BzMAG 3 ). The tetraflorophenol-activated ester of BzMAG 3 and the imidate of thiomannose were used to covalently attach BzMAG 3 and mannose to an amino-terminated conjugate of dextran. This yielded a 19-kilodalton macromolecule consisting of 3 BzMAG 3 and 21 mannose units per dextran. Dynamic light scattering was used to measure a mean diameter of 5.5 nanometers for BzMAG 3 -mannosyl-dextran and 0.28 microns for filtered Tc-99m sulfur colloid. A preliminary sentinel node detection study employing right fore and hind footpad injections of [ 99m Tc]MAG 3 -mannosyl-dextran and left fore and hind footpad injections of filtered Tc-99m sulfur colloid demonstrated greater sentinel lymph node uptake by the receptor-binding agent

  2. Binding constants of membrane-anchored receptors and ligands: A general theory corroborated by Monte Carlo simulations.

    Science.gov (United States)

    Xu, Guang-Kui; Hu, Jinglei; Lipowsky, Reinhard; Weikl, Thomas R

    2015-12-28

    Adhesion processes of biological membranes that enclose cells and cellular organelles are essential for immune responses, tissue formation, and signaling. These processes depend sensitively on the binding constant K2D of the membrane-anchored receptor and ligand proteins that mediate adhesion, which is difficult to measure in the "two-dimensional" (2D) membrane environment of the proteins. An important problem therefore is to relate K2D to the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in three dimensions (3D). In this article, we present a general theory for the binding constants K2D and K3D of rather stiff proteins whose main degrees of freedom are translation and rotation, along membranes and around anchor points "in 2D," or unconstrained "in 3D." The theory generalizes previous results by describing how K2D depends both on the average separation and thermal nanoscale roughness of the apposing membranes, and on the length and anchoring flexibility of the receptors and ligands. Our theoretical results for the ratio K2D/K3D of the binding constants agree with detailed results from Monte Carlo simulations without any data fitting, which indicates that the theory captures the essential features of the "dimensionality reduction" due to membrane anchoring. In our Monte Carlo simulations, we consider a novel coarse-grained model of biomembrane adhesion in which the membranes are represented as discretized elastic surfaces, and the receptors and ligands as anchored molecules that diffuse continuously along the membranes and rotate at their anchor points.

  3. Elucidation of relaxin-3 binding interactions in the extracellular loops of RXFP3

    Directory of Open Access Journals (Sweden)

    Ross eBathgate

    2013-02-01

    Full Text Available Relaxin-3 is a highly conserved neuropeptide in vertebrate species and binds to the Class A G protein-coupled receptor RXFP3. Relaxin-3 is involved in a wide range of behaviours, including feeding, stress responses, arousal and cognitive processes and therefore targeting of RXFP3 may be relevant for a range of neurological diseases. Structural knowledge of RXFP3 and its interaction with relaxin-3 would both increase our understanding of ligand recognition in GPCRs that respond to protein ligands and enable acceleration of the design of drug leads. In this study we have used comparative sequence analysis, molecular modelling and receptor mutagenesis to investigate the binding site of the native ligand human relaxin-3 (H3 relaxin on the human RXFP3 receptor. Previous structure function studies have demonstrated that arginine residues in the H3 relaxin B-chain are critical for binding interactions with the receptor extracellular loops and/or N-terminal domain. Hence we have concentrated on determining the ligand interacting sites in these domains and have focussed on glutamic (E and aspartic acid (D residues in these regions that may form electrostatic interactions with these critical arginine residues. Conserved D/E residues identified from vertebrate species multiple sequence alignments were mutated to Ala in human RXFP3 to test the effect of loss of amino acid side chain on receptor binding using both Eu-labelled relaxin-3 agonist. Finally data from mutagenesis experiments have been used in ligand docking simulations to a homology model of human RXFP3 based on the peptide-bound CXCR4 structure. These studies have resulted in a model of the relaxin-3 interaction with RXFP3 which will inform further interrogation of the agonist binding site.

  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. In Vivo Imaging of Cerebral Dopamine D3 Receptors in Alcoholism

    Science.gov (United States)

    Erritzoe, David; Tziortzi, Andri; Bargiela, David; Colasanti, Alessandro; Searle, Graham E; Gunn, Roger N; Beaver, John D; Waldman, Adam; Nutt, David J; Bani, Massimo; Merlo-Pich, Emilio; Rabiner, Eugenii A; Lingford-Hughes, Anne

    2014-01-01

    Animal studies support the role of the dopamine D3 receptor (DRD3) in alcohol reinforcement or liking. Sustained voluntary alcohol drinking in rats has been associated with an upregulation of striatal DRD3 gene expression and selective blockade of DRD3 reduces ethanol preference, consumption, and cue-induced reinstatement. In vivo measurement of DRD3 in the living human brain has not been possible until recently owing to a lack of suitable tools. In this study, DRD3 status was assessed for the first time in human alcohol addiction. Brain DRD3 availability was compared between 16 male abstinent alcohol-dependent patients and 13 healthy non-dependent age-matched males using the DRD3-preferring agonist positron emission tomography (PET) radioligand [11C]PHNO with and without blockade with a selective DRD3 antagonist (GSK598809 60 mg p.o.). In striatal regions of interest, where the [11C]PHNO PET signal represents primarily DRD2 binding, no differences were seen in [11C]PHNO binding between the groups at baseline. However, baseline [11C]PHNO binding was higher in alcohol-dependent patients in hypothalamus (VT: 16.5±4 vs 13.7±2.9, p=0.040), a region in which the [11C]PHNO signal almost entirely reflects DRD3 availability. The reductions in regional receptor binding (VT) following a single oral dose of GSK598809 (60 mg) were consistent with those observed in previous studies across all regions. There were no differences in regional changes in VT following DRD3 blockade between the two groups, indicating that the regional fractions of DRD3 are similar in the two groups, and the increased [11C]PHNO binding in the hypothalamus in alcohol-dependent patients is explained by elevated DRD3 in this group. Although we found no difference between alcohol-dependent patients and controls in striatal DRD3 levels, increased DRD3 binding in the hypothalamus of alcohol-dependent patients was observed. This may be relevant to the development of future therapeutic strategies to

  6. Opioid receptors in human neuroblastoma SH-SY5Y cells: evidence for distinct morphine (. mu. ) and enkephalin (delta) binding sites

    Energy Technology Data Exchange (ETDEWEB)

    Kazmi, S.M.I.; Mishra, R.K.

    1986-06-13

    Human neuroblastoma SH-SY5Y cells exhibited a heterogeneous population of ..mu.. and delta types of opioid binding sites. These specific binding sites displayed the characteristic saturability, stereospecificity and reversibility, expected of a receptor. Scatchard analysis of (/sup 3/H)-D-Ala/sup 2/-D-Leu/sup 5/-enkephalin (DADLE) in the presence of 10/sup -5/M D-Pro/sup 4/-morphiceptin (to block the ..mu.. receptors) and the competitive displacement by various highly selective ligands yielded the binding parameters of delta sites which closely resemble those of the delta receptors in brain and mouse neuroblastoma clones. Similarly, the high affinity binding of (/sup 3/H)-dihydromorphine, together with the higher potency of morphine analogues to displace (/sup 3/H)-naloxone binding established the presence of ..mu.. sites. Guanine nucleotides and NaCl significantly inhibited the association and increased the dissociation of (/sup 3/H)-DADLE binding.

  7. 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.

  8. Preliminary assessment of extrastriatal dopamine d-2 receptor binding in the rodent and nonhuman primate brains using the high affinity radioligand, {sup 18}F-fallypride

    Energy Technology Data Exchange (ETDEWEB)

    Mukherjee, Jogeshwar E-mail: jogeshwar-mukherjee@ketthealth.com; Yang, Z.-Y.; Brown, Terry; Lew, Robert; Wernick, Miles; Ouyang Xiaohu; Yasillo, Nicholas; Chen, C.-T.; Mintzer, Robert; Cooper, Malcolm

    1999-07-01

    We have identified the value of {sup 18}F-fallypride {l_brace}(S)-N-[(1-allyl-2-pyrrolidinyl)methyl]-5-(3-[{sup 18}F]fluoropropyl)-2,3-dim= ethoxybenzamide{r_brace}, as a dopamine D-2 receptor radiotracer for the study of striatal and extrastriatal receptors. Fallypride exhibits high affinities for D-2 and D-3 subtypes and low affinity for D-4 ({sup 3}H-spiperone IC{sub 50}s: D-2=0.05 nM [rat striata], D-3=0.30 nM [SF9 cell lines, rat recombinant], and D-4=240 nM [CHO cell lines, human recombinant]). Biodistribution in the rat brain showed localization of {sup 18}F-fallypride in striata and extrastriatal regions such as the frontal cortex, parietal cortex, amygdala, hippocampus, thalamus, and hypothalamus. In vitro autoradiographic studies in sagittal slices of the rat brain showed localization of {sup 18}F-fallypride in striatal and several extrastriatal regions, including the medulla. Positron emission tomography (PET) experiments with {sup 18}F-fallypride in male rhesus monkeys were carried out in a PET VI scanner. In several PET experiments, apart from the specific binding seen in the striatum, specific binding of {sup 18}F-fallypride was also identified in extracellular regions (in a lower brain slice, possibly the thalamus). Specific binding in the extrastriata was, however, significantly lower compared with that observed in the striata of the monkeys (extrastriata/cerebellum = 2, striata/cerebellum = 10). Postmortem analysis of the monkey brain revealed significant {sup 18}F-fallypride binding in the striata, whereas binding was also observed in extrastriatal regions such as the thalamus, cortical areas, and brain stem.

  9. Dopamine D3 receptors regulate reconsolidation of cocaine memory.

    Science.gov (United States)

    Yan, Y; Kong, H; Wu, E J; Newman, A H; Xu, M

    2013-06-25

    Memories of learned associations between the rewarding properties of drugs of abuse and environmental cues contribute to craving and relapse in humans. Disruption of reconsolidation dampens or even erases previous memories. Dopamine (DA) mediates the acquisition of reward memory and drugs of abuse can pathologically change related neuronal circuits in the mesolimbic DA system. Previous studies showed that DA D3 receptors are involved in cocaine-conditioned place preference (CPP) and reinstatement of cocaine-seeking behavior. However, the role of D3 receptors in reconsolidation of cocaine-induced reward memory remains unclear. In the present study, we combined genetic and pharmacological approaches to investigate the role of D3 receptors in reconsolidation of cocaine-induced CPP. We found that the mutation of the D3 receptor gene weakened reconsolidation of cocaine-induced CPP in mice triggered by a 3-min (min) retrieval. Furthermore, treatment of a selective D3 receptor antagonist PG01037 immediately following the 3-min retrieval disrupted reconsolidation of cocaine-induced CPP in wild-type mice and such disruption remained at least 1 week after the 3-min retrieval. These results suggest that D3 receptors play a key role in reconsolidation of cocaine-induced CPP in mice, and that pharmacological blockade of these receptors may be therapeutic for the treatment of cocaine craving and relapse in clinical settings. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  10. The effects of d-amphetamine on extrastriatal dopamine D{sub 2}/D{sub 3} receptors: a randomized, double-blind, placebo-controlled PET study with [{sup 11}C]FLB 457 in healthy subjects

    Energy Technology Data Exchange (ETDEWEB)

    Aalto, Sargo [University of Turku, Turku PET Centre, Turku (Finland); Aabo Akademi University, Department of Psychology, Turku (Finland); Hirvonen, Jussi; Kajander, Jaana; Naagren, Kjell; Rinne, Juha O. [University of Turku, Turku PET Centre, Turku (Finland); Kaasinen, Valtteri [University of Turku, Department of Neurology, P.O. Box 52, Turku (Finland); Hagelberg, Nora [University of Turku, Turku PET Centre, Turku (Finland); Turku University Central Hospital, Department of Anaesthesiology, Intensive Care, Emergency Care and Pain Medicine, Turku (Finland); Seppaelae, Timo [Drug Research Unit, National Public Health Institute, Helsinki (Finland); Scheinin, Harry [University of Turku, Turku PET Centre, Turku (Finland); University of Turku, Department of Pharmacology, Drug Development and Therapeutics, Turku (Finland); Hietala, Jarmo [University of Turku, Turku PET Centre, Turku (Finland); University of Turku, Department of Psychiatry, Turku (Finland)

    2009-03-15

    The dopamine D{sub 2}/D{sub 3} receptor ligand [{sup 11}C]FLB 457 and PET enable quantification of low-density extrastriatal D{sub 2}/D{sub 3} receptors, but it is uncertain whether [{sup 11}C]FLB 457 can be used for measuring extrastriatal dopamine release. We studied the effects of d-amphetamine (0.3 mg/kg i.v.) on extrastriatal [{sup 11}C]FLB 457 binding potential (BP{sub ND}) in a randomized, double-blind, placebo-controlled study including 24 healthy volunteers. The effects of d-amphetamine on [{sup 11}C]FLB 457 BP{sub ND} and distribution volume (V{sub T}) in the frontal cortex were not different from those of placebo. Small decreases in [{sup 11}C]FLB 457 BP{sub ND} were observed only in the posterior cingulate and hippocampus. The regional changes in [{sup 11}C]FLB 457 BP{sub ND} did not correlate with d-amphetamine-induced changes in subjective ratings of euphoria. This placebo-controlled study showed that d-amphetamine does not induce marked changes in measures of extrastriatal dopamine D{sub 2}/D{sub 3} receptor binding. Our results indicate that [{sup 11}C]FLB 457 PET is not a useful method for measuring extrastriatal dopamine release in humans. (orig.)

  11. A monoclonal antibody against PDGF B-chain inhibits PDGF-induced DNA synthesis in C3H fibroblasts and prevents binding of PDGF to its receptor.

    Science.gov (United States)

    Vassbotn, F S; Langeland, N; Hagen, I; Holmsen, H

    1990-09-01

    A monoclonal antibody (MAb 6D11) against platelet-derived growth factor (PDGF) was studied. We found that the MAb 6D11 in concentrations equimolar to PDGF blocked the [3H]thymidine incorporation in C3H/10T1/2 C18 fibroblasts stimulated by PDGF B-B and PDGF A-B. This inhibition was overcome by high doses of PDGF. The [3H]thymidine incorporation stimulated by other growth factors (aFGF, bFGF and bombesin) was not inhibited by the antibody. The MAb 6D11 blocked receptor binding of PDGF B-B, but not PDGF A-A. These findings suggest that the MAb 6D11 abolishes PDGF-induced DNA synthesis by blocking PDGF receptor binding. In this communication we demonstrate an isoform-specific monoclonal antibody against PDGF.

  12. Functionalized Ergot-alkaloids as potential dopamine D3 receptor agonists for treatment of schizophrenia

    Science.gov (United States)

    Ivanova, Bojidarka; Spiteller, Michael

    2012-12-01

    The relationship between the molecular structure and physical properties of functionalized naturally occurred Ergot-alkaloids as potential dopamine D3 receptor agonists is presented. The molecular modeling of the ergoline-skeleton is based on the comprehensive theoretical study of the binding affinity of the isolated chemicals towards the active sites of the D3 sub-type receptor (D3R) loops. The studied proton accepting ability under physiological conditions allows classifying four types of monocationics, characterizing with the different binding modes to D3R involving selected amino acid residues to the active sites. These results marked the pharmaceutical potential and clinical usage of the reported compounds as antipsychotic drugs for Schizophrenia treatment, since they allowed evaluating the highlights of the different hypothesizes of the biochemical causes the illness. The applied complex approach for theoretical and experimental elucidation, including quantum chemistry method, electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI) mass spectrometric (MS) methods, nuclear magnetic resonance and vibrational IR and Raman spectroscopy on the isolated fifteen novel derivatives (1)-(15) and their different protonated forms (1a)-(15a) evidenced a strong dependence of molecular conformation, physical properties and binding affinity. Thus, the semi-synthetic functionalization of the naturally occurred products (NPs), provided significant possibilities to further molecular drugs-design and development of novel derivatives with wanted biological function, using the established profile of selected classes/families of NPs. The work described chiefly the non-linear (NL) approach for the interpretation of the mass chromatograms on the performed hybrid high performance liquid chromatography (HPLC) tandem MS/MS and MS/MS/MS experiments, discussing the merits and great diversity of instrumentation flexibility, thus achieving fundamental

  13. Dopamine D2/D3 receptor binding of [123I]epidepride in risperidone-treatment chronic MK-801-induced rat schizophrenia model using nanoSPECT/CT neuroimaging

    International Nuclear Information System (INIS)

    Huang, Y.R.; Pai, C.W.; Cheng, K.H.; Kuo, W.I.; Chen, M.W.; Chang, K.W.

    2014-01-01

    Introduction: Epidepride is a compound with an affinity in picomolar range for D 2 /D 3 receptors. The aim of this work was designed to investigate the diagnostic possibility of [ 123 I]epidepride imaging platform for risperidone-treatment chronic MK-801-induced rat schizophrenia model. Methods: Rats received repeated administration of MK-801 (dissolved in saline, i.p., 0.3 mg/kg/day) or saline for 4 weeks. After 1-week administration of MK-801, rats in MK-801 + risperidone group received risperidone (0.5 mg/kg/day) intraperitoneally 15 min prior to MK-801 administration for the rest of 3-week treatment. We obtained serial [ 123 I]epidepride neuroimages from nanoSPECT/CT and evaluated the alteration of specific binding in striatum and midbrain. Results: Risperidone reversed chronic MK-801-induced decrease in social interaction duration. IHC and ELISA analysis showed consistent results that chronic MK-801 treatment significantly decreased striatal and midbrain D 2 R expression but repeated risperidone administration reversed the effect of MK-801 treatment. In addition, [ 123 I]epidepride nanoSPECT/CT neuroimaging revealed that low specific [ 123 I]epidepride binding ratios caused by MK-801 in striatum and midbrain were statistically alleviated after 1- and 2-week risperidone administration, respectively. Conclusions: We established a rat schizophrenia model by chronic MK-801 administration for 4 weeks. [ 123 I]Epidepride nanoSPECT neuroimaging can trace the progressive alteration of D 2 R expression in striatum and midbrain caused by long-lasting MK-801 treatment. Besides diagnosing illness stage of disease, [ 123 I]epidepride can be a useful tool to evaluate therapeutic effects of antipsychotic drug in chronic MK-801-induced rat schizophrenia model

  14. In vivo binding of [11C]nemonapride to sigma receptors in the cortex and cerebellum.

    Science.gov (United States)

    Ishiwata, K; Senda, M

    1999-08-01

    Radiolabeled nemonapride (NEM, YM-09151-2) is widely used as a representative dopamine D2-like receptor ligand in pharmacological and neurological studies, and 11C-labeled analog ([11C]NEM) has been developed for positron emission tomography (PET) studies. The aim of this study was to evaluate whether [11C]NEM binds in vivo to sigma receptors. [11C]NEM and one of six dopamine D2-like receptor ligands or seven sigma receptor ligands were co-injected into mice, and the regional brain uptake of [11C]NEM was measured by a tissue dissection method. The striatal uptake of [11C]NEM was reduced by D2-like receptor ligands, NEM, haloperidol, (+)-butaclamol, raclopride, and sulpiride, but not by a D4 receptor ligand clozapine. In the cortex and cerebellum the uptake was also reduced by D2-like receptor ligands with affinity for sigma receptors, but not by raclopride. Although none of seven sigma receptor ligands, SA6298, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine hydrochloride (NE-100), (+)-pentazocine, R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane hydrochloride ([-]-PPAP), (-)-pentazocine, R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ([+]-3-PPP), and (+)-N-allylnormetazocine hydrochloride ([+]-SKF 10047), blocked the striatal uptake, five of them with relatively higher affinity significantly reduced the [11C]NEM uptake by the cortex, and four of them reduced that by the cerebellum. We concluded that [11C]NEM binds in vivo not only to dopamine D2-like receptors in the striatum but also to sigma receptors in other regions such as cortex and cerebellum.

  15. In vivo binding of [11C]nemonapride to sigma receptors in the cortex and cerebellum

    International Nuclear Information System (INIS)

    Ishiwata, Kiichi; Senda, Michio

    1999-01-01

    Radiolabeled nemonapride (NEM, YM-09151-2) is widely used as a representative dopamine D 2 -like receptor ligand in pharmacological and neurological studies, and 11 C-labeled analog ([ 11 C]NEM) has been developed for positron emission tomography (PET) studies. The aim of this study was to evaluate whether [ 11 C]NEM binds in vivo to sigma receptors. [ 11 C]NEM and one of six dopamine D 2 -like receptor ligands or seven sigma receptor ligands were co-injected into mice, and the regional brain uptake of [ 11 C]NEM was measured by a tissue dissection method. The striatal uptake of [ 11 C]NEM was reduced by D 2 -like receptor ligands, NEM, haloperidol, (+)-butaclamol, raclopride, and sulpiride, but not by a D 4 receptor ligand clozapine. In the cortex and cerebellum the uptake was also reduced by D 2 -like receptor ligands with affinity for sigma receptors, but not by raclopride. Although none of seven sigma receptor ligands, SA6298, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine hydrochloride (NE-100), (+)-pentazocine, R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane hydrochloride ([-]-PPAP), (-)-pentazocine, R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ([+]-3-PPP), and (+)-N-allylnormetazocine hydrochloride ([+]-SKF 10047), blocked the striatal uptake, five of them with relatively higher affinity significantly reduced the [ 11 C]NEM uptake by the cortex, and four of them reduced that by the cerebellum. We concluded that [ 11 C]NEM binds in vivo not only to dopamine D 2 -like receptors in the striatum but also to sigma receptors in other regions such as cortex and cerebellum

  16. Syntheses and in vitro evaluation of fluorinated naphthoxazines as dopamine D2/D3 receptor agonists: radiosynthesis, ex vivo biodistribution and autoradiography of [18F]F-PHNO

    International Nuclear Information System (INIS)

    Vasdev, Neil; Seeman, Philip; Garcia, Armando; Stableford, Winston T.; Nobrega, Jose N.; Houle, Sylvain; Wilson, Alan A.

    2007-01-01

    Introduction: Carbon-11-labeled (+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ([ 11 C]-(+)-PHNO) is a dopamine D2/D3 agonist radioligand that is currently used to image the high-affinity state of dopamine receptors in humans with positron emission tomography (PET). The present study reports the preparation and evaluation of fluorinated (+)-PHNO derivatives. Methods: Five fluorinated (+)-PHNO derivatives were synthesized and tested in vitro for inhibition of binding of [ 3 H]domperidone in homogenates of rat striatum and inhibition of binding to [ 3 H]-(+)-PHNO in homogenates of human-cloned D2Long receptors in Chinese hamster ovary cells and rat striatum. Radiolabeling with fluorine-18 was carried out for the most promising candidate, N-fluoropropyl-(+)-HNO (F-PHNO), and ex vivo biodistribution and autoradiography studies with this radiopharmaceutical were performed in rodents. Results: (+)-PHNO and the fluorinated analogs inhibited binding of [ 3 H]domperidone and [ 3 H]-(+)-PHNO to the high- and low-affinity states of dopamine D2 receptors, consistent with D2 agonist behavior. The average dissociation constant at the high-affinity state of D2, K i High , was 0.4 nM for F-PHNO and proved to be equipotent with (+)-PHNO (0.7 nM). All other fluorinated derivatives were significantly less potent (K i High =2-102 nM). The most promising candidate, F-PHNO, was labeled with fluorine-18 in 5% uncorrected radiochemical yield, with respect to starting fluoride. Ex vivo biodistribution and autoradiography studies in rodents revealed that [ 18 F]F-PHNO rapidly enters the rodent brain. However, this radiotracer does not reveal specific binding in the brain and is rapidly cleared. Conclusions: Five novel dopamine D2/D3 agonists based on (+)-PHNO were synthesized and evaluated in vitro. F-PHNO was shown to behave as a potent D2 agonist in vitro and was therefore radiolabeled with fluorine-18. Despite the promising in vitro pharmacological profile, [ 18

  17. A case of Cotard syndrome: (123)I-IBZM SPECT imaging of striatal D(2) receptor binding.

    Science.gov (United States)

    De Risio, Sergio; De Rossi, Giuseppe; Sarchiapone, Marco; Camardese, Giovanni; Carli, Vladimir; Cuomo, Chiara; Satta, Maria Antonietta; Di Giuda, Daniela

    2004-01-15

    A case of 'dèlire de nègation' that suddenly appeared in a 43-year-old male is presented. No alteration in regional cerebral blood, as measured by (99m)Tc-HMPAO-SPECT, was found, but (123)I-IBZM-SPECT analysis showed reduced striatal D(2) receptor binding that further decreased after treatment.

  18. Guanine nucleotide regulatory protein co-purifies with the D2-dopamine receptor

    International Nuclear Information System (INIS)

    Senogles, S.E.; Caron, M.G.

    1986-01-01

    The D 2 -dopamine receptor from bovine anterior pituitary was purified ∼1000 fold by affinity chromatography on CMOS-Sepharose. Reconstitution of the affinity-purified receptor into phospholipid vesicles revealed the presence of high and low affinity agonist sites as detected by N-n-propylnorapomorphine (NPA) competition experiments with 3 H-spiperone. High affinity agonist binding could be converted to the low affinity form by guanine nucleotides, indicating the presence of an endogenous guanine nucleotide binding protein (N protein) in the affinity-purified D 2 receptor preparations. Furthermore, this preparation contained an agonist-sensitive GTPase activity which was stimulated 2-3 fold over basal by 10 μM NPA. 35 S-GTPγS binding to these preparations revealed a stoichiometry of 0.4-0.7 mole N protein/mole receptor, suggesting the N protein may be specifically coupled with the purified D 2 -dopamine receptor and not present as a contaminant. Pertussis toxin treatment of the affinity purified receptor preparations prevented high affinity agonist binding, as well as agonist stimulation of the GTPase activity, presumably by inactivating the associated N protein. Pertussis toxin lead to the ADP-ribosylation of a protein of 39-40K on SDS-PAGE. These findings indicate that an endogenous N protein, N/sub i/ or N/sub o/, co-purifies with the D 2 -dopamine receptor which may reflect a precoupling of this receptor with an N protein within the membranes

  19. Binding equilibrium and kinetics of membrane-anchored receptors and ligands in cell adhesion: Insights from computational model systems and theory

    Science.gov (United States)

    Weikl, Thomas R.; Hu, Jinglei; Xu, Guang-Kui; Lipowsky, Reinhard

    2016-01-01

    ABSTRACT The adhesion of cell membranes is mediated by the binding of membrane-anchored receptor and ligand proteins. In this article, we review recent results from simulations and theory that lead to novel insights on how the binding equilibrium and kinetics of these proteins is affected by the membranes and by the membrane anchoring and molecular properties of the proteins. Simulations and theory both indicate that the binding equilibrium constant K2D and the on- and off-rate constants of anchored receptors and ligands in their 2-dimensional (2D) membrane environment strongly depend on the membrane roughness from thermally excited shape fluctuations on nanoscales. Recent theory corroborated by simulations provides a general relation between K2D and the binding constant K3D of soluble variants of the receptors and ligands that lack the membrane anchors and are free to diffuse in 3 dimensions (3D). PMID:27294442

  20. Nigrostriatal and Mesolimbic D2/3 Receptor Expression in Parkinson's Disease Patients with Compulsive Reward-Driven Behaviors.

    Science.gov (United States)

    Stark, Adam J; Smith, Christopher T; Lin, Ya-Chen; Petersen, Kalen J; Trujillo, Paula; van Wouwe, Nelleke C; Kang, Hakmook; Donahue, Manus J; Kessler, Robert M; Zald, David H; Claassen, Daniel O

    2018-03-28

    The nigrostriatal and mesocorticolimbic dopamine networks regulate reward-driven behavior. Regional alterations to mesolimbic dopamine D 2/3 receptor expression are described in drug-seeking and addiction disorders. Parkinson's disease (PD) patients are frequently prescribed D 2 -like dopamine agonist (DAgonist) therapy for motor symptoms, yet a proportion develop clinically significant behavioral addictions characterized by impulsive and compulsive behaviors (ICBs). Until now, changes in D 2/3 receptor binding in both striatal and extrastriatal regions have not been concurrently quantified in this population. We identified 35 human PD patients (both male and female) receiving DAgonist therapy, with ( n = 17) and without ( n = 18) ICBs, matched for age, disease duration, disease severity, and dose of dopamine therapy. In the off-dopamine state, all completed PET imaging with [ 18 F]fallypride, a high affinity D 2 -like receptor ligand that can measure striatal and extrastriatal D 2/3 nondisplaceable binding potential (BP ND ). Striatal differences between ICB+/ICB- patients localized to the ventral striatum and putamen, where ICB+ subjects had reduced BP ND In this group, self-reported severity of ICB symptoms positively correlated with midbrain D 2/3 receptor BP ND Group differences in regional D 2/3 BP ND relationships were also notable: ICB+ (but not ICB-) patients expressed positive correlations between midbrain and caudate, putamen, globus pallidus, and amygdala BP ND s. These findings support the hypothesis that compulsive behaviors in PD are associated with reduced ventral and dorsal striatal D 2/3 expression, similar to changes in comparable behavioral disorders. The data also suggest that relatively preserved ventral midbrain dopaminergic projections throughout nigrostriatal and mesolimbic networks are characteristic of ICB+ patients, and may account for differential DAgonist therapeutic response. SIGNIFICANCE STATEMENT The biologic determinants of

  1. Characteristics of stably expressed human dopamine D1a and D1b receptors: atypical behavior of the dopamine D1b receptor

    DEFF Research Database (Denmark)

    Pedersen, U B; Norby, B; Jensen, Anders A.

    1994-01-01

    Human dopamine D1a and D1b receptors were stably expressed in Baby Hamster Kidney (BHK) or Chinese Hamster Ovary (CHO) cells. [3H]SCH23390 saturation experiments indicated the presence of only a single binding site in the D1a expressing cell line with a Kd of 0.5 nM. In D1b expressing cell lines...

  2. Kinetic studies on binding of thyroid hormones (L-T3 and L-T4) to the receptors of lymphocyte cells isolated from uraemia subjects

    International Nuclear Information System (INIS)

    Al-Sultani, A.S.J.

    1989-01-01

    The levels of L-T 3 , L-T 4 and TSH in uremic sera have been measured by (RIA), and shown to have a decrease in both L-T 3 and L-T 4 levels with normal level of TSH for most specimens used in this study. Kinetics properties for binding of thyroid hormones L-T 3 and L-T 4 with nuclear receptors of human lymphocyte cells extracted from uremic patient have been studied and compared this result with control and hypothyroidism subjects and we obtained that uremic condition have a large effect on these nuclear receptors properties. Dissociation constant (K d ) and maximal binding capacity (MBC) of both L-T 3 and L-T 4 with these nuclear receptors have been determined, and we obtained that uremic condition did not affect on (K d ) values for both L-T 3 and L-T 4 but it affected on (MBC) values compared with normal subject. 8 tabs.; 25 figs.; 203 refs

  3. Predicting treatment response from dopamine D2/3 receptor bnding potential? - A study in antipsychotic-naïve patients with schizophrenia

    DEFF Research Database (Denmark)

    Wulff, Sanne; Pinborg, Lars Hageman; Svarer, Claus

    of antipsychotic compounds on the positive symptoms. Furthermore, blockade of striatal dopamine D2 receptors have in studies shown to associate negatively with subjective well-being. Our main aim was to explore a possible predictive value of striatal dopamine D2/3 receptor binding potential (BPp) for treatment...... of 29 antipsychotic-naïve patients with schizophrenia and 26 matched healthy controls, SPECT with [123l]-IBZM was used to examine the BPP of striatal dopamine D2/3 receptors. The participants were examined at baseline and after 6 weeks of treatment with a selective D2/3 receptor antagonist, amisulpride....... Results: We found a significant inverse correlation between the striatal BPp at baseline and improvement of positive symptoms (p=0.046; R squared = 0.152) after six weeks of treatment with amisulpride. There was no association between the blockade of the D2/3 receptors and improvement of positive symptoms...

  4. Dopamine D2 receptors in the cerebral cortex: Distribution and pharmacological characterization with [3H]raclopride

    International Nuclear Information System (INIS)

    Lidow, M.S.; Goldman-Rakic, P.S.; Rakic, P.; Innis, R.B.

    1989-01-01

    An apparent involvement of dopamine in the regulation of cognitive functions and the recognition of a widespread dopaminergic innervation of the cortex have focused attention on the identity of cortical dopamine receptors. However, only the presence and distribution of dopamine D 1 receptors in the cortex have been well documented. Comparable information on cortical D 2 sites is lacking. The authors report here the results of binding studied in the cortex and neostriatum of rat and monkey using the D 2 selective antagonist [ 3 H]raclopride. In both structures [ 3 H]raclopride bound in a sodium-dependent and saturable manner to a single population of sites with pharmacological profiles of dopamine D 2 receptors. D 2 sites were present in all regions of the cortex, although their density was much lower than in the neostriatum. The density of these sites in both monkey and, to a lesser extent, rat cortex displayed a rostral-caudal gradient with highest concentrations in the prefrontal and lowest concentrations in the occipital cortex, corresponding to dopamine levels in these areas. Thus, the present study established the presence and widespread distribution of dopamine D 2 receptors in the cortex

  5. Dopamine D(3) receptors contribute to methamphetamine-induced alterations in dopaminergic neuronal function: role of hyperthermia.

    Science.gov (United States)

    Baladi, Michelle G; Newman, Amy H; Nielsen, Shannon M; Hanson, Glen R; Fleckenstein, Annette E

    2014-06-05

    Methamphetamine administration causes long-term deficits to dopaminergic systems that, in humans, are thought to be associated with motor slowing and memory impairment. Methamphetamine interacts with the dopamine transporter (DAT) and increases extracellular concentrations of dopamine that, in turn, binds to a number of dopamine receptor subtypes. Although the relative contribution of each receptor subtype to the effects of methamphetamine is not fully known, non-selective dopamine D2/D3 receptor antagonists can attenuate methamphetamine-induced changes to dopamine systems. The present study extended these findings by testing the role of the dopamine D3 receptor subtype in mediating the long-term dopaminergic, and for comparison serotonergic, deficits caused by methamphetamine. Results indicate that the dopamine D3 receptor selective antagonist, PG01037, attenuated methamphetamine-induced decreases in striatal DAT, but not hippocampal serotonin (5HT) transporter (SERT), function, as assessed 7 days after treatment. However, PG01037 also attenuated methamphetamine-induced hyperthermia. When methamphetamine-induced hyperthermia was maintained by treating rats in a warm ambient environment, PG01037 failed to attenuate the effects of methamphetamine on DAT uptake. Furthermore, PG01037 did not attenuate methamphetamine-induced decreases in dopamine and 5HT content. Taken together, the present study demonstrates that dopamine D3 receptors mediate, in part, the long-term deficits in DAT function caused by methamphetamine, and that this effect likely involves an attenuation of methamphetamine-induced hyperthermia. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Dopamine D3 receptors contribute to methamphetamine-induced alterations in dopaminergic neuronal function: Role of hyperthermia

    Science.gov (United States)

    Baladi, Michelle G.; Newman, Amy H.; Nielsen, Shannon M.; Hanson, Glen R.; Fleckenstein, Annette E.

    2014-01-01

    Methamphetamine administration causes long-term deficits to dopaminergic systems that, in humans, are thought to be associated with motor slowing and memory impairment. Methamphetamine interacts with the dopamine transporter (DAT) and increases extracellular concentrations of dopamine that, in turn, binds to a number of dopamine receptor subtypes. Although the relative contribution of each receptor subtype to the effects of methamphetamine is not fully known, non-selective dopamine D2/D3 receptor antagonists can attenuate methamphetamine-induced changes to dopamine systems. The present study extended these findings by testing the role of the dopamine D3 receptor subtype in mediating the long-term dopaminergic, and for comparison serotonergic, deficits caused by methamphetamine. Results indicate that the dopamine D3 receptor selective antagonist, PG01037, attenuated methamphetamine-induced decreases in striatal DAT, but not hippocampal serotonin (5HT) transporter (SERT), function, as assessed 7 days after treatment. However, PG01037 also attenuated methamphetamine-induced hyperthermia. When methamphetamine-induced hyperthermia was maintained by treating rats in a warm ambient environment, PG01037 failed to attenuate the effects of methamphetamine on DAT uptake. Furthermore, PG01037 did not attenuate methamphetamine-induced decreases in dopamine and 5HT content. Taken together, the present study demonstrates that dopamine D3 receptors mediate, in part, the long-term deficits in DAT function caused by methamphetamine, and that this effect likely involves an attenuation of methamphetamine-induced hyperthermia. PMID:24685638

  7. MDMA-evoked changes in the binding of dopamine D(2) receptor ligands in striatum of rats with unilateral serotonin depletion

    DEFF Research Database (Denmark)

    Ostergaard, Søren Dinesen; Alstrup, Aage Kristian Olsen; Gramsbergen, Jan Bert

    2010-01-01

    We earlier reported an anomalous 50% decrease in [(11)C]N-methylspiperone ([(11)C]NMSP) binding to dopamine D(2)-like receptors in living pig striatum after challenge with 3,4-methylenedioxymethamphetamine (MDMA, "Ecstasy"), suggesting either (1) a species peculiarity in the vulnerability...... lesions, later verified by [(125)I]RTI-55 autoradiography. Baseline [(11)C]NMSP microPET recordings were followed by either saline or MDMA-HCl (4 mg/kg) injections (i.v.), and a second [(11)C]NMSP recording, culminating with injection of [(3)H]raclopride for autoradiography ex vivo. Neither MDMA......-challenge nor serotonin lesion had any detectable effect on [(11)C]NMSP binding. In contrast, MDMA challenge increased receptor occupancy by [(3)H]raclopride ex vivo (relative to the B(max) in vitro) from 8% to 12%, and doubled the free ligand concentration in cerebral cortex, apparently by blocking hepatic CYP...

  8. Pharmacology and Structural Analysis of Ligand Binding to the Orthosteric Site of Glutamate-Like GluD2 Receptors

    DEFF Research Database (Denmark)

    Kristensen, Anders S; Hansen, Kasper B; Naur, Peter

    2016-01-01

    -term depression. Here, we investigate the pharmacology of the orthosteric binding site in GluD2 by examining the activity of analogs of D-Ser and GluN1 glycine site competitive antagonists at GluD2 receptors containing the lurcher mutation (GluD2(LC)), which promotes spontaneous channel activation. We identify...

  9. Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

    Science.gov (United States)

    Cai, Minying; Marelli, Udaya Kiran; Mertz, Blake; Beck, Johannes G; Opperer, Florian; Rechenmacher, Florian; Kessler, Horst; Hruby, Victor J

    2017-08-15

    Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His 6 -d-Nal(2') 7 -NMe-Arg 8 -Trp 9 -Lys]-NH 2 (15) and Ac-Nle-c[Asp-His 6 -d-Nal(2') 7 -NMe-Arg 8 -NMe-Trp 9 -NMe-Lys]-NH 2 (17). It is known that the pharmacophore (His 6 -DNal 7 -Arg 8 -Trp 9 ) of the SHU-9119 peptides occupies a β II-turn-like region with the turn centered about DNal 7 -Arg 8 . The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp 9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg 8 and Trp 9 side chains are involved in a majority of the interactions with the receptor. While Arg 8 forms polar contacts with D154 and D158 of hMC3R, Trp 9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp 9 -hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

  10. Structural basis for ligand recognition at the benzodiazepine binding site of GABAA alpha 3 receptor, and pharmacophore-based virtual screening approach.

    Science.gov (United States)

    Vijayan, R S K; Ghoshal, Nanda

    2008-10-01

    Given the heterogeneity of GABA(A) receptor, the pharmacological significance of identifying subtype selective modulators is increasingly being recognized. Thus, drugs selective for GABA(A) alpha(3) receptors are expected to display fewer side effects than the drugs presently in clinical use. Hence we carried out 3D QSAR (three-dimensional quantitative structure-activity relationship) studies on a series of novel GABA(A) alpha(3) subtype selective modulators to gain more insight into subtype affinity. To identify the 3D functional attributes required for subtype selectivity, a chemical feature-based pharmacophore, primarily based on selective ligands representing diverse structural classes was generated. The obtained pseudo receptor model of the benzodiazepine binding site revealed a binding mode akin to "Message-Address" concept. Scaffold hopping was carried out across multi-conformational May Bridge database for the identification of novel chemotypes. Further a focused data reduction approach was employed to choose a subset of enriched compounds based on "Drug likeness" and "Similarity-based" methods. These results taken together could provide impetus for rational design and optimization of more selective and high affinity leads with a potential to have decreased adverse effects.

  11. The multiplicity of the D-1 dopamine receptor

    International Nuclear Information System (INIS)

    Mailman, R.B.; Klits, C.D.; Lewis, M.H.; Rollema, H.; Schulz, D.W.; Wyrick, S.

    1986-01-01

    The authors have sought to address two questions of some neuropharmacological importance in this chapter. First, they examine the nature of mechanisms by which dopamine initiates many psychopharmacological effects and, second, they study the possibility of designing highly specific drugs targeted only at a selected subpopulation of dopamine receptors. Effects of SCH23390 and haloperidol on concentrations of dopamine, DOPAC, and HVA in various rat brain regions are shown. In addition, the effects of SCH23390 on the in vivo binding of dipropyl-5, 6-ADTN are shown. Differential distribution of a dopamine sensitive adenylate cyclase and ( 3 H)-SCH23390 binding sites are examined. A model is presented of D 1 dopamine receptors in membrane, illustrating the lack of identity of some of the ( 3 H)-SCH23390 binding sites with the dopamine receptor linked to stimulation of cAMP synthesis

  12. Comparison of D2 receptor binding (123I-IBZM) and rCBF (99mTc-HMPAO) in extrapyramidal disorders

    International Nuclear Information System (INIS)

    Saur, H.B.; Bartenstein, P.; Schober, O.; Oberwittler, C.; Lerch, H.; Masur, H.

    1994-01-01

    The aim of this SPECT study was to determine whether there is a correlation between rCBF ( 99m Tc-HMPAO) and D2 receptor binding ( 123 I-IBZM) in disorders of the extrapyramidal system and in which situation the 99 MTc-HMPAO scan could predict the outcome of the 123 I-IBZM study. 13 patients with Parkinson's syndrome and 13 patients with hyperkinetic extrapyramidal disorders were studied. In all patients the two SPECT studies were performed within 2-7 days. ROIs were placed over the basal ganglia (BG), the frontal cortex (FC) and the cerebellum (CE). The ratios BG/FC and BG/CE were calculated. In both groups the scatter was lower when the frontal cortex was used as reference region. Among the patients with hyperkinetic extrapyramidal hyperkinetic extrapyramidal disorders the two patients with Huntington's chorea had lower rCBF and D2 receptor binding compared to other hyperkinetic extrapyramidal disorders. There was no correlation between D2 receptor binding and rCBF in the basal ganglia. The 99 MTc-HMPAO studies did not provide clinically useful information, except in Huntington's chorea. (orig.) [de

  13. MDM2 binds and inhibits vitamin D receptor

    OpenAIRE

    Heyne, Kristina; Heil, Tessa-Carina; Bette, Birgit; Reichrath, Jörg; Roemer, Klaus

    2015-01-01

    The E3 ubiquitin ligase and transcriptional repressor MDM2 is a potent inhibitor of the p53 family of transcription factors and tumor suppressors. Herein, we report that vitamin D receptor (VDR), another transcriptional regulator and probably, tumor suppressor, is also bound and inhibited by MDM2. This interaction was not affected by vitamin D ligand. VDR was ubiquitylated in the cell and its steady-state level was controlled by the proteasome. Strikingly, overproduced MDM2 reduced the level ...

  14. Syntheses and in vitro evaluation of fluorinated naphthoxazines as dopamine D2/D3 receptor agonists: radiosynthesis, ex vivo biodistribution and autoradiography of [{sup 18}F]F-PHNO

    Energy Technology Data Exchange (ETDEWEB)

    Vasdev, Neil [PET Centre for Addiction and Mental Health, Toronto, Ontario, Canada, M5T-1R8 (Canada) and Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T-1R8 (Canada)]. E-mail: neil.vasdev@camhpet.ca; Seeman, Philip [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T-1R8 (Canada); Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S-1A8 (Canada); Garcia, Armando [PET Centre for Addiction and Mental Health, Toronto, Ontario, M5T-1R8 (Canada); Stableford, Winston T. [PET Centre for Addiction and Mental Health, Toronto, Ontario, M5T-1R8 (Canada); Nobrega, Jose N. [PET Centre for Addiction and Mental Health, Toronto, Ontario, M5T-1R8 (Canada); Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T-1R8 (Canada); Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S-1A8 (Canada); Houle, Sylvain [PET Centre for Addiction and Mental Health, Toronto, Ontario, M5T-1R8 (Canada); Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T-1R8 (Canada); Wilson, Alan A. [PET Centre for Addiction and Mental Health, Toronto, Ontario, M5T-1R8 (Canada); Department of Psychiatry, University of Toronto, Toronto, Ontario, M5T-1R8 (Canada)

    2007-02-15

    Introduction: Carbon-11-labeled (+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4]oxazin-9-ol ([{sup 11}C]-(+)-PHNO) is a dopamine D2/D3 agonist radioligand that is currently used to image the high-affinity state of dopamine receptors in humans with positron emission tomography (PET). The present study reports the preparation and evaluation of fluorinated (+)-PHNO derivatives. Methods: Five fluorinated (+)-PHNO derivatives were synthesized and tested in vitro for inhibition of binding of [{sup 3}H]domperidone in homogenates of rat striatum and inhibition of binding to [{sup 3}H]-(+)-PHNO in homogenates of human-cloned D2Long receptors in Chinese hamster ovary cells and rat striatum. Radiolabeling with fluorine-18 was carried out for the most promising candidate, N-fluoropropyl-(+)-HNO (F-PHNO), and ex vivo biodistribution and autoradiography studies with this radiopharmaceutical were performed in rodents. Results: (+)-PHNO and the fluorinated analogs inhibited binding of [{sup 3}H]domperidone and [{sup 3}H]-(+)-PHNO to the high- and low-affinity states of dopamine D2 receptors, consistent with D2 agonist behavior. The average dissociation constant at the high-affinity state of D2, K {sub i} {sup High}, was 0.4 nM for F-PHNO and proved to be equipotent with (+)-PHNO (0.7 nM). All other fluorinated derivatives were significantly less potent (K {sub i} {sup High}=2-102 nM). The most promising candidate, F-PHNO, was labeled with fluorine-18 in 5% uncorrected radiochemical yield, with respect to starting fluoride. Ex vivo biodistribution and autoradiography studies in rodents revealed that [{sup 18}F]F-PHNO rapidly enters the rodent brain. However, this radiotracer does not reveal specific binding in the brain and is rapidly cleared. Conclusions: Five novel dopamine D2/D3 agonists based on (+)-PHNO were synthesized and evaluated in vitro. F-PHNO was shown to behave as a potent D2 agonist in vitro and was therefore radiolabeled with fluorine-18. Despite the

  15. Photoaffinity labeling of opiate receptors using intrinsically photoactive 3H-opiates

    International Nuclear Information System (INIS)

    Kooper, G.N.; Levinson, N.R.; Copeland, C.F.; Bowen, W.D.

    1988-01-01

    Opiate receptors in rat and cow brain membranes have been labeled irreversibly using the intrinsic photolability of 3H-opiates. Membranes were incubated with 3H-ligand and then irradiated with UV light of 254 nm. Nonspecific binding was determined in the presence of 10 microM unlabeled levallorphan. Irreversible binding was defined as binding which survived heat or acid denaturation of membranes. Specific incorporation of label into denatured samples was observed only when unbound or loosely bound 3H-ligand was washed free from the membranes prior to irradiation. There was a general correlation between photosensitivity of the 3H-ligand and its ability to photolabel receptors. Hence, photolabeling presumably results by covalent attachment of highly reactive species generated during photochemical decomposition of ligand. With 3H-etorphine, optimal irradiation time was 5 min. In addition to 3H-etorphine, receptors could be labeled irreversibly with 3H-oxymorphone, 3H-dihydromorphine, and 3H-ethylketocyclazocine. Of the specific binding present in irradiated, nondenatured samples, 45-60% remained attached to receptors upon denaturation. 3H-Ethylketocyclazocine exhibited an 86% yield of incorporation. Signal-to-noise levels of 50-80% could be achieved in denatured samples. Therefore, this method provides a means of covalently labeling opiate receptors in high yield and with high signal-to-noise ratios. The opioid peptides, 3H-D-Ala2,D-Leu5-enkephalin, 3H-D-Ser2,Leu5,Thr6-enkephalin, 3H-D-Ala2,Met5-enkephalin amide, and 3H-D-Ala2,N-MePhe4,Gly-ol5-enkephalin, as well as the benzomorphan, 3H-bremazocine, apparently lack the structural characteristics which allow photolabeling

  16. Different receptors binding to distinct interfaces on herpes simplex virus gD can trigger events leading to cell fusion and viral entry

    International Nuclear Information System (INIS)

    Spear, Patricia G.; Manoj, Sharmila; Yoon, Miri; Jogger, Cheryl R.; Zago, Anna; Myscofski, Dawn

    2006-01-01

    One of the herpes simplex virus envelope glycoproteins, designated gD, is the principal determinant of cell recognition for viral entry. Other viral glycoproteins, gB, gH and gL, cooperate with gD to mediate the membrane fusion that is required for viral entry and cell fusion. Membrane fusion is triggered by the binding of gD to one of its receptors. These receptors belong to three different classes of cell surface molecules. This review summarizes recent findings on the structure and function of gD. The results presented indicate that gD may assume more than one conformation, one in the absence of receptor, another when gD is bound to the herpesvirus entry mediator, a member of the TNF receptor family, and a third when gD is bound to nectin-1, a cell adhesion molecule in the immunoglobulin superfamily. Finally, information and ideas are presented about a membrane-proximal region of gD that is required for membrane fusion, but not for receptor binding, and that may have a role in activating the fusogenic activity of gB, gH and gL

  17. New detection of brain dopamine receptors with [3H]dihydroergocryptine

    International Nuclear Information System (INIS)

    Tittler, M.; Weinreich, P.; Seeman, P.

    1977-01-01

    Because dihydroergocryptine (DHE) and closely related ergots are dopamine-mimetic agonists, we tested [ 3 H]DHE as a possible ligand for [ 3 H]dopamine receptors in the calf caudate. In order to avoid [ 3 H]DHE from tagging α-adrenergic receptors, an excess of 500 nM phentolamine was used to block these sites, permitting the dopamine receptors to be measured separately. Specific binding of [ 3 H]DHE was defined as total binding minus that occurring in the presence of 1 μM (+)-butaclamol. Excess phentolamine reduced the specific binding of [ 3 H]DHE from 328 down to 138 fmol/mg, the difference presumably representing α-receptors. The K/sub D/ for [ 3 H]DHE was 0.55 nM (with or without phentolamine), and this high affinity site was blocked (in the presence of phentolamine) by 250 nM apomorphine, 650 nM dopamine, and 1200 nM (-)-norepinephrine, indicating that [ 3 H]DHE was binding to dopamine receptors. All neuroleptics blocked specific [ 3 H]DHE binding in direct relation to the clinical potency of the neuroleptic. The displacement of specific [ 3 H]DHE binding by dopamine or by norepinephrine (in the presence of phentolamine) revealed two subsets of dopamine receptors

  18. The binding of [3H]AF-DX 384 to rat ileal smooth muscle muscarinic receptors

    International Nuclear Information System (INIS)

    Entzeroth, M.; Mayer, N.

    1991-01-01

    The tritiated cardioselective muscarinic antagonist AF-DX 384 (5,11-dihydro-11-[2-[-(8-dipropylamino)methyl]-1-piperidinyl-ethyl-amino-carbonyl]-6H-pyrido [2,3-b] [1,4]benzodiazepin-6-one) was used to label muscarinic receptors in the rat ileum. Saturation binding to membrane suspensions revealed a high affinity binding site with a Kd of 9.2 nM. The maximal number of binding sites labeled in this tissue (Bmax) is 237 fmol/mg protein. The association and dissociation kinetics were well represented by single exponential reactions, and the dissociation constant obtained from the ratio of rate constants was in agreement with that derived from saturation experiments. Specific binding was inhibited by muscarinic antagonists with a rank order of potencies of atropine (pKi: 8.80) greater than 4-DAMP (pKi: 8.23) = AF-DX 384 (pKi: 8.20) greater than AF-DX 116 (pKi: 7.09) = hexahydro-sila-difenidol (pKi: 6.97) greater than pirenzepine (pKi: 6.49) and is consistent with the interaction of [3H]AF-DX 384 with muscarinic receptors of the M2 subtype. It can be concluded that [3H]AF-DX 384 can be used to selectively label M2 muscarinic receptors in heterogeneous receptor populations

  19. The potent opioid agonist, (+)-cis-3-methylfentanyl binds pseudoirreversibly to the opioid receptor complex in vitro and in vivo: Evidence for a novel mechanism of action

    Energy Technology Data Exchange (ETDEWEB)

    Band, L.; Xu, Heng; Bykov, V.; Rothman, R.B.; Kim, Chongho; Newman, A.; Jacobson, A.E.; Rice, K.C. (NIDDK, Bethesda, MD (USA)); Greig, N. (NIA, Bethesda, MD (USA))

    1990-01-01

    The present study demonstrates that pretreatment of rat brain membranes with (+)-cis-3-methylfentanyl ((+)-cis-MF), followed by extensive washing of the membranes, produces a wash-resistant decreasing in the binding of ({sup 3}H)-(D-ala{sup 2}, D-leu{sup 5})enkephalin to the d binding site of the opioid receptor complex ({delta}{sub cx} binding site). Intravenous administration of (+)-cis-MF (50 {mu}g/kg) to rats produced a pronounced catalepsy and also produced a wash-resistant masking of {delta}{sub cx} and {mu} binding sites in membranes prepared 120 min post-injection. Administration of 1 mg/kg i.v. of the opioid antagonist, 6-desoxy-6{beta}-fluoronaltrexone (cycloFOXY), 100 min after the injection of (+)-cis-MF (20 min prior to the preparation of membranes) completely reversed the catatonia and restored masked {delta}{sub cx} binding sites to control levels. This was not observed with (+)-cycloFOXY. The implications of these and other findings for the mechanism of action of (+)-cis-MF and models of the opioid receptors are discussed.

  20. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    International Nuclear Information System (INIS)

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-01-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent [ 3 H]acetylcholine release from rabbit retina labeled in vitro with [ 3 H]choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of [ 3 H]acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of [ 3 H]acetylcholine with the following order of potency: apomorphine ≤ SKF(R)82526 3 H]acetylcholine: SCH 23390 (IC50 = 1 nM) 3 H]acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by [ 3 H]SCH 23390, or as determined by adenylate cyclase activity. [ 3 H]SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of [ 3 H]SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate [ 3 H]acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at [ 3 H]SCH 23390 binding sites (r = 0.755, P < .05, n = 8)

  1. Effects of the dopamine D2 allosteric modulator, PAOPA, on the expression of GRK2, arrestin-3, ERK1/2, and on receptor internalization.

    Directory of Open Access Journals (Sweden)

    Dipannita Basu

    Full Text Available The activity of G protein-coupled receptors (GPCRs is intricately regulated by a range of intracellular proteins, including G protein-coupled kinases (GRKs and arrestins. Understanding the effects of ligands on these signaling pathways could provide insights into disease pathophysiologies and treatment. The dopamine D2 receptor is a GPCR strongly implicated in the pathophysiology of a range of neurological and neuropsychiatric disorders, particularly schizophrenia. Previous studies from our lab have shown the preclinical efficacy of a novel allosteric drug, 3(R-[(2(S-pyrrolidinylcarbonylamino]-2-oxo-1-pyrrolidineacetamide (PAOPA, in attenuating schizophrenia-like behavioural abnormalities in rodent models of the disease. As an allosteric modulator, PAOPA binds to a site on the D2 receptor, which is distinct from the endogenous ligand-binding site, in order to modulate the binding of the D2 receptor ligand, dopamine. The exact signaling pathways affected by this allosteric modulator are currently unknown. The objectives of this study were to decipher the in vivo effects, in rats, of chronic PAOPA administration on D2 receptor regulatory and downstream molecules, including GRK2, arrestin-3 and extracellular receptor kinase (ERK 1/2. Additionally, an in vitro cellular model was also used to study PAOPA's effects on D2 receptor internalization. Results from western immunoblots showed that chronic PAOPA treatment increased the striatal expression of GRK2 by 41%, arrestin-3 by 34%, phospho-ERK1 by 51% and phospho-ERK2 by 36%. Results also showed that the addition of PAOPA to agonist treatment in cells increased D2 receptor internalization by 33%. This study provides the foundational evidence of putative signaling pathways, and changes in receptor localization, affected by treatment with PAOPA. It improves our understanding on the diverse mechanisms of action of allosteric modulators, while advancing PAOPA's development into a novel drug for the

  2. Pituitary and brain D2 receptor density measured in vitro and in vivo in EEDQ treated male rats

    International Nuclear Information System (INIS)

    Ekman, A.; Eriksson, E.

    1991-01-01

    The effect of the alkylating compound N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) on dopamine D2 receptor density in rat pituitary and brain was measured using in vitro and in vivo radioligand binding techniques. In the in vitro radioligand binding experiments EEDQ was found to reduce the density (B max ) of [ 3 H]-spiperone binding sites in the striatum by 86% while in the pituitary the corresponding decrease was only 37%. The affinity (K D ) of the remaining striatal and pituitary D2 receptors was not different in EEDQ treated animals as compared to controls. When D2 receptor density was measured in vivo the effect of EEDQ was less pronounced. Thus, in rats given EEDQ the specific binding of either of the two D2 ligands [ 3 H]-raclopride or [ 3 H]-spiperone in striatum and in the limbic forebrain was reduced by 45-62%; moreover, no significant decrease in pituitary D2 receptor density was observed. The data are discussed in relation to the finding that the same dose of EEDQ that failed to influence pituitary D2 receptor density as measured in vivo effectively antagonizes the prolactin decreasing effect of the partial D2 agonist (-)-3-(3-hydroxyphenyl)-N-n-propyl-piperidine [(-)-3-PPP

  3. 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.

  4. B700, a murine melanoma-specific antigen, binds Vitamin D3; conservation of binding among albuminoid molecules

    International Nuclear Information System (INIS)

    Farzaneh, N.K.; Walden, T.L. Jr.; Hearing, V.J.; Gersten, D.M.

    1990-01-01

    B700, a murine melanoma-specific antigen, is a member of the serum albumin protein family. Other members of this family include serum albumin (SMA), a-fetoprotein (AFP), vitamin D binding protein (DBP), and C700. The primary structure and biochemical functions of B700, as well as its in vivo metabolic fate are largely unknown. The authors examined the functional characteristics of MSA, AFP, and DBP, and for their ability to specifically bind [ 3 H]-1,25-dihydroxy-vitamin D 3 . Scatchard analysis revealed a single binding site for B700 with a Kd of 51,000 M and a Bmax of 4.51 x 10 -7 . There is no significant difference between the Kd and Bmax values among the albuminoid proteins. However, differences in the binding sites could be distinguished by competition of the 1,25-dihydroxy vitamin D 3 with other steroids. 2nM of vitamin D 3 , vitamin D 2 , or estrogen competed for the specific binding of 1,25-dihydroxy vitamin D 3 by B700 but not by DBP. The MSA binding site for 1,25 dihydroxy vitamin D 3 more closely resembles that of DBP than B700. These data indicate that the binding function of the albuminoid proteins has been conserved in the B700 melanoma antigen

  5. Hereditary 1,25-dihydroxyvitamin D-resistant rickets with alopecia resulting from a novel missense mutation in the DNA-binding domain of the vitamin D receptor

    Science.gov (United States)

    Malloy, Peter J.; Wang, Jining; Srivastava, Tarak; Feldman, David

    2009-01-01

    The rare genetic recessive disease, hereditary vitamin D resistant rickets (HVDRR), is caused by mutations in the vitamin D receptor (VDR) that result in resistance to the active hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3 or calcitriol). In this study, we examined the VDR from a young boy with clinical features of HVDRR including severe rickets, hypocalcemia, hypophosphatemia and partial alopecia. The pattern of alopecia was very unusual with areas of total baldness, adjacent to normal hair and regions of scant hair. The child failed to improve on oral calcium and vitamin D therapy but his abnormal chemistries and his bone x-rays normalized with intravenous calcium therapy. We found that the child was homozygous for a unique missense mutation in the VDR gene that converted valine to methionine at amino acid 26 (V26M) in the VDR DNA-binding domain (DBD). The mutant VDR was studied in the patient’s cultured skin fibroblasts and found to exhibit normal [3H]1,25-(OH)2D3 binding and protein expression. However, the fibroblasts were unresponsive to treatment with high concentrations of 1,25(OH)2D3 as demonstrated by their failure to induce CYP24A1 gene expression, a marker of 1,25(OH)2D3 responsiveness. We recreated the V26M mutation in the WT VDR and showed that in transfected COS-7 cells the mutation abolished 1,25(OH)2D3-mediated transactivation. The mutant VDR exhibited normal ligand-induced binding to RXRα and to the coactivator DRIP205. However, the V26M mutation inhibited VDR binding to a consensus vitamin D response element (VDRE). In summary, we have identified a novel V26M mutation in the VDR DBD as the molecular defect in a patient with HVDRR and an unusual pattern of alopecia. PMID:19815438

  6. 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.

  7. 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

  8. Striatal D2/3 Binding Potential Values in Drug-Naïve First-Episode Schizophrenia Patients Correlate With Treatment Outcome

    Science.gov (United States)

    Wulff, Sanne; Pinborg, Lars Hageman; Svarer, Claus; Jensen, Lars Thorbjørn; Nielsen, Mette Ødegaard; Allerup, Peter; Bak, Nikolaj; Rasmussen, Hans; Frandsen, Erik; Rostrup, Egill; Glenthøj, Birte Yding

    2015-01-01

    One of best validated findings in schizophrenia research is the association between blockade of dopamine D2 receptors and the effects of antipsychotics on positive psychotic symptoms. The aim of the present study was to examine correlations between baseline striatal D2/3 receptor binding potential (BPp) values and treatment outcome in a cohort of antipsychotic-naïve first-episode schizophrenia patients. Additionally, we wished to investigate associations between striatal dopamine D2/3 receptor blockade and alterations of negative symptoms as well as functioning and subjective well-being. Twenty-eight antipsychotic-naïve schizophrenia patients and 26 controls were included in the study. Single-photon emission computed tomography (SPECT) with [123I]iodobenzamide ([123I]-IBZM) was used to examine striatal D2/3 receptor BPp. Patients were examined before and after 6 weeks of treatment with the D2/3 receptor antagonist amisulpride. There was a significant negative correlation between striatal D2/3 receptor BPp at baseline and improvement of positive symptoms in the total group of patients. Comparing patients responding to treatment to nonresponders further showed significantly lower baseline BPp in the responders. At follow-up, the patients demonstrated a negative correlation between the blockade and functioning, whereas no associations between blockade and negative symptoms or subjective well-being were observed. The results show an association between striatal BPp of dopamine D2/3 receptors in antipsychotic-naïve first-episode patients with schizophrenia and treatment response. Patients with a low BPp have a better treatment response than patients with a high BPp. The results further suggest that functioning may decline at high levels of dopamine receptor blockade. PMID:25698711

  9. Regulation of CYP3A4 by pregnane X receptor: The role of nuclear receptors competing for response element binding

    Energy Technology Data Exchange (ETDEWEB)

    Istrate, Monica A., E-mail: monicai@scripps.edu [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Nussler, Andreas K., E-mail: nuessler@uchir.me.tum.de [Department of Traumatology, Technical University Munich, Ismaningerstr. 22, 81675 Munich (Germany); Eichelbaum, Michel, E-mail: michel.eichelbaum@ikp-stuttgart.de [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany); Burk, Oliver, E-mail: oliver.burk@ikp-stuttgart.de [Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Auerbachstr. 112, D-70376 Stuttgart (Germany)

    2010-03-19

    Induction of the major drug metabolizing enzyme CYP3A4 by xenobiotics contributes to the pronounced interindividual variability of its expression and often results in clinically relevant drug-drug interactions. It is mainly mediated by PXR, which regulates CYP3A4 expression by binding to several specific elements in the 5' upstream regulatory region of the gene. Induction itself shows a marked interindividual variability, whose underlying determinants are only partly understood. In this study, we investigated the role of nuclear receptor binding to PXR response elements in CYP3A4, as a potential non-genetic mechanism contributing to interindividual variability of induction. By in vitro DNA binding experiments, we showed that several nuclear receptors bind efficiently to the proximal promoter ER6 and distal xenobiotic-responsive enhancer module DR3 motifs. TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII further demonstrated dose-dependent repression of PXR-mediated CYP3A4 enhancer/promoter reporter activity in transient transfection in the presence and absence of the PXR inducer rifampin, while VDR showed this effect only in the absence of treatment. By combining functional in vitro characterization with hepatic expression analysis, we predict that TR{alpha}1, TR{beta}1, COUP-TFI, and COUP-TFII show a strong potential for the repression of PXR-mediated activation of CYP3A4 in vivo. In summary, our results demonstrate that nuclear receptor binding to PXR response elements interferes with PXR-mediated expression and induction of CYP3A4 and thereby contributes to the interindividual variability of induction.

  10. Antagonism by supidimide of haloperidol-induced augmentation of [3H]-spiperone binding in rat striatum

    International Nuclear Information System (INIS)

    Hennies, H.H.; Hess, V.; Flohe, L.

    1984-01-01

    Rats were treated for two weeks with haloperidol alone or with additional test drugs and the D 2 receptor density in the striatum was investigated after a drug-free period of five days. The D 2 receptor system as analysed by [ 3 H]-spinerone binding was best characterized by a model with two binding sites of different affinity. Chronic haloperidol treatment substantially augmented the total binding capacity 2-(2-Oxo-3-piperidyl)-1,2-benzisothiazoline-3-one-1,1-dioxide (supidimide), a functional synergist of neuroleptics in acute experiments, surprisingly antagonized the haloperidol-induced receptor augmentation, whereas other CNS depressants (diazepam and phenobarbital) were ineffective. (orig./MG) [de

  11. Presence of D4 dopamine receptors in human prefrontal cortex: a postmortem study Presença de receptores dopaminérgicos D4 em córtex cerebral humano: um estudo post-mortem

    Directory of Open Access Journals (Sweden)

    Donatella Marazziti

    2007-06-01

    Full Text Available OBJECTIVE: The aim of our study was to explore the presence and the distribution of D4 dopamine receptors in postmortem human prefrontal cortex, by means of the binding of [³H]YM-09151-2, an antagonist that has equal affinity for D2, D3 and D4 receptors. It was therefore necessary to devise a unique assay method in order to distinguish and detect the D4 component. METHOD: Frontal cortex samples were harvested postmortem, during autopsy sessions, from 5 subjects. In the first assay, tissue homogenates were incubated with increasing concentrations of [³H]YM-09151-2, whereas L-745870, which has a high affinity for D4 and a low affinity for D2/D3 receptors, was used as the displacer. In the second assay, raclopride, which has a high affinity for D2/D3 receptors and a low affinity for D4 receptors, was used to block D2/D3. The L-745870 (500 nM was added to both assays in order to determine the nonspecific binding. RESULTS: Our experiments revealed the presence of specific and saturable binding of [³H]YM-09151-2. The blockade of D2 and D3 receptors with raclopride ensured that the D4 receptors were labeled. The mean maximum binding capacity was 88 ± 25 fmol/mg protein, and the dissociation constant was 0.8 ± 0.4 nM. DISCUSSION AND CONCLUSIONS: Our findings, although not conclusive, suggest that the density of D4 receptors is low in the human prefrontal cortex.OBJETIVO: O objetivo deste estudo foi quantificar a presença e a distribuição de receptores dopaminérgicos do tipo 4 (D4 no córtex cerebral humano em amostras post-mortem através do bloqueio com ³H-YM-09151-2 - um antagonista com afinidade equivalente pelos receptores D2, D3 e D4 - e do desenvolvimento de um método para a detecção específica do componente D4. MÉTODO: Foram obtidas amostras de córtex cerebral de cinco cadáveres. Em um primeiro ensaio, os homogeneizados de tecido cerebral foram incubados em concentrações crescentes de ³H-YM-09151-2, enquanto que o L-745

  12. Effects of egg-adaptation on receptor-binding and antigenic properties of recent influenza A (H3N2) vaccine viruses.

    Science.gov (United States)

    Parker, Lauren; Wharton, Stephen A; Martin, Stephen R; Cross, Karen; Lin, Yipu; Liu, Yan; Feizi, Ten; Daniels, Rodney S; McCauley, John W

    2016-06-01

    Influenza A virus (subtype H3N2) causes seasonal human influenza and is included as a component of influenza vaccines. The majority of vaccine viruses are isolated and propagated in eggs, which commonly results in amino acid substitutions in the haemagglutinin (HA) glycoprotein. These substitutions can affect virus receptor-binding and alter virus antigenicity, thereby, obfuscating the choice of egg-propagated viruses for development into candidate vaccine viruses. To evaluate the effects of egg-adaptive substitutions seen in H3N2 vaccine viruses on sialic acid receptor-binding, we carried out quantitative measurement of virus receptor-binding using surface biolayer interferometry with haemagglutination inhibition (HI) assays to correlate changes in receptor avidity with antigenic properties. Included in these studies was a panel of H3N2 viruses generated by reverse genetics containing substitutions seen in recent egg-propagated vaccine viruses and corresponding cell culture-propagated wild-type viruses. These assays provide a quantitative approach to investigating the importance of individual amino acid substitutions in influenza receptor-binding. Results show that viruses with egg-adaptive HA substitutions R156Q, S219Y, and I226N, have increased binding avidity to α2,3-linked receptor-analogues and decreased binding avidity to α2,6-linked receptor-analogues. No measurable binding was detected for the viruses with amino acid substitution combination 156Q+219Y and receptor-binding increased in viruses where egg-adaptation mutations were introduced into cell culture-propagated virus. Substitutions at positions 156 and 190 appeared to be primarily responsible for low reactivity in HI assays with post-infection ferret antisera raised against 2012-2013 season H3N2 viruses. Egg-adaptive substitutions at position 186 caused substantial differences in binding avidity with an insignificant effect on antigenicity.

  13. Stereocontrolled dopamine receptor binding and subtype selectivity of clebopride analogues synthesized from aspartic acid.

    Science.gov (United States)

    Einsiedel, Jürgen; Weber, Klaus; Thomas, Christoph; Lehmann, Thomas; Hübner, Harald; Gmeiner, Peter

    2003-10-06

    Employing the achiral 4-aminopiperidine derivative clebopride as a lead compound, chiral analogues were developed displaying dopamine receptor binding profiles that proved to be strongly dependent on the stereochemistry. Compared to the D1 receptor, the test compounds showed high selectivity for the D2-like subtypes including D2(long), D2(short), D3 and D4. The highest D4 and D3 affinities were observed for the cis-3-amino-4-methylpyrrolidines 3e and the enantiomer ent3e resulting in K(i) values of 0.23 and 1.8 nM, respectively. The benzamides of type 3 and 5 were synthesized in enantiopure form starting from (S)-aspartic acid and its unnatural optical antipode.

  14. Sex differences in hippocampal estradiol-induced N-methyl-D-aspartic acid binding and ultrastructural localization of estrogen receptor-alpha.

    Science.gov (United States)

    Romeo, Russell D; McCarthy, J Brian; Wang, Athena; Milner, Teresa A; McEwen, Bruce S

    2005-01-01

    Estradiol increases dendritic spine density and synaptogenesis in the CA1 region of the female hippocampus. This effect is specific to females, as estradiol-treated males fail to show increases in hippocampal spine density. Estradiol-induced spinogenesis in the female is dependent upon upregulation of the N-methyl-D-aspartic acid (NMDA) receptor as well as on non-nuclear estrogen receptors (ER), including those found in dendrites. Thus, in the male, the inability of estradiol to induce spinogenesis may be related to a failure of estradiol to increase hippocampal NMDA receptors as well as a paucity of dendritic ER. In the first experiment, we sought to investigate this possibility by assessing NMDA receptor binding, using [(3)H]-glutamate autoradiography, in estradiol-treated males and females. We found that while estradiol increases NMDA binding in gonadectomized females, estradiol fails to modulate NMDA binding in gonadectomized males. To further investigate sex differences in the hippocampus, we conducted a second separate, but related, ultrastructural study in which we quantified ERalpha-immunoreactivity (ERalpha-ir) in neuronal profiles in the CA1 region of the hippocampus in intact males and females in diestrus and proestrus. Consistent with previous reports in the female, we found ERalpha-ir in several extranuclear sites including dendrites, spines, terminals and axons. Statistical analyses revealed that females in proestrus had a 114.3% increase in ERalpha-labeled dendritic spines compared to females in diestrus and intact males. Taken together, these studies suggest that both the ability of estrogen to increase NMDA binding in the hippocampus and the presence of ERalpha in dendritic spines may contribute to the observed sex difference in estradiol-induced hippocampal spinogenesis. Copyright (c) 2005 S. Karger AG, Basel.

  15. 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

  16. In vivo binding of [{sup 11}C]nemonapride to sigma receptors in the cortex and cerebellum

    Energy Technology Data Exchange (ETDEWEB)

    Ishiwata, Kiichi E-mail: ishiwata@pet.tmig.or.jp; Senda, Michio

    1999-08-01

    Radiolabeled nemonapride (NEM, YM-09151-2) is widely used as a representative dopamine D{sub 2}-like receptor ligand in pharmacological and neurological studies, and {sup 11}C-labeled analog ([{sup 11}C]NEM) has been developed for positron emission tomography (PET) studies. The aim of this study was to evaluate whether [{sup 11}C]NEM binds in vivo to sigma receptors. [{sup 11}C]NEM and one of six dopamine D{sub 2}-like receptor ligands or seven sigma receptor ligands were co-injected into mice, and the regional brain uptake of [{sup 11}C]NEM was measured by a tissue dissection method. The striatal uptake of [{sup 11}C]NEM was reduced by D{sub 2}-like receptor ligands, NEM, haloperidol, (+)-butaclamol, raclopride, and sulpiride, but not by a D{sub 4} receptor ligand clozapine. In the cortex and cerebellum the uptake was also reduced by D{sub 2}-like receptor ligands with affinity for sigma receptors, but not by raclopride. Although none of seven sigma receptor ligands, SA6298, N,N-dipropyl-2-[4-methoxy-3-(2-phenylethoxy)phenyl]ethylamine hydrochloride (NE-100), (+)-pentazocine, R(-)-N-(3-phenyl-1-propyl)-1-phenyl-2-aminopropane hydrochloride ([-]-PPAP), (-)-pentazocine, R(+)-3-(3-hydroxyphenyl)-N-propylpiperidine hydrochloride ([+]-3-PPP), and (+)-N-allylnormetazocine hydrochloride ([+]-SKF 10047), blocked the striatal uptake, five of them with relatively higher affinity significantly reduced the [{sup 11}C]NEM uptake by the cortex, and four of them reduced that by the cerebellum. We concluded that [{sup 11}C]NEM binds in vivo not only to dopamine D{sub 2}-like receptors in the striatum but also to sigma receptors in other regions such as cortex and cerebellum.

  17. 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

  18. Dopamine D2 receptors photolabeled by iodo-azido-clebopride.

    Science.gov (United States)

    Niznik, H B; Dumbrille-Ross, A; Guan, J H; Neumeyer, J L; Seeman, P

    1985-04-19

    Iodo-azido-clebopride, a photoaffinity compound for dopamine D2 receptors, had high affinity for canine brain striatal dopamine D2 receptors with a dissociation constant (Kd) of 14 nM. Irradiation of striatal homogenate with iodo-azido-clebopride irreversibly inactivated 50% of dopamine D2 receptors at 20 nM (as indicated by subsequent [3H]spiperone binding). Dopamine agonists and antagonists prevented this photo-inactivation with the appropriate rank-order of potency. Striatal dopamine D1, serotonin (S2), alpha 1- and beta-adrenoceptors were not significantly inactivated following irradiation with iodo-azido-clebopride. Thus, iodo-azido-clebopride is a selective photoaffinity probe for dopamine D2 receptors, the radiolabelled form of which may aid in the molecular characterization of these proteins.

  19. 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

  20. Synthesis of the possible receptor Ligand [125I]-spiperone for D2-dopamine receptor and in-vivo biodistribution

    International Nuclear Information System (INIS)

    Amin, A.M.; Shoukry, M.; Abd EL-Bary, A.

    2009-01-01

    The spiperone is a selective D2-dopamine receptor antagonist radioiodination of spiperone is of interest for dopamine (DA) receptor studies both in vivo and in vitro. The labeling of spiperone with iodine-125 was extremely done in a neutral ph 7, using chloramine-T as oxidizing agent via heating the reaction mixture at 70 C (degree) for 10 - 15 minutes producing radiochemical yield of 97 %. In vivo biodistribution studies showed that the initial brain uptake correlated fairly well with the brain uptake index and that the kinetics of the radioactivity specifically bound to the striatum were strongly influenced by the dopamine receptor binding affinity of the compound. The brain uptake of 125 I-Spiperone was high and equal to 3.5, 3.25,2.75 and 1.7 % per gram tissue at 5, 30, 60 and 120 minutes post injection, respectively. 125 I-Spiperone binds with high affinity to dopamine receptors in vivo. Specific binding is about 65% of the total binding as is displaced stereo-specifically by clozapine. 125 I-spiperone may prove to be a useful ligand in studies examining D2-dopamine receptors. Furthermore iodinated spiperone may be useful in radioreceptor assays of neuroleptic drug levels and, in a 123 I-labeled form, for imaging of dopamine receptors, in vivo, using single photon tomography.

  1. Cordycepin (3'-deoxyadenosine) inhibits the growth of B16-BL6 mouse melanoma cells through the stimulation of adenosine A3 receptor followed by glycogen synthase kinase-3beta activation and cyclin D1 suppression.

    Science.gov (United States)

    Yoshikawa, Noriko; Yamada, Shizuo; Takeuchi, Chihiro; Kagota, Satomi; Shinozuka, Kazumasa; Kunitomo, Masaru; Nakamura, Kazuki

    2008-06-01

    Cordyceps sinensis, a parasitic fungus on the larvae of Lepidoptera, has been used as a traditional Chinese medicine. We previously reported that the growth of B16-BL6 mouse melanoma (B16-BL6) cells was inhibited by cordycepin (3'-deoxyadenosine), an active ingredient of C. sinensis, and its effect was antagonized by MRS1191, a selective adenosine A3 receptor antagonist. In this study, the radioligand binding assay using [125I]-AB-MECA (a selective adenosine A3 receptor agonist) has shown that B16-BL6 cells express adenosine A3 receptors and that cordycepin binds to these receptors. We also confirmed the involvement of adenosine A3 receptors in the action of cordycepin using MRS1523 and MRS1220, specific adenosine A3 receptor antagonists. Next, indirubin, a glycogen synthase kinase-3beta (GSK-3beta) inhibitor, antagonized the growth suppression induced by cordycepin. Furthermore, the level of cyclin D1 protein in B16-BL6 cells was decreased by cordycepin using Western blot analysis. In conclusion, this study demonstrated that cordycepin inhibits the proliferation of B16-BL6 cells by stimulating adenosine A3 receptors followed by the Wnt signaling pathway, including GSK-3beta activation and cyclin D1 inhibition.

  2. 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

  3. 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.

  4. Presence of dopamine D-2 receptors in human tumoral cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Sokoloff, P.; Riou, J.F.; Martres, M.P.; Schwartz, J.C. (Centre Paul Broca, Paris (France))

    1989-07-31

    ({sup 125}I) Iodosulpride binding was examined on eight human cell lines derived from lung, breast and digestive tract carcinomas, neuroblastomas and leukemia. Specific binding was detected in five of these cell lines. In the richest cell line N417, derived from small cell lung carcinoma, ({sup 125}I) iodosulpride bound with a high affinity (Kd = 1.3 nM) to an apparently homogeneous population of binding site (Bmax = 1,606 sites per cell). These sites displayed a typical D-2 specificity, established with several dopaminergic agonists and antagonists selective of either D-1 or D-2 receptor subtypes. In addition, dopamine, apomorphine and RU 24926 distinguished high- and low-affinity sites, suggesting that the binding sites are associated with a G-protein. The biological significance and the possible diagnostic implication of the presence of D-2 receptors on these cell lines are discussed.

  5. (/sup 3/H)MK-801 labels a site on the N-methyl-D-aspartate receptor channel complex in rat brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Wong, E H; Knight, A R; Woodruff, G N

    1988-01-01

    The potent noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist (/sup 3/H)MK-801 bound with nanomolar affinity to rat brain membranes in a reversible, saturable, and stereospecific manner. The affinity of (/sup 3/H)MK-801 was considerably higher in 5 mM Tris-HCl (pH 7.4) than in previous studies using Krebs-Henseleit buffer. (/sup 3/H)MK-801 labels a homogeneous population of sites in rat cerebral cortical membranes with KD of 6.3 nM and Bmax of 2.37 pmol/mg of protein. This binding was unevenly distributed among brain regions, with hippocampus greater than cortex greater than olfactory bulb = striatum greater than medulla-pons, and the cerebellum failing to show significant binding. Detailed pharmacological characterization indicated (/sup 3/H)MK-801 binding to a site which was competitively and potently inhibited by known noncompetitive NMDA receptor antagonists, such as phencyclidine, thienylcyclohexylpiperidine (TCP), ketamine, N-allylnormetazocine (SKF 10,047), cyclazocine, and etoxadrol, a specificity similar to sites labelled by (/sup 3/H)TCP. These sites were distinct from the high-affinity sites labelled by the sigma receptor ligand (+)-(/sup 3/H)SKF 10,047. (/sup 3/H)MK-801 binding was allosterically modulated by the endogenous NMDA receptor antagonist Mg2+ and by other active divalent cations. These data suggest that (/sup 3/H)MK-801 labels a high-affinity site on the NMDA receptor channel complex, distinct from the NMDA recognition site, which is responsible for the blocking action of MK-801 and other noncompetitive NMDA receptor antagonists.

  6. (/sup 3/H)Spiperone binding sites in brain: autoradiographic localization of multiple receptors

    Energy Technology Data Exchange (ETDEWEB)

    Palacios, J M; Niehoff, D L; Kuhar, M J [Johns Hopkins Univ., Baltimore, MD (USA). School of Medicine

    1981-01-01

    (/sup 3/H)Spiperone ((/sup 3/H)SP) binding sites were localized by light microscopic autoradiography, after in vitro labelling. The kinetic and pharmacological characteristics of these binding sites were studied in slide-mounted sections of rat forebrain, and optimal labeling conditions were defined. Autoradiograms were obtained by apposing emulsion-coated coverslips to labeled sections. Differential drug sensitivity allowed the selective displacement of (/sup 3/H)SP from dopamine receptors by ADTN, from serotonin receptors by cinanserin, from both by haloperidol and from unique spiperone sites by unlabeled spiperone. The various sites presented a differential anatomical localization. For example, only dopaminergic sites were found in the glomerular layer of the olfactory bulb; only serotonergic sites were found in lamina IV of the neocortex, and a high concentration of unique spiperone sites were found in parts of the hippocampus.

  7. Antagonism by supidimide of haloperidol-induced augmentation of (/sup 3/H)-spiperone binding in rat striatum

    Energy Technology Data Exchange (ETDEWEB)

    Hennies, H H; Hess, V; Flohe, L

    1984-01-01

    Rats were treated for two weeks with haloperidol alone or with additional test drugs and the D/sub 2/ receptor density in the striatum was investigated after a drug-free period of five days. The D/sub 2/ receptor system as analysed by (/sup 3/H)-spinerone binding was best characterized by a model with two binding sites of different affinity. Chronic haloperidol treatment substantially augmented the total binding capacity 2-(2-Oxo-3-piperidyl)-1,2-benzisothiazoline-3-one-1,1-dioxide (supidimide), a functional synergist of neuroleptics in acute experiments, surprisingly antagonized the haloperidol-induced receptor augmentation, whereas other CNS depressants (diazepam and phenobarbital) were ineffective.

  8. On the clinical impact of cerebral dopamine D2 receptor scintigraphy

    International Nuclear Information System (INIS)

    Larisch, R.; Klimke, A.

    1998-01-01

    The present review describes findings and clinical indications for the dopamine D 2 receptor scintigraphy. Methods for the examination of D 2 receptors are positron emission tomography (PET) using 11 C- or 18 F-labelled butyrophenones or benzamides or single photon emission tomography (SPECT) using 123 I-iodobenzamide (IBZM) respectively. The most important indication in neurology is the differential diagnosis of Parkinsonism: Patients with early Parkinson's disease show an increased D 2 receptor binding (D 2 -RB) compared to control subjects. However, patients suffering from Steele-Richardson-Olszewski-Syndrome or Multiple System Atrophy show a decreased D 2 -RB and are generally non-responsive to treatment. Postsynaptic blockade of D 2 receptors results in a drug induced Parkinsonian syndrome, which can be diagnosed by D 2 scintigraphy. Further possible indications occur in psychiatry: The assessment of receptor occupancy is useful in schizophrenic patients treated with neuroleptics. Additionally, D 2 receptor scintigraphy might help to clarify the differential diagnosis between neuroleptic malignant syndrome and lethal catatonia. The method might be useful for supervising neurobiochemical changes in drug dependency and during withdrawal. Assessment of dopamine D 2 receptor binding can simplify the choice of therapy in depressive disorder: Patients showing a low D 2 binding are likely to improve following an antidepressive drug treatment whereas sleep deprivation is promising in patients with high D 2 binding. (orig.) [de

  9. 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....

  10. Effects of pilocarpine and kainate-induced seizures on N-methyl-d-aspartate receptor gene expression in the rat hippocampus

    Energy Technology Data Exchange (ETDEWEB)

    Przewlocka, B.; Labuz, D.; Machelska, H.; Przewlocki, R.; Turchan, J.; Lason, W. [Department of Molecular Neuropharmacology, Institute of Pharmacology, Polish Academy of Sciences, 12 Smetna Street, 31-343 Krakow (Poland)

    1997-04-14

    The effects of pilocarpine- and kainate-induced seizures on N-methyl-d-aspartate receptor subunit-1 messenger RNA and [{sup 3}H]dizocilpine maleate binding were studied in the rat hippocampal formation. Pilocarpine- but not kainate-induced seizures decreased N-methyl-d-aspartate receptor subunit-1 messenger RNA level in dentate gyrus at 24 and 72 h after drug injection. Both convulsants decreased the messenger RNA level in CA1 pyramidal cells at 24 and 72 h, the effects of kainate being more profound. Kainate also decreased the N-methyl-d-aspartate receptor subunit-1 messenger RNA level in CA3 region after 24 and 72 h, whereas pilocarpine decreased the messenger RNA level at 72 h only. At 3 h after kainate, but not pilocarpine, an increased binding of [{sup 3}H]dizocilpine maleate in several apical dendritic fields of pyramidal cells was found. Pilocarpine reduced the [{sup 3}H]dizocilpine maleate binding in stratum lucidum only at 3 and 24 h after the drug injection. Pilocarpine but not kainate induced prolonged decrease in N-methyl-d-aspartate receptor subunit-1 gene expression in dentate gyrus. However, at the latest time measured, kainate had the stronger effect in decreasing both messenger RNA N-methyl-d-aspartate receptor subunit-1 and [{sup 3}H]dizocilpine maleate binding in CA1 and CA3 hippocampal pyramidal cells. The latter changes corresponded, however, to neuronal loss and may reflect higher neurotoxic potency of kainate.These data point to some differences in hippocampal N-methyl-d-aspartate receptor regulation in pilocarpine and kainate models of limbic seizures. Moreover, our results suggest that the N-methyl-d-aspartate receptor subunit-1 messenger RNA level is more susceptible to limbic seizures than is [{sup 3}H]dizocilpine maleate binding in the rat hippocampal formation. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  11. Effects of pilocarpine and kainate-induced seizures on N-methyl-d-aspartate receptor gene expression in the rat hippocampus

    International Nuclear Information System (INIS)

    Przewlocka, B.; Labuz, D.; Machelska, H.; Przewlocki, R.; Turchan, J.; Lason, W.

    1997-01-01

    The effects of pilocarpine- and kainate-induced seizures on N-methyl-d-aspartate receptor subunit-1 messenger RNA and [ 3 H]dizocilpine maleate binding were studied in the rat hippocampal formation. Pilocarpine- but not kainate-induced seizures decreased N-methyl-d-aspartate receptor subunit-1 messenger RNA level in dentate gyrus at 24 and 72 h after drug injection. Both convulsants decreased the messenger RNA level in CA1 pyramidal cells at 24 and 72 h, the effects of kainate being more profound. Kainate also decreased the N-methyl-d-aspartate receptor subunit-1 messenger RNA level in CA3 region after 24 and 72 h, whereas pilocarpine decreased the messenger RNA level at 72 h only. At 3 h after kainate, but not pilocarpine, an increased binding of [ 3 H]dizocilpine maleate in several apical dendritic fields of pyramidal cells was found. Pilocarpine reduced the [ 3 H]dizocilpine maleate binding in stratum lucidum only at 3 and 24 h after the drug injection. Pilocarpine but not kainate induced prolonged decrease in N-methyl-d-aspartate receptor subunit-1 gene expression in dentate gyrus. However, at the latest time measured, kainate had the stronger effect in decreasing both messenger RNA N-methyl-d-aspartate receptor subunit-1 and [ 3 H]dizocilpine maleate binding in CA1 and CA3 hippocampal pyramidal cells. The latter changes corresponded, however, to neuronal loss and may reflect higher neurotoxic potency of kainate.These data point to some differences in hippocampal N-methyl-d-aspartate receptor regulation in pilocarpine and kainate models of limbic seizures. Moreover, our results suggest that the N-methyl-d-aspartate receptor subunit-1 messenger RNA level is more susceptible to limbic seizures than is [ 3 H]dizocilpine maleate binding in the rat hippocampal formation. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  12. Identification and characterization of luekotriene C4 and D4 receptors on a cultured smooth muscle cell line, BC3H-1

    International Nuclear Information System (INIS)

    Tamura, N.; Agrawal, D.K.; Townley, R.G.

    1987-01-01

    The authors studied the characteristics of the leukotriene (LT) C 4 and D 4 receptors on a cultured smooth muscle cell line, BC3H-1. Specific [ 3 H]LTC 4 binding to the cell membrane was greater than 80% of total binding and saturable at a density of 3.96 +/- 0.39 pmol/mg protein, with an apparent dissociation constant(Kd) of 14.3 +/- 2.0 nM (n=9). The association and dissociation of [ 3 H]LTC 4 binding were rapid and apparent equilibrium conditions were established within 5 min. Calculated Kd value of [ 3 H]LTC binding from the kinetic analysis was 9.9 nM. From the competition analysis, calculated Ki value of unlabeled LTC 4 to compete for the specific binding of [ 3 H]LTC 4 was 9.2 nM and was in good agreement with the Kd value obtained from the Scatchard plots or kinetic analysis. The maximum number of binding sites (Bmax) of [ 3 H]LTD 4 in the membrane of BC3H-1 cell line was about 11 times lower than that of the [ 3 H]LTC 4 . The calculated values of Kd and Bmax of [ 3 H]LTD 4 binding were 9.3 +/- 0.8 nM and 0.37 +/- 0.04 pmol/mg proteins, respectively (n=3). These findings demonstrate that BC3H-1 cell line possess both LTC 4 and LTD 4 receptors with a predominance of LTC 4 receptors. Thus, BC3H-1 cell line is a good model to study the regulation of LTC 4 and LTD 4 receptors. 34 references, 5 figures, 1 table

  13. Regulation of dopamine D2 receptors in a novel cell line (SUP1)

    International Nuclear Information System (INIS)

    Ivins, K.J.; Luedtke, R.R.; Artymyshyn, R.P.; Molinoff, P.B.

    1991-01-01

    A prolactin-secreting cell line, SUP1, has been established from rat pituitary tumor 7315a. In radioligand binding experiments, the D2 receptor antagonist (S)-(-)-3- 125 I iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2- pyrrolidinyl)methyl]benzamide ( 125 I IBZM) labeled a single class of sites in homogenates of SUP1 cells (Kd = 0.6 nM; Bmax = 45 fmol/mg of protein). The sites displayed a pharmacological profile consistent with that of D2 receptors. Inhibition of the binding of 125 I IBZM by dopamine was sensitive to GTP, suggesting that D2 receptors in SUP1 cells are coupled to guanine nucleotide-binding protein(s). In the presence of isobutylmethylxanthine, dopamine decreased the level of cAMP accumulation in SUP1 cells. Dopamine also inhibited prolactin secretion from SUP1 cells. Both the inhibition of cAMP accumulation and the inhibition of prolactin secretion were blocked by D2 receptor antagonists, suggesting that these effects of dopamine were mediated by an interaction with D2 receptors. The regulation of D2 receptors in SUP1 cells by D2 receptor agonists was investigated. Exposure of SUP1 cells to dopamine or to the D2 receptor agonist N-propylnorapomorphine led to increased expression of D2 receptors, with no change in the affinity of the receptors for 125 I IBZM. An increase in the density of D2 receptors in SUP1 cells was evident within 7 hr of exposure to dopamine. Spiroperidol, a D2 receptor antagonist, blocked the effect of dopamine on receptor density. These results suggest that exposure of D2 receptors in SUP1 cells to agonists leads to an up-regulation of D2 receptors. Dopamine retained the ability to inhibit cAMP accumulation in SUP1 cells exposed to dopamine for 24 hr, suggesting that D2 receptors in SUP1 cells are not desensitized by prolonged exposure to agonist

  14. Peptide displacement of [3H]5-hydroxytryptamine binding to bovine cortical membranes

    International Nuclear Information System (INIS)

    Takeuchi, Y.; Root-Bernstein, R.S.; Shih, J.C.

    1990-01-01

    Chemical studies have demonstrated that peptides such as the encephalitogenic (EAE) peptide of myelin basic protein (MBP) and luteinizing hormone-releasing hormone (LHRH) can bind serotonin (5-hydroxytryptamine, 5-HT) in vitro. The present research was undertaken to determine whether such binding interferes with 5-HT binding to its 5-HT1 receptors on bovine cerebral cortical membranes. EAE peptide and LHRH displaced [ 3 H]5-HT with IC50s of 4.0 x 10(-4) and 1.8 x 10(-3) M respectively. MBP itself also showed apparent displacing ability with an IC50 of 6.0 x 10(-5) M, though it also caused aggregation of cortical membranes that might have interfered with normal receptor binding. These results support previous suggestions that the tryptophan peptide region of MBP may act as a 5-HT receptor in the neural system. We also tested the effects of muramyl dipeptide (N-acetyl-muramyl-L-Ala-D-isoGln, MD), a bacterial cell-wall breakdown product that acts as a slow-wave sleep promoter, binds to LHRH and EAE peptide, and competes for 5-HT binding sites on macrophages. It showed no significant displacement of 5-HT binding to cortical membranes (IC50 greater than 10(-1) M), but its D-Ala analogue did (IC50 = 1.7 x 10(-3) M). Thus, it seems likely that the 5-HT-related effects of naturally occurring muramyl peptides are physiologically limited by receptor types

  15. Lack of specific (3H) prazosin binding sites in dog and rabbit cerebral arteries

    International Nuclear Information System (INIS)

    Ferron, P.M.; Banner, W. Jr.; Duckles, S.P.

    1984-01-01

    In order to explore the characteristics of alpha adrenergic receptors on cerebrovascular smooth muscle, specific binding sites for the alpha 1 adrenergic ligand, ( 3 H) prazosin, were studied in blood vessel homogenates. No specific ( 3 H) prazosin binding was found in either rabbit or dog cerebral arteries, but specific binding was demonstrated in the rabbit saphenous and ear arteries. In the ear artery 3 H-prazosin binding was saturable with a K/sub d/ of 0.51 +/- 0.20 nM and a Bmax of 89 +/- 29 fmoles/mg protein. To confirm the adequacy of our membrane preparation, homogenates of both dog and rabbit cerebral arteries showed saturable specific binding with two different ligands: one for muscarinic receptors, [ 3 H](-) quinuclidinyl benzilate (QNB) and one for alpha 2 adrenergic receptors, ( 3 H) yohimbine. The results of these studies demonstrate a lack of alpha 1 adrenergic receptors on cerebral blood vessels, confirming functional studies showing only a weak contractile response to norepinephrine. 29 references, 3 figures, 2 tables

  16. 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.

  17. Isolation of lymphocyte membrane complement receptor type two (the C3d receptor) and preparation of receptor-specific antibody.

    OpenAIRE

    Lambris, J D; Dobson, N J; Ross, G D

    1981-01-01

    A glycoprotein binding complement component C3d was isolated from media used for culture of Raji human lymphoblastoid cells. Analysis by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and gas/liquid chromatography indicated that the C3d-binding glycoprotein consisted of a single polypeptide chain with extensive intrachain disulfide bonds, a molecular weight of 72,000, and several different bound carbohydrates. Several lines of evidence indicated that this medium-derived C3d-binding...

  18. Anti-idiotypes against a monoclonal anti-haloperidol antibody bind to dopamine receptor

    International Nuclear Information System (INIS)

    Elazar, Z.; Kanety, H.; Schreiber, M.; Fuchs, S.

    1988-01-01

    Anti-idiotypic antibodies were raised in rabbits by immunization with a monoclonal anti-haloperidol antibody. Some of these anti-idiotypic antibodies bind in a concentration dependent manner to bovine striatal membranes. Following affinity purification, these antibodies inhibit haloperidol binding to striatal membranes and deplete [ 3 H]-spiperone binding sites from a solubilized preparation of striatal membranes. It is thus concluded that these anti-idiotypic antibodies are an internal image of haloperidol and as such can interact with D 2 -dopamine receptors

  19. 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

  20. 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

  1. [3H]52770 RP, a platelet-activating factor receptor antagonist, and tritiated platelet-activating factor label a common specific binding site in human polymorphonuclear leukocytes

    International Nuclear Information System (INIS)

    Marquis, O.; Robaut, C.; Cavero, I.

    1988-01-01

    In human polymorphonuclear leukocytes (PMNs), the tritiated platelet activating factor ([ 3 H]PAF) labels in a saturable manner a single class of binding sites with a Kd of 3.5 +/- 0.5 nM (n = 7) and a maximum binding capacity (Bmax) of 206 +/- 13 fmol/2.5 X 10(6) PMNs (n = 7). 52770 RP, a nonphospholipid antagonist of PAF receptors, fully and competitively displaced the [ 3 H]PAF from its binding sites with a Ki of 7.0 +/- 0.7 nM (n = 4). The high potency and the low solubility in cellular membranes of this compound led us to prepare [ 3 H]52770 RP. This ligand was characterized by a binding which was rapid, reversible, confined to a single site, saturable, specific and stereoselective. Its Kd and Bmax were 4.2 +/- 0.3 nM and 181 +/- 11 fmol/2.5 X 10(6) PMNs, respectively. The stereoselectivity of the binding was suggested by the 600- and 1050-fold higher potency of the d-enantiomer with respect to l-52770 RP in displacing [ 3 H]52770 RP or [ 3 H]PAF, respectively. Several PAF analogs (e.g., lyso-PAF, 2-O-methyl-lyso-PAF), which are poorly active as PAF receptor agonists in functional tests, were weak displacers of [ 3 H]PAF and [ 3 H]52770 RP. Furthermore, for a series of 14 known PAF receptor agonists or antagonists belonging to different chemical families, there was an excellent correlation (r = 0.98) between their ability to displace [ 3 H]PAF and [ 3 H]52770 RP. Thus, [ 3 H]52770 RP and [ 3 H]PAF appear to interact with the same binding site on human PMNs which is proposed to be the PAF receptor mediating functional responses

  2. Glycine receptors in the human substantia nigra as defined by (3H)strychnine binding

    Energy Technology Data Exchange (ETDEWEB)

    de Montis, G; Beaumont, K; Javoy-Agid, F; Agid, Y; Constandinidis, J; Lowenthal, A; Lloyd, K G

    1982-03-01

    Specific (3H)strychnine binding was used to identify the glycine receptor macromolecular complex in human spinal cord, substantia nigra, inferior olivary nucleus, and cerebral cortex. In material from control patients a high-affinity KD (3--8 nM) was observed in the spinal cord and the substantia nigra, both the pars compacta and the pars reticulata. This is very similar to the values observed in the rat and bovine spinal cord (8 and 3 nM, respectively) and rat substantia nigra (12 nM). In the human brain the distribution of (3H)strychnine binding (at 10 nM) was: spinal cord . substantia nigra, pars compacta greater than substantia nigra, pars reticulata . inferior olivary nucleus greater than cerebral cortex. The binding capacity (Bmax) of the rat brain (substantia nigra or spinal cord) was approximately 10-fold that of the human brain. (3H)Strychnine binding was significantly decreased in the substantia nigra from Parkinson's disease patients, both in the pars compacta (67% of control) and the pars reticulata (50% of control), but not in the inferior olivary nucleus. The results were reproduced in preliminary experiment in rats with unilateral 6-hydroxydopamine lesions of the medial forebrain bundle. In the substantia nigra from patients who died with Huntington's disease, (3H)strychnine binding tended to be high (150% of control, NS) in both the pars compacta and the reticulata. (3H)Strychnine binding was unaltered in the substantia nigra of patients with senile dementia. Together with previous neurophysiological and neuropharmacological findings, those results support the hypothesis of glycine receptors occurring on dopamine cell bodies and/or dendrites in the substantia nigra.

  3. 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.

  4. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors.

    Science.gov (United States)

    McGlade, C J; Ellis, C; Reedijk, M; Anderson, D; Mbamalu, G; Reith, A D; Panayotou, G; End, P; Bernstein, A; Kazlauskas, A

    1992-01-01

    The binding of cytoplasmic signaling proteins such as phospholipase C-gamma 1 and Ras GTPase-activating protein to autophosphorylated growth factor receptors is directed by their noncatalytic Src homology region 2 (SH2) domains. The p85 alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase, which associates with several receptor protein-tyrosine kinases, also contains two SH2 domains. Both p85 alpha SH2 domains, when expressed individually as fusion proteins in bacteria, bound stably to the activated beta receptor for platelet-derived growth factor (PDGF). Complex formation required PDGF stimulation and was dependent on receptor tyrosine kinase activity. The bacterial p85 alpha SH2 domains recognized activated beta PDGF receptor which had been immobilized on a filter, indicating that SH2 domains contact autophosphorylated receptors directly. Several receptor tyrosine kinases within the PDGF receptor subfamily, including the colony-stimulating factor 1 receptor and the Steel factor receptor (Kit), also associate with PI 3-kinase in vivo. Bacterially expressed SH2 domains derived from the p85 alpha subunit of PI 3-kinase bound in vitro to the activated colony-stimulating factor 1 receptor and to Kit. We infer that the SH2 domains of p85 alpha bind to high-affinity sites on these receptors, whose creation is dependent on receptor autophosphorylation. The SH2 domains of p85 are therefore primarily responsible for the binding of PI 3-kinase to activated growth factor receptors. Images PMID:1372092

  5. Detailed mapping of serotonin 5-HT1B and 5-HT1D receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

    International Nuclear Information System (INIS)

    Leysen, J.E.; Schotte, A.; Jurzak, M.; Luyten, W.H.M.L.; Voorn, P.; Bonaventure, P.

    1997-01-01

    The similar pharmacology of the 5-HT 1B and 5-HT 1D receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [ 35 S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [ 3 H]alniditan).The anatomical patterns of 5-HT 1B and 5-HT 1D receptor messenger RNA were quite different. While 5-HT 1B receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT 1D receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT 1B/1D binding sites (combined) obtained with [ 3 H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT 1B receptor labelling in the presence of ketanserin under conditions to occlude 5-HT 1D receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT 1B and 5-HT 1D receptors. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  6. 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

  7. Subtype-selective regulation of IP(3) receptors by thimerosal via cysteine residues within the IP(3)-binding core and suppressor domain.

    Science.gov (United States)

    Khan, Samir A; Rossi, Ana M; Riley, Andrew M; Potter, Barry V L; Taylor, Colin W

    2013-04-15

    IP(3)R (IP(3) [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca(2+) channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP(3)R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP(3)-evoked Ca(2+) release via IP(3)R1 and IP(3)R2, but inhibited IP(3)R3. Activation of IP(3)R is initiated by IP(3) binding to the IBC (IP(3)-binding core; residues 224-604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1-223). Thimerosal (100 μM) stimulated IP(3) binding to the isolated NT (N-terminal; residues 1-604) of IP(3)R1 and IP(3)R2, but not to that of IP(3)R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP(3)) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP(3)R activation. IP(3) binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP(3)R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP(3) binding to the chimaeric NT and IP(3)-evoked Ca(2+) release from the chimaeric IP(3)R. This is the first systematic analysis of the effects of a thiol reagent on each IP(3)R subtype. We conclude that thimerosal selectively sensitizes IP(3)R1 and IP(3)R2 to IP(3) by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor.

  8. Subtype-selective regulation of IP3 receptors by thimerosal via cysteine residues within the IP3-binding core and suppressor domain

    Science.gov (United States)

    Khan, Samir A.; Rossi, Ana M.; Riley, Andrew M.; Potter, Barry V. L.; Taylor, Colin W.

    2013-01-01

    IP3R (IP3 [inositol 1,4,5-trisphosphate] receptors) and ryanodine receptors are the most widely expressed intracellular Ca2+ channels and both are regulated by thiol reagents. In DT40 cells stably expressing single subtypes of mammalian IP3R, low concentrations of thimerosal (also known as thiomersal), which oxidizes thiols to form a thiomercurylethyl complex, increased the sensitivity of IP3-evoked Ca2+ release via IP3R1 and IP3R2, but inhibited IP3R3. Activation of IP3R is initiated by IP3 binding to the IBC (IP3-binding core; residues 224–604) and proceeds via re-arrangement of an interface between the IBC and SD (suppressor domain; residues 1–223). Thimerosal (100 μM) stimulated IP3 binding to the isolated NT (N-terminal; residues 1–604) of IP3R1 and IP3R2, but not to that of IP3R3. Binding of a competitive antagonist (heparin) or partial agonist (dimeric-IP3) to NT1 was unaffected by thiomersal, suggesting that the effect of thimerosal is specifically related to IP3R activation. IP3 binding to NT1 in which all cysteine residues were replaced by alanine was insensitive to thimerosal, so too were NT1 in which cysteine residues were replaced in either the SD or IBC. This demonstrates that thimerosal interacts directly with cysteine in both the SD and IBC. Chimaeric proteins in which the SD of the IP3R was replaced by the structurally related A domain of a ryanodine receptor were functional, but thimerosal inhibited both IP3 binding to the chimaeric NT and IP3-evoked Ca2+ release from the chimaeric IP3R. This is the first systematic analysis of the effects of a thiol reagent on each IP3R subtype. We conclude that thimerosal selectively sensitizes IP3R1 and IP3R2 to IP3 by modifying cysteine residues within both the SD and IBC and thereby stabilizing an active conformation of the receptor. PMID:23282150

  9. Occupancy of pramipexole (Sifrol at cerebral dopamine D2/3 receptors in Parkinson's disease patients

    Directory of Open Access Journals (Sweden)

    Angela Deutschländer

    2016-01-01

    Full Text Available Whereas positron emission tomography (PET with the antagonist ligand [18F]fallypride reveals the composite of dopamine D2 and D3 receptors in brain, treatment of Parkinson's disease (PD patients with the D3-prefering agonist pramipexole should result in preferential occupancy in the nucleus accumbens, where the D3-subtype is most abundant. To test this prediction we obtained pairs of [18F]fallypride PET recordings in a group of nine PD patients, first in a condition of treatment as usual with pramipexole (ON-Sifrol; 3 × 0.7 mg p.d., and again at a later date, after withholding pramipexole 48–72 h (OFF-Sifrol; in that condition the serum pramipexole concentration had declined by 90% and prolactin levels had increased four-fold, in conjunction with a small but significant worsening of PD motor symptoms. Exploratory comparison with historical control material showed 14% higher dopamine D2/3 availability in the more-affected putamen of patients OFF medication. On-Sifrol there was significant (p ˂ 0.01 occupancy at [18F]fallypride binding sites in globus pallidus (8% thalamus (9% and substantia nigra (19%, as well as marginally significant occupancy in frontal and temporal cortex of patients. Contrary to expectation, comparison of ON- and OFF-Sifrol results did not reveal any discernible occupancy in nucleus accumbens, or elsewhere in the extended striatum; present methods should be sensitive to a 10% change in dopamine D2/3 receptor availability in striatum; the significant findings elsewhere in the basal ganglia and in cerebral cortex are consistent with a predominance of D3 receptors in those structures, especially in substantia nigra, and imply that therapeutic effects of pramipexole may be obtained at sites outside the extended striatum.

  10. 5D-QSAR for spirocyclic sigma1 receptor ligands by Quasar receptor surface modeling.

    Science.gov (United States)

    Oberdorf, Christoph; Schmidt, Thomas J; Wünsch, Bernhard

    2010-07-01

    Based on a contiguous and structurally as well as biologically diverse set of 87 sigma(1) ligands, a 5D-QSAR study was conducted in which a quasi-atomistic receptor surface modeling approach (program package Quasar) was applied. The superposition of the ligands was performed with the tool Pharmacophore Elucidation (MOE-package), which takes all conformations of the ligands into account. This procedure led to four pharmacophoric structural elements with aromatic, hydrophobic, cationic and H-bond acceptor properties. Using the aligned structures a 3D-model of the ligand binding site of the sigma(1) receptor was obtained, whose general features are in good agreement with previous assumptions on the receptor structure, but revealed some novel insights since it represents the receptor surface in more detail. Thus, e.g., our model indicates the presence of an H-bond acceptor moiety in the binding site as counterpart to the ligands' cationic ammonium center, rather than a negatively charged carboxylate group. The presented QSAR model is statistically valid and represents the biological data of all tested compounds, including a test set of 21 ligands not used in the modeling process, with very good to excellent accuracy [q(2) (training set, n=66; leave 1/3 out) = 0.84, p(2) (test set, n=21)=0.64]. Moreover, the binding affinities of 13 further spirocyclic sigma(1) ligands were predicted with reasonable accuracy (mean deviation in pK(i) approximately 0.8). Thus, in addition to novel insights into the requirements for binding of spirocyclic piperidines to the sigma(1) receptor, the presented model can be used successfully in the rational design of new sigma(1) ligands. Copyright (c) 2010 Elsevier Masson SAS. All rights reserved.

  11. Manipulation of Very Few Receptor Discriminator Residues Greatly Enhances Receptor Specificity of Non-visual Arrestins*

    Science.gov (United States)

    Gimenez, Luis E.; Vishnivetskiy, Sergey A.; Baameur, Faiza; Gurevich, Vsevolod V.

    2012-01-01

    Based on the identification of residues that determine receptor selectivity of arrestins and the analysis of the evolution in the arrestin family, we introduced 10 mutations of “receptor discriminator” residues in arrestin-3. The recruitment of these mutants to M2 muscarinic (M2R), D1 (D1R) and D2 (D2R) dopamine, and β2-adrenergic receptors (β2AR) was assessed using bioluminescence resonance energy transfer-based assays in cells. Seven of 10 mutations differentially affected arrestin-3 binding to individual receptors. D260K and Q262P reduced the binding to β2AR, much more than to other receptors. The combination D260K/Q262P virtually eliminated β2AR binding while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arrestin-3 binding to β2AR and reduced the binding to M2R, D1R, and D2R, whereas Q256Y selectively reduced recruitment to D2R. The Y239T/Q256Y combination virtually eliminated the binding to D2R and reduced the binding to β2AR and M2R, yielding a mutant with high selectivity for D1R. Eleven of 12 mutations significantly changed the binding to light-activated phosphorhodopsin. Thus, manipulation of key residues on the receptor-binding surface modifies receptor preference, enabling the construction of non-visual arrestins specific for particular receptor subtypes. These findings pave the way to the construction of signaling-biased arrestins targeting the receptor of choice for research or therapeutic purposes. PMID:22787152

  12. Manipulation of very few receptor discriminator residues greatly enhances receptor specificity of non-visual arrestins.

    Science.gov (United States)

    Gimenez, Luis E; Vishnivetskiy, Sergey A; Baameur, Faiza; Gurevich, Vsevolod V

    2012-08-24

    Based on the identification of residues that determine receptor selectivity of arrestins and the analysis of the evolution in the arrestin family, we introduced 10 mutations of "receptor discriminator" residues in arrestin-3. The recruitment of these mutants to M2 muscarinic (M2R), D1 (D1R) and D2 (D2R) dopamine, and β(2)-adrenergic receptors (β(2)AR) was assessed using bioluminescence resonance energy transfer-based assays in cells. Seven of 10 mutations differentially affected arrestin-3 binding to individual receptors. D260K and Q262P reduced the binding to β(2)AR, much more than to other receptors. The combination D260K/Q262P virtually eliminated β(2)AR binding while preserving the interactions with M2R, D1R, and D2R. Conversely, Y239T enhanced arrestin-3 binding to β(2)AR and reduced the binding to M2R, D1R, and D2R, whereas Q256Y selectively reduced recruitment to D2R. The Y239T/Q256Y combination virtually eliminated the binding to D2R and reduced the binding to β(2)AR and M2R, yielding a mutant with high selectivity for D1R. Eleven of 12 mutations significantly changed the binding to light-activated phosphorhodopsin. Thus, manipulation of key residues on the receptor-binding surface modifies receptor preference, enabling the construction of non-visual arrestins specific for particular receptor subtypes. These findings pave the way to the construction of signaling-biased arrestins targeting the receptor of choice for research or therapeutic purposes.

  13. Photoaffinity ligand for dopamine D2 receptors: azidoclebopride

    International Nuclear Information System (INIS)

    Niznik, H.B.; Guan, J.H.; Neumeyer, J.L.; Seeman, P.

    1985-01-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [ 3 H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol

  14. Multiple [3H]-nemonapride binding sites in calf brain.

    Science.gov (United States)

    Helmeste, D M; Tang, S W; Li, M; Fang, H

    1997-07-01

    [3H]-Nemonapride has been the ligand of choice to label D4 dopamine receptors. Its specificity was questioned when it was discovered that sigma (sigma) sites were also labeled by [3H]-nemonapride. To further characterize the binding of [3H]-nemonapride, three areas of calf brain (striatum, frontal cortex and cerebellum) were examined. In all three areas, [3H]-nemonapride labeled multiple sites. Dopaminergic and sigma sites were the most prominent. The sigma binding profile was sigma-1 like with a Ki binding profile as follows (in order of decreasing potency): haloperidol, PPAP, pentazocine, DTG, U-50488, R(+)-3-PPP. Experiments using sulpiride and pentazocine to block striatal dopaminergic and sigma sites, respectively, revealed additional, not previously characterized binding sites for [3H]-nemonapride. One component which was present in striatum but not in frontal cortex or cerebellum, had affinity for some neuroleptics and WB-4101, but not for typical serotonergic agents. Thus, [3H]-nemonapride has no selectivity for dopamine receptors unless stringent experimental conditions are met.

  15. Insect Ryanodine Receptor: Distinct But Coupled Insecticide Binding Sites for [N-C3H3]Chlorantraniliprole, Flubendiamide, and [3H]Ryanodine

    OpenAIRE

    Isaacs, André K.; Qi, Suzhen; Sarpong, Richmond; Casida, John E.

    2012-01-01

    Radiolabeled anthranilic diamide insecticide [N-C3H3]chlorantraniliprole was synthesized at high specific activity and compared with phthalic diamide insecticide flubendiamide and [3H]ryanodine in radioligand binding studies with house fly muscle membranes to provide the first direct evidence with a native insect ryanodine receptor that the major anthranilic and phthalic diamide insecticides bind at different allosterically coupled sites, i.e. there are three distinct Ca2+-release channel tar...

  16. Functional antagonistic properties of clozapine at the 5-HT3 receptor.

    Science.gov (United States)

    Hermann, B; Wetzel, C H; Pestel, E; Zieglgänsberger, W; Holsboer, F; Rupprecht, R

    1996-08-23

    The atypical neuroleptic clozapine is thought to exert its psychopharmacological actions through a variety of neurotransmitter receptors. It binds preferentially to D4 and 5-HT2 receptors; however, little is known on it's interaction with the 5-HT3 receptor. Using a cell line stably expressing the 5-HT3 receptor, whole-cell voltage-clamp analysis revealed functional antagonistic properties of clozapine at low nanomolar concentrations in view of a binding affinity in the upper nanomolar range. Because the concentration of clozapine required for an interaction with the 5-HT3 receptor can be achieved with therapeutical doses, functional antagonistic properties at this ligand-gated ion channel may contribute to its unique psychopharmacological profile.

  17. Microbial methodological artifacts in [3H]glutamate receptor binding assays

    International Nuclear Information System (INIS)

    Yoneda, Y.; Ogita, K.

    1989-01-01

    Incubation of radiolabeled L-glutamic acid, a putative central excitatory neurotransmitter, in 50 mM Tris-acetate buffer (pH 7.4) at 30 degrees C in the absence of brain synaptic membranes resulted in a significant adsorption of the radioactivity to glass fiber filters routinely employed to trap the bound ligand in receptor binding assays. The adsorption was not only eliminated by the inclusion of L-isomers of structurally related amino acids, but also inhibited by that of most presumed agonists and antagonists for the brain glutamate receptors. This displaceable adsorption was a temperature-dependent nonreversible, and saturable phenomenon. Scatchard analysis of these data revealed that the adsorption consisted of a single component with an apparent dissociation constant of 73 nM. The displaceable adsorption was significantly attenuated by a concurrent incubation with papain, pronase E, and phospholipase C. A significant amount of the radioactivity was detected in the pass-through fraction of the Dowex column following an application of the reaction mixture incubated with purified [ 3 H]glutamate at 30 degree C for 60 min in the absence of membranous proteins added. Complete abolition of the displaceable adsorption resulted from the use of incubation buffer boiled at 100 degrees C as well as filtered through a nitrocellulose membrane filter with a pore size of 0.45 micron immediately before use. These results suggest that the displaceable adsorption may be attributable to the radioactive metabolite of [ 3 H]glutamate by microorganisms contaminating the Tris-acetate buffer. This might in part contribute to some of the controversial results with regard to receptor binding studies on acidic amino acids

  18. Interactions of L-3,5,3'-Triiodothyronine, Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes

    Science.gov (United States)

    Westergard, Thomas; Salari, Reza; Martin, Joseph V.; Brannigan, Grace

    2015-01-01

    Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3’-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone. PMID:26421724

  19. Molecular mechanism of ligand recognition by NR3 subtype glutamate receptors

    Energy Technology Data Exchange (ETDEWEB)

    Yao, Yongneng; Harrison, Chris B.; Freddolino, Peter L.; Schulten, Klaus; Mayer, Mark L. (UIUC); (NIH)

    2008-10-27

    NR3 subtype glutamate receptors have a unique developmental expression profile, but are the least well-characterized members of the NMDA receptor gene family, which have key roles in synaptic plasticity and brain development. Using ligand binding assays, crystallographic analysis, and all atom MD simulations, we investigate mechanisms underlying the binding by NR3A and NR3B of glycine and D-serine, which are candidate neurotransmitters for NMDA receptors containing NR3 subunits. The ligand binding domains of both NR3 subunits adopt a similar extent of domain closure as found in the corresponding NR1 complexes, but have a unique loop 1 structure distinct from that in all other glutamate receptor ion channels. Within their ligand binding pockets, NR3A and NR3B have strikingly different hydrogen bonding networks and solvent structures from those found in NR1, and fail to undergo a conformational rearrangement observed in NR1 upon binding the partial agonist ACPC. MD simulations revealed numerous interdomain contacts, which stabilize the agonist-bound closed-cleft conformation, and a novel twisting motion for the loop 1 helix that is unique in NR3 subunits.

  20. Positron emission tomography with (18F)methylspiperone demonstrates D2 dopamine receptor binding differences of clozapine and haloperidol

    International Nuclear Information System (INIS)

    Karbe, H.; Wienhard, K.; Huber, M.; Herholz, K.; Heiss, W.D.; Hamacher, K.; Coenen, H.H.; Stoecklin, G.; Loevenich, A.

    1991-01-01

    Four schizophrenic patients were investigated with dynamic positron emission tomography (PET) using ( 18 F)fluorodeoxyglucose (FDG) and ( 18 F)methylspiperone (MSP) as tracers. Two schizophrenics were on haloperidol therapy at the time of MSP PET. The other two schizophrenics were treated with clozapine, in one of them MSP PET was carried out twice with different daily doses (100 mg and 450mg respectively). Neuroleptic serum levels were measured in all patients. Results were compared with MSP PET of two drugfree male control subjects and with a previous fluoroethylspiperone (FESP) study of normals. Three hours after tracer injection specific binding of MSP was observed in the striatum in all cases. The striatum to cerebellum ratio was used to estimate the degree of neuroleptic-caused striatal D 2 dopamine receptor occupancy. In the haloperidol treated patients MSP binding was significantly decreased, whereas in the clozapine treated patients striatum to cerebellum ratio was normal. Even the increase of clozapine dose in the same patient had no influence on this ratio. Despite the smaller number of patients the study shows for the first time in humans that striatal MSP binding reflects the different D 2 dopamine receptor affinities of clozapine and haloperidol. (authors)

  1. Structure of unliganded HSV gD reveals a mechanism for receptor-mediated activation of virus entry

    Energy Technology Data Exchange (ETDEWEB)

    Krummenacher, Claude; Supekar, Vinit M.; Whitbeck, J. Charles; Lazear, Eric; Connolly, Sarah A.; Eisenberg, Roselyn J.; Cohen, Gary H.; Wiley, Don C.; Carfi, Andrea (UPENN); (IRBM); (CHLMM)

    2010-07-19

    Herpes simplex virus (HSV) entry into cells requires binding of the envelope glycoprotein D (gD) to one of several cell surface receptors. The 50 C-terminal residues of the gD ectodomain are essential for virus entry, but not for receptor binding. We have determined the structure of an unliganded gD molecule that includes these C-terminal residues. The structure reveals that the C-terminus is anchored near the N-terminal region and masks receptor-binding sites. Locking the C-terminus in the position observed in the crystals by an intramolecular disulfide bond abolished receptor binding and virus entry, demonstrating that this region of gD moves upon receptor binding. Similarly, a point mutant that would destabilize the C-terminus structure was nonfunctional for entry, despite increased affinity for receptors. We propose that a controlled displacement of the gD C-terminus upon receptor binding is an essential feature of HSV entry, ensuring the timely activation of membrane fusion.

  2. Insulin, Central Dopamine D2 Receptors, and Monetary Reward Discounting in Obesity.

    Science.gov (United States)

    Eisenstein, Sarah A; Gredysa, Danuta M; Antenor-Dorsey, Jo Ann; Green, Leonard; Arbeláez, Ana Maria; Koller, Jonathan M; Black, Kevin J; Perlmutter, Joel S; Moerlein, Stephen M; Hershey, Tamara

    2015-01-01

    Animal research finds that insulin regulates dopamine signaling and reward behavior, but similar research in humans is lacking. We investigated whether individual differences in body mass index, percent body fat, pancreatic β-cell function, and dopamine D2 receptor binding were related to reward discounting in obese and non-obese adult men and women. Obese (n = 27; body mass index>30) and non-obese (n = 20; body mass indexmonetary rewards relative to immediate, certain smaller monetary rewards was measured using delayed and probabilistic reward discounting tasks. Positron emission tomography using a non-displaceable D2-specific radioligand, [11C](N-methyl)benperidol quantified striatal D2 receptor binding. Groups differed in body mass index, percent body fat, and disposition index, but not in striatal D2 receptor specific binding or reward discounting. Higher percent body fat in non-obese women related to preference for a smaller, certain reward over a larger, less likely one (greater probabilistic discounting). Lower β-cell function in the total sample and lower insulin sensitivity in obese related to stronger preference for an immediate and smaller monetary reward over delayed receipt of a larger one (greater delay discounting). In obese adults, higher striatal D2 receptor binding related to greater delay discounting. Interestingly, striatal D2 receptor binding was not significantly related to body mass index, percent body fat, or β-cell function in either group. Our findings indicate that individual differences in percent body fat, β-cell function, and striatal D2 receptor binding may each contribute to altered reward discounting behavior in non-obese and obese individuals. These results raise interesting questions about whether and how striatal D2 receptor binding and metabolic factors, including β-cell function, interact to affect reward discounting in humans.

  3. Radiochemical synthesis and biological evaluation of 3-[4-(4-[{sup 18}F]fluorobenzyl)piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine as dopamine D{sub 4} receptor radioligand

    Energy Technology Data Exchange (ETDEWEB)

    Li, Gu-Cai; Zhang, Ru; Jiang, Kai-Jun; Chen, Bo [Hunan Institute of Engineering, Xiangtan (China). College of Chemistry and Chemical Engineering

    2014-09-01

    A potential dopamine D{sub 4} receptor radioligand, 3-[4-(4-[{sup 18}F]fluorobenzyl)piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine was synthesized through a one-pot two-step procedure with total yield 18.5% (decay corrected). The molar radioactivity was 115 GBq/μmol and the radiochemical purity was greater than 95.5%. Its affinity and selectivity for dopamine D{sub 2}-like receptors were measured through in vitro receptor binding experiments and the K{sub i} for D{sub 4} receptor was determined to be 17 ± 0.5 nM. The partition coefficient (Log P) of it was determined to be 2.80 ± 0.10 through octanol experiment. The in vivo biodistribution of it in rat brain exposed that the radioligand penetrates through blood-brain- barrier (BBB) and may specifically bind to dopamine D{sub 4} receptor. The results indicated that the radioligand shows promise for the in vivo study of dopamine D{sub 4} receptor. (orig.)

  4. Acute effect of intravenously applied alcohol in the human striatal and extrastriatal D2 /D3 dopamine system.

    Science.gov (United States)

    Pfeifer, Philippe; Tüscher, Oliver; Buchholz, Hans Georg; Gründer, Gerhard; Vernaleken, Ingo; Paulzen, Michael; Zimmermann, Ulrich S; Maus, Stephan; Lieb, Klaus; Eggermann, Thomas; Fehr, Christoph; Schreckenberger, Mathias

    2017-09-01

    Investigations on the acute effects of alcohol in the human mesolimbic dopamine D 2 /D 3 receptor system have yielded conflicting results. With respect to the effects of alcohol on extrastriatal D 2 /D 3 dopamine receptors no investigations have been reported yet. Therefore we applied PET imaging using the postsynaptic dopamine D 2 /D 3 receptor ligand [ 18 F]fallypride addressing the question, whether intravenously applied alcohol stimulates the extrastriatal and striatal dopamine system. We measured subjective effects of alcohol and made correlation analyses with the striatal and extrastriatal D 2 /D 3 binding potential. Twenty-four healthy male μ-opioid receptor (OPRM1)118G allele carriers underwent a standardized intravenous and placebo alcohol administration. The subjective effects of alcohol were measured with a visual analogue scale. For the evaluation of the dopamine response we calculated the binding potential (BP ND ) by using the simplified reference tissue model (SRTM). In addition, we calculated distribution volumes (target and reference regions) in 10 subjects for which metabolite corrected arterial samples were available. In the alcohol condition no significant dopamine response in terms of a reduction of BP ND was observed in striatal and extrastriatal brain regions. We found a positive correlation for 'liking' alcohol and the BP ND in extrastriatal brain regions (Inferior frontal cortex (IFC) (r = 0.533, p = 0.007), orbitofrontal cortex (OFC) (r = 0.416, p = 0.043) and prefrontal cortex (PFC) (r = 0.625, p = 0.001)). The acute alcohol effects on the D 2 /D 3 dopamine receptor binding potential of the striatal and extrastriatal system in our experiment were insignificant. A positive correlation of the subjective effect of 'liking' alcohol with cortical D 2 /D 3 receptors may hint at an addiction relevant trait. © 2016 Society for the Study of Addiction.

  5. 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

  6. 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

  7. [123I]epidepride binding to cerebellar dopamine D2/D3 receptors is displaceable: implications for the use of cerebellum as a reference region

    DEFF Research Database (Denmark)

    Pinborg, Lars H; Videbaek, Charlotte; Ziebell, Morten

    2007-01-01

    The low density of cerebellar dopamine D(2)/D(3) receptors provides the basis for using the cerebellum as a representation of free- and non-specifically bound radioligand in positron emission tomography (PET) and single photon emission computed tomography (SPECT) studies. With the development of ...

  8. Synthesis and in vitro evaluation of no-carrier-added 2-(3-(4-(4-[{sup 18}F]fluorobenzyl)piperazin-1-yl)propyl)benzo[d]thiazole, a potential dopamine D{sub 4} receptor radioligand

    Energy Technology Data Exchange (ETDEWEB)

    Li, Gu-Cai; Zhang, Ru [Hunan Institute of Engineering, Hunan Xiangtan (China). College of Chemistry and Chemical Engineering; Xia, Jiao-yun [Changsha Univ. of Science and Technology (China). School of Chemistry and Biology Engineering

    2016-07-01

    The dopamine D{sub 4} receptor has been shown to play important roles in some central nervous system pathologies. Specific radioligands for the D{sub 4} receptor may be useful to understand the function of the D{sub 4} receptor and its correlations with various disorders. 2-(3-(4-(4-[{sup 18}F]Fluorobenzyl)piperazin-1-yl)propyl)benzo[d]thiazole ([{sup 18}F]4) was synthesized through a one-pot two-step procedure with total yield 18.6% (decay corrected). The specific activity of the radioligand was 112 GBq/μmol and its radiochemical purity was >95.0%. Its affinity and selectivity for dopamine D{sub 2}-like receptors were measured through in vitro receptor binding evaluation and the K{sub i} value for the D{sub 4} receptor was determined to be 2.9±0.2 nM, and its selectivity for the dopamine D{sub 4} receptor is 709-fold versus D{sub 2long} receptor, 823-fold versus D{sub 3} receptor. The partition coefficient (Log D) of it was determined to be 2.6±0.1 through octanol-water partition experiment. The ligand presents desirable combination of lipophilicity, affinity and selectivity for the dopamine D{sub 4} receptor. The results suggested that the radioligand shows promises for the in vivo study of the dopamine D{sub 4} receptor.

  9. Varenicline increases in vivo striatal dopamine D2/3 receptor binding: an ultra-high-resolution pinhole [123I]IBZM SPECT study in rats

    International Nuclear Information System (INIS)

    Crunelle, Cleo L.; Wit, Tim C. de; Bruin, Kora de; Ramakers, Ruud M.; Have, Frans van der; Beekman, Freek J.; Brink, Wim van den; Booij, Jan

    2012-01-01

    Introduction: Ex vivo storage phosphor imaging rat studies reported increased brain dopamine D 2/3 receptor (DRD 2/3 ) availability following treatment with varenicline, a nicotinergic drug. However, ex vivo studies can only be performed using cross-sectional designs. Small-animal imaging offers the opportunity to perform serial assessments. We evaluated whether high-resolution pinhole single photon emission computed tomography (SPECT) imaging in rats was able to reproduce previous ex vivo findings. Methods: Rats were imaged for baseline striatal DRD 2/3 availability using ultra-high-resolution pinhole SPECT (U-SPECT-II) and [ 123 I]IBZM as a radiotracer, and randomized to varenicline (n=7; 2 mg/kg) or saline (n=7). Following 2 weeks of treatment, a second scan was acquired. Results: Significantly increased striatal DRD 2/3 availability was found following varenicline treatment compared to saline (time⁎treatment effect): posttreatment difference in binding potential between groups corrected for initial baseline differences was 2.039 (P=.022), indicating a large effect size (d=1.48). Conclusions: Ultra-high-resolution pinhole SPECT can be used to assess varenicline-induced changes in DRD 2/3 availability in small laboratory animals over time. Future small-animal studies should include imaging techniques to enable repeated within-subjects measurements and reduce the amount of animals.

  10. Ligand binding to the human MT2 melatonin receptor: The role of residues in transmembrane domains 3, 6, and 7

    Czech Academy of Sciences Publication Activity Database

    Mazna, Petr; Berka, K.; Jelínková, Irena; Balík, Aleš; Svoboda, Petr; Obšilová, Veronika; Obšil, T.; Teisinger, Jan

    2005-01-01

    Roč. 332, č. 3 (2005), s. 726-734 ISSN 0006-291X R&D Projects: GA AV ČR(CZ) KJB5011308; GA ČR(CZ) GA309/02/1479; GA ČR(CZ) GA204/03/0714; GA ČR(CZ) GA309/04/0496 Institutional research plan: CEZ:AV0Z5011922 Keywords : MT2 melatonin receptor * homology modeling * binding study Subject RIV: FR - Pharmacology ; Medidal Chemistry Impact factor: 3.000, year: 2005

  11. 6-hydroxydopamine-induced degeneration of nigral dopamine neurons: differential effect on nigral and striatal D-1 dopamine receptors

    International Nuclear Information System (INIS)

    Porceddu, M.L.; Giorgi, O.; De Montis, G.; Mele, S.; Cocco, L.; Ongini, E.; Biggio, G.

    1987-01-01

    Dopamine-sensitive adenylate cyclase and 3 H-SCH 23390 binding parameters were measured in the rat substantia nigra and striatum 15 days after the injection of 6-hydroxydopamine into the medial forebrain bundle. The activity of nigral dopamine-sensitive adenylate cyclase and the binding of 3 H-SCH 23390 to rat nigral D-1 dopamine receptors were markedly decreased after the lesion. On the contrary, 6-hydroxydopamine-induced degeneration of the nigrostriatal dopamine pathway enhanced both adenylate cyclase activity and the density of 3 H-SCH 23390 binding sites in striatal membrane preparations. The changes in 3 H-SCH 23390 binding found in both nigral and striatal membrane preparations were associated with changes in the total number of binding sites with no modifications in their apparent affinity. The results indicate that: a) within the substantia nigra a fraction (30%) of D-1 dopamine receptors coupled to the adenylate cyclase is located on cell bodies and and/or dendrites of dopaminergic neurons; b) striatal D-1 dopamine receptors are tonically innervated by nigrostriatal afferent fibers. 24 references, 1 figure, 1 table

  12. Functional selectivity of allosteric interactions within G protein-coupled receptor oligomers: the dopamine D1-D3 receptor heterotetramer.

    Science.gov (United States)

    Guitart, Xavier; Navarro, Gemma; Moreno, Estefania; Yano, Hideaki; Cai, Ning-Sheng; Sánchez-Soto, Marta; Kumar-Barodia, Sandeep; Naidu, Yamini T; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I; Casadó, Vicent; McCormick, Peter J; Ferré, Sergi

    2014-10-01

    The dopamine D1 receptor-D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa-induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R-D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists. U.S. Government work not protected by U.S. copyright.

  13. Rostrocaudal gradients of dopamine D2/3 receptor binding in striatal subregions measured with [11C]raclopride and high-resolution positron emission tomography

    DEFF Research Database (Denmark)

    Alakurtti, Kati; Johansson, Jarkko J; Tuokkola, Terhi

    2013-01-01

    scanned with brain-dedicated high-resolution research tomography (HRRT, Siemens Medical Solutions, Knoxville, TN, USA) and [(11)C]raclopride. Coronally defined regions of interest (ROIs) of the caudate nucleus, putamen and ventral striatum (VST) were sampled plane-by-plane, 1.5mm apart, on spatially...... observed in the VST. The novelty of this study lies in the presentation, for the first time, of the D2/3 receptor binding gradients in each striatal subregion in the brains of living healthy humans. The high spatial resolution provided by HRRT enables frequent sampling of BPND along the longitudinal extent...

  14. 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.

  15. Piracetam defines a new binding site for allosteric modulators of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors.

    Science.gov (United States)

    Ahmed, Ahmed H; Oswald, Robert E

    2010-03-11

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators.

  16. Binding of 7-dehydrocholesterol to sterol carrier protein and vitamin D3 effect

    International Nuclear Information System (INIS)

    Takase, Sachiko; Oizumi, Kumiko; Moriuchi, Sachiko; Hosoya, Norimasa

    1975-01-01

    It was confirmed that deltasup(5,7)-sterol delta 7 -reductase activity was suppressed by cholecalciferol (vitamin D 3 ) in the enzyme system consisted of microsomes and sterol carrier protein (SCP). The enzyme activity was significantly decreased in the combination with microsomes obtained from either vitamin D-deficient or vitamin D 3 -treated rat liver and with SCP obtained from vitamin D 3 -treated rat. It was also demonstrated by the binding assay of the dextran-charcoal technique that 7-dehydrocholesterol binding to SCP could be specifically displaced by vitamin D 3 . The inhibition of cholecalciferol on 7-dehydro-cholesterol binding to liver SCP was confirmed to be non-competitive inhibition. (auth.)

  17. Vitamin D receptor signaling and its therapeutic implications: Genome-wide and structural view.

    Science.gov (United States)

    Carlberg, Carsten; Molnár, Ferdinand

    2015-05-01

    Vitamin D3 is one of the few natural compounds that has, via its metabolite 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) and the transcription factor vitamin D receptor (VDR), a direct effect on gene regulation. For efficiently applying the therapeutic and disease-preventing potential of 1,25(OH)2D3 and its synthetic analogs, the key steps in vitamin D signaling need to be understood. These are the different types of molecular interactions with the VDR, such as (i) the complex formation of VDR with genomic DNA, (ii) the interaction of VDR with its partner transcription factors, (iii) the binding of 1,25(OH)2D3 or its synthetic analogs within the ligand-binding pocket of the VDR, and (iv) the resulting conformational change on the surface of the VDR leading to a change of the protein-protein interaction profile of the receptor with other proteins. This review will present the latest genome-wide insight into vitamin D signaling, and will discuss its therapeutic implications.

  18. Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: Resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

    International Nuclear Information System (INIS)

    Tiberi, M.; Magnan, J.

    1990-01-01

    The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid [3H]D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid [3H]D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid [3H]U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by [3H]U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan [3H]ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, [3H]ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of [3H]ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with [3H]U69593. Saturation studies using the nonselective opioid [3H]bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue)

  19. Quantitative analysis of multiple kappa-opioid receptors by selective and nonselective ligand binding in guinea pig spinal cord: Resolution of high and low affinity states of the kappa 2 receptors by a computerized model-fitting technique

    Energy Technology Data Exchange (ETDEWEB)

    Tiberi, M.; Magnan, J. (Universite de Montreal, Quebec (Canada))

    1990-05-01

    The binding characteristics of selective and nonselective opioids have been studied in whole guinea pig spinal cord, using a computer fitting method to analyze the data obtained from saturation and competition studies. The delineation of specific binding sites labeled by the mu-selective opioid (3H)D-Ala2,MePhe4,Gly-ol5-enkephalin (Kd = 2.58 nM, R = 4.52 pmol/g of tissue) and by the delta-selective opioid (3H)D-Pen2, D-Pen5-enkephalin (Kd = 2.02 nM, R = 1.47 pmol/g of tissue) suggests the presence of mu and delta-receptors in the spinal cord tissue. The presence of kappa receptors was probed by the kappa-selective opioid (3H)U69593 (Kd = 3.31 nM, R = 2.00 pmol/g of tissue). The pharmacological characterization of the sites labeled by (3H)U69593 confirms the assumption that this ligand discriminates kappa receptors in guinea pig spinal cord. The benzomorphan (3H)ethylketazocine labels a population of receptors with one homogeneous affinity state (Kd = 0.65 nM, R = 7.39 pmol/g of tissue). The total binding capacity of this ligand was not different from the sum of the binding capacities of mu, delta-, and kappa-selective ligands. Under mu- and delta-suppressed conditions, (3H)ethylketazocine still binds to receptors with one homogeneous affinity state (Kd = 0.45 nM, R = 1.69 pmol/g of tissue). Competition studies performed against the binding of (3H)ethylketazocine under these experimental conditions reveal that the pharmacological profile of the radiolabeled receptors is similar to the profile of the kappa receptors labeled with (3H)U69593. Saturation studies using the nonselective opioid (3H)bremazocine demonstrate that this ligand binds to spinal cord membranes with heterogeneous affinities (Kd1 = 0.28 nM, R1 = 7.91 pmol/g of tissue; Kd2 = 3.24 nM, R2 = 11.2 pmol/g of tissue).

  20. Spinal cord dopamine D2/D3 receptors: in vivo and ex vivo imaging in the rat using 18F/11C-fallypride

    International Nuclear Information System (INIS)

    Kaur, Jasmeet; Khararjian, Armen; Coleman, Robert A.; Constantinescu, Cristian C.; Pan, Min-Liang; Mukherjee, Jogeshwar

    2014-01-01

    Objectives: The spinal cord is known to be innervated with dopaminergic cells with catecholaminergic projections arising from the medulla and pons and dopaminergic transmission in the spinal cord is vital for sensory and motor function. Our goal was to evaluate and compare the imaging capability of dopamine D2/D3 receptors in the rat spinal cord using PET ligands 18 F-fallypride and 11 C-fallypride. Methods: Male Sprague–Dawley rats were used in all in vitro and in vivo studies. Spinal cord and brain sections were used for in vitro autoradiography and ex vivo autoradiography. For in vivo studies animals received a 18 F-fallypride scan or a 11 C-fallypride PET scan. The spinal cord and the brain were then harvested, flash-frozen and imaged ex vivo. For in vivo analysis Logan plots with cerebellum as a reference was used to evaluate binding potentials (BP). Tissue ratios were used for ex vivo analysis. Drug effects were evaluated using clozapine, haloperidol and dopamine were evaluated on spinal cord sections in vitro. Results: In vitro studies showed 18 F-fallypride binding to superficial dorsal horn (SDH), dorsal horn (DH), ventral horn (VH) and the pars centralis (PC). In the cervical section, the greatest amount of binding appeared to be in the SDH. Ex vivo studies showed approximately 6% of 18 F-fallypride in SDH compared to that observed in the striatum. In vivo analysis of both 18 F-fallypride and 11 C-fallypride in the spinal cord were comparable to that in the extrastriatal regions. Haloperidol and clozapine displaced more than 75% of the 18 F-fallypride in spinal cord sections. Conclusions: Our studies showed 18 F-fallypride and 11 C-fallypride binding in the spinal cord in vitro and in vivo. The binding pattern correlates well with the known distribution of dopamine D2/D3 receptors in the spinal cord

  1. Interactions of L-3,5,3'-Triiodothyronine [corrected], Allopregnanolone, and Ivermectin with the GABAA Receptor: Evidence for Overlapping Intersubunit Binding Modes.

    Science.gov (United States)

    Westergard, Thomas; Salari, Reza; Martin, Joseph V; Brannigan, Grace

    2015-01-01

    Structural mechanisms of modulation of γ-aminobutyric acid (GABA) type A receptors by neurosteroids and hormones remain unclear. The thyroid hormone L-3,5,3'-triiodothyronine (T3) inhibits GABAA receptors at micromolar concentrations and has common features with neurosteroids such as allopregnanolone (ALLOP). Here we use functional experiments on α2β1γ2 GABAA receptors expressed in Xenopus oocytes to detect competitive interactions between T3 and an agonist (ivermectin, IVM) with a crystallographically determined binding site at subunit interfaces in the transmembrane domain of a homologous receptor (glutamate-gated chloride channel, GluCl). T3 and ALLOP also show competitive effects, supporting the presence of both a T3 and ALLOP binding site at one or more subunit interfaces. Molecular dynamics (MD) simulations over 200 ns are used to investigate the dynamics and energetics of T3 in the identified intersubunit sites. In these simulations, T3 molecules occupying all intersubunit sites (with the exception of the α-β interface) display numerous energetically favorable conformations with multiple hydrogen bonding partners, including previously implicated polar/acidic sidechains and a structurally conserved deformation in the M1 backbone.

  2. Dopamine D1 and D3 receptor polypharmacology as a potential treatment approach for substance use disorder.

    Science.gov (United States)

    Galaj, Ewa; Ewing, Scott; Ranaldi, Robert

    2018-06-01

    In the search for efficacious pharmacotherapies to treat cocaine addiction much attention has been given to agents targeting dopamine D1 or D3 receptors because of the involvement of these receptors in drug-related behaviors. D1-like and D3 receptor partial agonists and antagonists have been shown to reduce drug reward, reinstatement of drug seeking and conditioned place preference in rodents and non-human primates. However, translation of these encouraging results to clinical settings has been limited due to a number of factors including toxicity, poor pharmacokinetic properties and extrapyramidal and sedative side effects. This review highlights the role of D1 and D3 receptors in drug reward and seeking, the discovery of D1-D3 heteromers and their potential as targets in the treatment of addiction. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. 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.

  4. Identification of critical residues in loop E in the 5-HT3ASR binding site

    Directory of Open Access Journals (Sweden)

    Muthalagi Mani

    2002-06-01

    Full Text Available Abstract Background The serotonin type 3 receptor (5-HT3R is a member of a superfamily of ligand gated ion channels. All members of this family share a large degree of sequence homology and presumably significant structural similarity. A large number of studies have explored the structure-function relationships of members of this family, particularly the nicotinic and GABA receptors. This information can be utilized to gain additional insights into specific structural and functional features of other receptors in this family. Results Thirteen amino acids in the mouse 5-HT3ASR that correspond to the putative E binding loop of the nicotinic α7 receptor were chosen for mutagenesis. Due to the presence of a highly conserved glycine in this region, it has been suggested that this binding loop is comprised of a hairpin turn and may form a portion of the ligand-binding site in this ion channel family. Mutation of the conserved glycine (G147 to alanine eliminated binding of the 5-HT3R antagonist [3H]granisetron. Three tyrosine residues (Y140, Y142 and Y152 also significantly altered the binding of 5-HT3R ligands. Mutations in neighboring residues had little or no effect on binding of these ligands to the 5-HT3ASR. Conclusion Our data supports a role for the putative E-loop region of the 5-HT3R in the binding of 5-HT, mCPBG, d-tc and lerisetron. 5-HT and mCPBG interact with Y142, d-tc with Y140 and lerisetron with both Y142 and Y152. Our data also provides support for the hypothesis that this region of the receptor is present in a loop structure.

  5. Insulin, Central Dopamine D2 Receptors, and Monetary Reward Discounting in Obesity.

    Directory of Open Access Journals (Sweden)

    Sarah A Eisenstein

    Full Text Available Animal research finds that insulin regulates dopamine signaling and reward behavior, but similar research in humans is lacking. We investigated whether individual differences in body mass index, percent body fat, pancreatic β-cell function, and dopamine D2 receptor binding were related to reward discounting in obese and non-obese adult men and women. Obese (n = 27; body mass index>30 and non-obese (n = 20; body mass index<30 adults were assessed for percent body fat with dual-energy X-ray absorptiometry and for β-cell function using disposition index. Choice of larger, but delayed or less certain, monetary rewards relative to immediate, certain smaller monetary rewards was measured using delayed and probabilistic reward discounting tasks. Positron emission tomography using a non-displaceable D2-specific radioligand, [11C](N-methylbenperidol quantified striatal D2 receptor binding. Groups differed in body mass index, percent body fat, and disposition index, but not in striatal D2 receptor specific binding or reward discounting. Higher percent body fat in non-obese women related to preference for a smaller, certain reward over a larger, less likely one (greater probabilistic discounting. Lower β-cell function in the total sample and lower insulin sensitivity in obese related to stronger preference for an immediate and smaller monetary reward over delayed receipt of a larger one (greater delay discounting. In obese adults, higher striatal D2 receptor binding related to greater delay discounting. Interestingly, striatal D2 receptor binding was not significantly related to body mass index, percent body fat, or β-cell function in either group. Our findings indicate that individual differences in percent body fat, β-cell function, and striatal D2 receptor binding may each contribute to altered reward discounting behavior in non-obese and obese individuals. These results raise interesting questions about whether and how striatal D2 receptor binding

  6. Dopamine D(3) receptor antagonists. 1. Synthesis and structure-activity relationships of 5,6-dimethoxy-N-alkyl- and N-alkylaryl-substituted 2-aminoindans.

    Science.gov (United States)

    Haadsma-Svensson, S R; Cleek, K A; Dinh, D M; Duncan, J N; Haber, C L; Huff, R M; Lajiness, M E; Nichols, N F; Smith, M W; Svensson, K A; Zaya, M J; Carlsson, A; Lin, C H

    2001-12-20

    5,6-Dimethoxy-2-(N-dipropyl)-aminoindan (3, PNU-99194A) was found to be a selective dopamine D(3) receptor antagonist with potential antipsychotic properties in animal models. To investigate the effects of nitrogen substitution on structure-activity relationships, a series of 5,6-dimethoxy-N-alkyl- and N-alkylaryl-substituted 2-aminoindans were synthesized and evaluated in vitro for binding affinity and metabolic stability. The results indicate that substitution at the amine nitrogen of the 2-aminoindans is fairly limited to the di-N-propyl group in order to achieve selective D(3) antagonists. Thus, combinations of various alkyl groups were generally inactive at the D(3) receptor. Although substitution with an N-alkylaryl or N-alkylheteroaryl group yields compounds with potent D(3) binding affinity, the D(2) affinity is also enhanced, resulting in a less than 4-fold preference for the D(3) receptor site, and no improvements in metabolic stability were noted. A large-scale synthesis of the D(3) antagonist 3 has been developed that has proven to be reproducible with few purification steps. The improvements include the use of 3,4-dimethoxybenzaldehyde as a low-cost starting material to provide the desired 5,6-dimethoxy-1-indanone 5c in good overall yield (65%) and the formation of a soluble silyl oxime 17 that was reduced efficiently with BH(3).Me(2)S. The resulting amino alcohol was alkylated and then deoxygenated using a Lewis acid and Et(3)SiH to give the desired product 3 in good overall yield of ( approximately 65%) from the indanone 5c.

  7. Kinetic analysis of transport and opioid receptor binding of [3H](-)-cyclofoxy in rat brain in vivo: Implications for human studies

    International Nuclear Information System (INIS)

    Sawada, Y.; Kawai, R.; McManaway, M.; Otsuki, H.; Rice, K.C.; Patlak, C.S.; Blasberg, R.G.

    1991-01-01

    [3H]Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB); estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using [18F]CF and positron emission tomography

  8. Effects of alkylating agents on dopamine D(3) receptors in rat brain: selective protection by dopamine.

    Science.gov (United States)

    Zhang, K; Weiss, N T; Tarazi, F I; Kula, N S; Baldessarini, R J

    1999-11-13

    Dopamine D(3) receptors are structurally highly homologous to other D(2)-like dopamine receptors, but differ from them pharmacologically. D(3) receptors are notably resistant to alkylation by 1-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ), which readily alkylates D(2) receptors. We compared EEDQ with N-(p-isothiocyanatophenethyl)spiperone (NIPS), a selective D(2)-like receptor alkylating agent, for effects on D(3) and D(2) receptors in rat brain using autoradiographic analysis. Neither agent occluded D(3) receptors in vivo at doses that produced substantial blockade of D(2) receptors, even after catecholamine-depleting pretreatments. In vitro, however, D(3) receptors were readily alkylated by both NIPS (IC(50)=40 nM) and EEDQ (IC(50)=12 microM). These effects on D(3) sites were blocked by nM concentrations of dopamine, whereas microM concentrations were required to protect D(2) receptors from the alkylating agents. The findings are consistent with the view that alkylation of D(3) receptors in vivo is prevented by its high affinity for even minor concentrations of endogenous dopamine.

  9. Expression and Purification of Functional Ligand-binding Domains of T1R3 Taste Receptors

    Energy Technology Data Exchange (ETDEWEB)

    Nie,Y.; Hobbs, J.; Vigues, S.; Olson, W.; Conn, G.; Munger, S.

    2006-01-01

    Chemosensory receptors, including odor, taste, and vomeronasal receptors, comprise the largest group of G protein-coupled receptors (GPCRs) in the mammalian genome. However, little is known about the molecular determinants that are critical for the detection and discrimination of ligands by most of these receptors. This dearth of understanding is due in part to difficulties in preparing functional receptors suitable for biochemical and biophysical analyses. Here we describe in detail two strategies for the expression and purification of the ligand-binding domain of T1R taste receptors, which are constituents of the sweet and umami taste receptors. These class C GPCRs contain a large extracellular N-terminal domain (NTD) that is the site of interaction with most ligands and that is amenable to expression as a separate polypeptide in heterologous cells. The NTD of mouse T1R3 was expressed as two distinct fusion proteins in Escherichia coli and purified by column chromatography. Spectroscopic analysis of the purified NTD proteins shows them to be properly folded and capable of binding ligands. This methodology should not only facilitate the characterization of T1R ligand interactions but may also be useful for dissecting the function of other class C GPCRs such as the large family of orphan V2R vomeronasal receptors.

  10. Challenges in the development of dopamine D2- and D3-selective radiotracers for PET imaging studies.

    Science.gov (United States)

    Mach, Robert H; Luedtke, Robert R

    2018-03-01

    The dopamine D2-like receptors (ie, D2/3 receptors) have been the most extensively studied CNS receptor with Positron Emission Tomography (PET). The 3 different radiotracers that have been used in these studies are [ 11 C]raclopride, [ 18 F]fallypride, and [ 11 C]PHNO. Because these radiotracers have a high affinity for both dopamine D2 and D3 receptors, the density of dopamine receptors in the CNS is reported as the D2/3 binding potential, which reflects a measure of the density of both receptor subtypes. Although the development of D2- and D3-selective PET radiotracers has been an active area of research for many years, this by and large presents an unmet need in the area of translational PET imaging studies. This article discusses some of the challenges that have inhibited progress in this area of research and the current status of the development of subtype selective radiotracers for imaging D3 and D2 dopamine receptors with PET. Copyright © 2017 John Wiley & Sons, Ltd.

  11. 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

  12. Purification and characterization of the recombinant human dopamine D2S receptor from Pichia pastoris

    NARCIS (Netherlands)

    de Jong, Lutea; Grünewald, S; Franke, J.P.; Uges, Donald; Bischoff, Rainer

    The human dopamine D2S receptor was expressed in the methylotrophic yeast Pichia pastoris, where the receptor with a molecular mass of approximately 40 kDa exhibited specific and saturable binding properties. The dopamine antagonist [H-3]spiperone showed an average dissociation constant K-d of 0.6

  13. Selective labelling of dopamine (D2) receptors in rat striatum by [3H]domperidone but not by [3H]spiperone

    International Nuclear Information System (INIS)

    Lazareno, S.; Nahorski, S.R.

    1982-01-01

    Specific binding of [ 3 H]spiperone and [ 3 H]domperidone, displaceable by 1 μM d-butaclamol, was examined in rat striatal membranes. Initial saturation and displacement experiments indicated that [ 3 H]spiperone bound to more sites than [ 3 H]domperidone and that, whilst all displacing drugs were more potent against [ 3 H]domperidone, this difference in potency was greatest for dopamine agonists and specific antagonists and least for 5HT-related drugs. Sulpiride displaced [ 3 H]spiperone biphasically, and was used at a concentration of 50 μM to examine two classes of [ 3 H]spiperone binding: site 1 displaceable by sulpiride, and site 2 displaceable by butaclamol but not by sulpiride. Site 1 had twice the capacity of site 2 and ten times the affinity for [ 3 H]spiperone. Dopaminergic drugs displaced preferentially from site 1, whilst 5HT-related drugs were more potent against site 2. GTP reduced the potency of dopamine, noradrenaline and, to a lesser extent, 5HT at site 1, but had no effect at site 2. [ 3 H]Domperidone sites had the same capacity as [ 3 H]spiperone site 1, and dopamine, noradrenaline and 5HT, in the absence or presence of GTP, and sulpiride had essentially identical affinities for [ 3 H]domperidone sites and [ 3 H]spiperone site 1. It is concluded that [ 3 H]domperidone and [ 3 H]spiperone label an identical population of dopamine (D 2 ) receptors, whilst [ 3 H]spiperone also labels a substantial number of non-dopamine sites, at least some of which are 5TH-related. [ 3 H]Domperidone is the better radioligand for dopamine receptors. (Auth.)

  14. Lessons learned from participating in D3R 2016 Grand Challenge 2: compounds targeting the farnesoid X receptor

    Science.gov (United States)

    Duan, Rui; Xu, Xianjin; Zou, Xiaoqin

    2018-01-01

    D3R 2016 Grand Challenge 2 focused on predictions of binding modes and affinities for 102 compounds against the farnesoid X receptor (FXR). In this challenge, two distinct methods, a docking-based method and a template-based method, were employed by our team for the binding mode prediction. For the new template-based method, 3D ligand similarities were calculated for each query compound against the ligands in the co-crystal structures of FXR available in Protein Data Bank. The binding mode was predicted based on the co-crystal protein structure containing the ligand with the best ligand similarity score against the query compound. For the FXR dataset, the template-based method achieved a better performance than the docking-based method on the binding mode prediction. For the binding affinity prediction, an in-house knowledge-based scoring function ITScore2 and MM/PBSA approach were employed. Good performance was achieved for MM/PBSA, whereas the performance of ITScore2 was sensitive to ligand composition, e.g. the percentage of carbon atoms in the compounds. The sensitivity to ligand composition could be a clue for the further improvement of our knowledge-based scoring function.

  15. Characterisation of the interaction of the C-terminus of the dopamine D2 receptor with neuronal calcium sensor-1.

    Directory of Open Access Journals (Sweden)

    Lu-Yun Lian

    Full Text Available NCS-1 is a member of the neuronal calcium sensor (NCS family of EF-hand Ca(2+ binding proteins which has been implicated in several physiological functions including regulation of neurotransmitter release, membrane traffic, voltage gated Ca(2+ channels, neuronal development, synaptic plasticity, and learning. NCS-1 binds to the dopamine D2 receptor, potentially affecting its internalisation and controlling dopamine D2 receptor surface expression. The D2 receptor binds NCS-1 via a short 16-residue cytoplasmic C-terminal tail. We have used NMR and fluorescence spectroscopy to characterise the interactions between the NCS-1/Ca(2+ and D2 peptide. The data show that NCS-1 binds D2 peptide with a K(d of ∼14.3 µM and stoichiometry of peptide binding to NCS-1 of 2:1. NMR chemical shift mapping confirms that D2 peptide binds to the large, solvent-exposed hydrophobic groove, on one face of the NCS-1 molecule, with residues affected by the presence of the peptide spanning both the N and C-terminal portions of the protein. The NMR and mutagenesis data further show that movement of the C-terminal helix 11 of NCS-1 to fully expose the hydrophobic groove is important for D2 peptide binding. Molecular docking using restraints derived from the NMR chemical shift data, together with the experimentally-derived stoichiometry, produced a model of the complex between NCS-1 and the dopamine receptor, in which two molecules of the receptor are able to simultaneously bind to the NCS-1 monomer.

  16. N-fluoroalkylated and N-alkylated analogues of the dopaminergic D-2 receptor antagonist raclopride

    International Nuclear Information System (INIS)

    Lannoye, G.S.; Moerlein, S.M.; Parkinson, D.; Welch, M.J.

    1990-01-01

    A series of raclopride [(S)-2-[(3,5-dichloro-6-methoxy-2- hydroxybenzamido)methyl]-1-ethylpyrrolidine] derivatives bearing pyrrolidino N-fluoroalkyl or -alkyl substituents were synthesized and evaluated as potential dopaminergic receptor-based positron tomography radiopharmaceuticals. Radiosynthetic procedures for producing the corresponding N-[18F]fluoroalkylated analogues of raclopride from 18F- (beta+, t1/2 = 110 min) in high specific activity were also developed. In vitro binding assays using competitive displacement of [3H]spiperone from primate caudate tissue indicated that the N-alkylated analogues of raclopride had Ki values of 5-40 nM, whereas the corresponding values for analogous N-fluoroalkylated derivatives ranged from 90-160 nM. The relatively low D-2 binding affinity of these fluorinated salicylamides was corroborated by in vivo tissue biodistribution results in rodents. On the basis of structure-binding correlations, the impact of intramolecular hydrogen bonding, ligand basicity, and steric bulk on the affinity of the benzamides for D-2 receptor binding are discussed. Strategies are presented for the development of alternative fluorinated salicylamides that are both receptor active and metabolically stable

  17. Single event monitoring system based on Java 3D and XML data binding

    International Nuclear Information System (INIS)

    Wang Liang; Chinese Academy of Sciences, Beijing; Zhu Kejun; Zhao Jingwei

    2007-01-01

    Online single event monitoring is important to BESIII DAQ System. Java3D is extension of Java Language in 3D technology, XML data binding is more efficient to handle XML document than SAX and DOM. This paper mainly introduce the implementation of BESIII single event monitoring system with Java3D and XML data binding, and interface for track fitting software with JNI technology. (authors)

  18. Interleukin-1 interaction with neuroregulatory systems: selective enhancement by recombinant human and mouse interleukin-1 of in vitro opioid peptide receptor binding in rat brain

    Energy Technology Data Exchange (ETDEWEB)

    Wiedermann, C.J.

    1989-02-01

    Interleukin-1 (IL-1) exerts a wide variety of biological effects on various cell types and may be regarded as a pleiotropic peptide hormone. Biological evidence suggests that IL-1 participates in the modulation of central nervous system physiology and behavior in a fashion characteristic of neuroendocrine hormones. In this investigation, recombinant (r) human (h) IL-1 and r mouse (m) IL-1 were examined for their modulation of opioid peptide receptor binding in vitro. Experiments were performed on frozen sections of rat brain. Receptor binding of radiolabeled substance P and of radiolabeled neurotensin were not significantly affected by the presence of rIL-1s. Recombinant IL-1s, however, significantly enhanced specific binding of 125I-beta-endorphin (125I-beta-END) and of D-ala2-(tyrosyl-3,5-3H)enkephalin-(5-D-leucine) (3H-D-ALA), equipotently and in a concentration-dependent manner with maximal activity occurring at a concentration of 10 LAF units/ml. The increased binding of 125I-beta-END and 3H-D-ALA was blocked steroselectively by (-)-naloxone and by etorphine, suggesting detection of opiate receptors. In addition, brain distribution patterns of receptors labeled in the presence of rIL-1s corresponded to patterns previously published for opiate receptors. Autoradiographic visualization of receptors revealed that rIL-1s in the different areas of the brain exert their effect on opioid binding with comparable potencies. The data suggest that certain central nervous system effects of IL-1s may be mediated by their selective interaction with opiatergic systems at the receptor level.

  19. Direct demonstration of D1 dopamine receptors in the bovine parathyroid gland using the D1 selective antagonist [125I]-SCH 23982

    International Nuclear Information System (INIS)

    Monsma, F.J. Jr.; Sibley, D.R.

    1989-01-01

    The presence of D1 dopamine receptors in the parathyroid gland has been proposed based on the demonstration of dopaminergic regulation of adenylate cyclase activity and parathyroid hormone release in dispersed bovine parathyroid cells. Using a radioiodinated D1 selective antagonist [125I]-SCH 23982, we have now directly labeled and characterized the D1 dopamine receptors in bovine parathyroid gland membranes. [125I]-SCH 23982 binds in a saturable manner with high affinity and low nonspecific binding to membranes prepared from bovine parathyroid glands. D1 dopamine receptors are present in this preparation at a concentration of approximately 130 fMoles/mg protein and [125I]-SCH 23982 binding increases with increasing protein concentration in a linear fashion. Determination of the Kd using the association (k1) and dissociation (k-1) rate constants revealed good agreement with the Kd determined by saturation analysis (390 pM vs. 682 pM, respectively). Inhibition of 0.3 nM [125I]-SCH 23982 binding by a series of dopaminergic antagonists verified the D1 nature of this binding site, exhibiting appropriate affinities and rank order of potency. The competition curves of all antagonists exhibited Hill coefficients that were not significantly different from 1. Inhibition of [125I]-SCH 23982 binding by dopamine and other dopaminergic agonists revealed the presence of high and low affinity agonist binding sites. Addition of 200 microM GppNHp effected a complete conversion of high affinity dopamine binding sites to a homogeneous population of low affinity dopamine sites. The D1 receptors identified in the parathyroid gland with [125I]-SCH 23982 appear to be pharmacologically identical with those previously characterized in the central nervous system

  20. Characterization of D1 dopamine receptors in the central nervous system

    International Nuclear Information System (INIS)

    Hess, E.J.

    1987-01-01

    Several lines of evidence suggest an association of central nervous system dopaminergic systems in the etiology of the schizophrenia. Interest in the role of D 1 dopamine receptors has revived with the advent of selective drugs for this dopamine receptor, particularly the D 1 dopamine receptor antagonists, SCH23390. [ 3 H]SCH23390 represents a superior radioligand for labeling the two-state striatal D 1 dopamine receptor in that its high percent specific binding makes it especially suitable for detailed mechanistic studies of this receptor. Striatal D 1 dopamine receptors have been shown to mediate the stimulation of adenylate cyclase activity via a guanine nucleotide regulatory subunit. Forskolin acts in a synergistic manner with dopamine agonists, guanine nucleotides or sodium fluoride to potentiate the stimulation of rat striatal adenylate cyclase activity mediated by these reagents. By using the aforementioned reagents and the irreversible receptor modifying reagent N-ethoxycarbonyl-2-ethoxy-1,2,-dihydroquinoline, we demonstrated that the D 1 dopamine receptor population in rat striatum is not a stoichiometrically-limiting factor in agonist stimulation of adenylate cyclase activity

  1. Parkinson's disease treatment may cause impulse-control disorder via dopamine D3 receptors.

    Science.gov (United States)

    Seeman, Philip

    2015-04-01

    In treating Parkinson's disease with dopaminergic agonists, such as pramipexole, ropinirole, pergolide, rotigotine, apomorphine, or bromocriptine, it has been observed that a significant number of patients develop impulse-control disorders, such as compulsive shopping, pathological gambling, or hypersexuality. Because the dopamine agonists have high affinities for the dopamine D2 and D3 receptors, the drug dissociation constants of these drugs at the functional high-affinity states of these receptors, namely D2High and D3High, were compared. The data show that, compared to the other dopamine agonist drugs, pramipexole has a relatively high selectivity for the dopamine D3 receptor, as compared to D2, suggesting that the D3 receptor may be a primary target for pramipexole. There is a trend showing that the proportion of impulse-control disorders is related to the selectivity for D3 receptors over D2 receptors, with pramipexole having the highest association with, or frequency of, impulse-control disorders. While the number of studies are limited, the proportion of patients with impulse-control disorder in Parkinson patients treated with an add-on agonist were 32% for pramipexole, 25% for ropinirole, 16% for pergolide, 22% for rotigotine, 10% for apomorphine, and 6.8% for bromocriptine. Clinically, temporary replacement of pramipexole by bromocriptine may provide relief or reversal of the impulsive behavior associated with selective D3 stimulation by either pramipexole or ropinirole, while maintaining D2 stimulation needed for the anti-Parkinson action. © 2015 Wiley Periodicals, Inc.

  2. An interspecies comparison of mercury inhibition on muscarinic acetylcholine receptor binding in the cerebral cortex and cerebellum

    International Nuclear Information System (INIS)

    Basu, Niladri; Stamler, Christopher J.; Loua, Kovana Marcel; Chan, H.M.

    2005-01-01

    Mercury (Hg) is a ubiquitous pollutant that can disrupt neurochemical signaling pathways in mammals. It is well documented that inorganic Hg (HgCl 2 ) and methyl Hg (MeHg) can inhibit the binding of radioligands to the muscarinic acetylcholine (mACh) receptor in rat brains. However, little is known concerning this relationship in specific anatomical regions of the brain or in other species, including humans. The purpose of this study was to explore the inhibitory effects of HgCl 2 and MeHg on [ 3 H]-quinuclidinyl benzilate ([ 3 H]-QNB) binding to the mACh receptor in the cerebellum and cerebral cortex regions from human, rat, mouse, mink, and river otter brain tissues. Saturation binding curves were obtained from each sample to calculate receptor density (B max ) and ligand affinity (K d ). Subsequently, samples were exposed to HgCl 2 or MeHg to derive IC50 values and inhibition constants (K i ). Results demonstrate that HgCl 2 is a more potent inhibitor of mACh receptor binding than MeHg, and the receptors in the cerebellum are more sensitive to Hg-mediated mACh receptor inhibition than those in the cerebral cortex. Species sensitivities, irrespective of Hg type and brain region, can be ranked from most to least sensitive: river otter > rat > mink > mouse > humans. In summary, our data demonstrate that Hg can inhibit the binding [ 3 H]-QNB to the mACh receptor in a range of mammalian species. This comparative study provides data on interspecies differences and a framework for interpreting results from human, murine, and wildlife studies

  3. 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

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    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, ...

  4. Activation of dopamine D3 receptors inhibits reward-related learning induced by cocaine.

    Science.gov (United States)

    Kong, H; Kuang, W; Li, S; Xu, M

    2011-03-10

    Memories of learned associations between the rewarding properties of drugs and environmental cues contribute to craving and relapse in humans. The mesocorticolimbic dopamine (DA) system is involved in reward-related learning induced by drugs of abuse. DA D3 receptors are preferentially expressed in mesocorticolimbic DA projection areas. Genetic and pharmacological studies have shown that DA D3 receptors suppress locomotor-stimulant effects of cocaine and reinstatement of cocaine-seeking behaviors. Activation of the extracellular signal-regulated kinase (ERK) induced by acute cocaine administration is also inhibited by D3 receptors. How D3 receptors modulate cocaine-induced reward-related learning and associated changes in cell signaling in reward circuits in the brain, however, have not been fully investigated. In the present study, we show that D3 receptor mutant mice exhibit potentiated acquisition of conditioned place preference (CPP) at low doses of cocaine compared to wild-type mice. Activation of ERK and CaMKIIα, but not the c-Jun N-terminal kinase and p38, in the nucleus accumbens, amygdala and prefrontal cortex is also potentiated in D3 receptor mutant mice compared to that in wild-type mice following CPP expression. These results support a model in which D3 receptors modulate reward-related learning induced by low doses of cocaine by inhibiting activation of ERK and CaMKIIα in reward circuits in the brain. Copyright © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  5. 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

  6. 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.

  7. 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

  8. Clinical and treatment effects on 3H-clonidine and 3H-imipramine binding in elderly depressed patients

    International Nuclear Information System (INIS)

    Georgotas, A.; Schweitzer, J.; McCue, R.E.; Armour, M.; Friedhoff, A.J.

    1987-01-01

    3 H-clonidine and 3 H-imipramine binding were measured in depressed patients, 55 years and older. There was no significant difference in either 3 H-clonidine or 3 H-imipramine binding between depressed patients and age- and sex-matched controls. There was no significant correlation between 3 H-clonidine or 3 H-imipramine binding and severity of depression before treatment. There was a significant negative correlation between the K/sub D/ of 3 H-imipramine binding sites and Hamilton score over seven weeks of antidepressant treatment. There was no significant difference between receptor data of responders and nonresponders to antidepressant treatment. 19 references, 2 tables

  9. Binding of [3H]MSX-2 (3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargylxanthine) to rat striatal membranes--a new, selective antagonist radioligand for A(2A) adenosine receptors.

    Science.gov (United States)

    Müller, C E; Maurinsh, J; Sauer, R

    2000-01-01

    The present study describes the preparation and binding properties of a new, potent, and selective A(2A) adenosine receptor (AR) antagonist radioligand, [3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxystyryl)-1-propargy lxanth ine ([3H]MSX-2). [3H]MSX-2 binding to rat striatal membranes was saturable and reversible. Saturation experiments showed that [3H]MSX-2 labeled a single class of binding sites with high affinity (K(d)=8.0 nM) and limited capacity (B(max)=1.16 fmol.mg(-1) of protein). The presence of 100 microM GTP, or 10 mM magnesium chloride, respectively, had no effect on [3H]MSX-2 binding. AR agonists competed with the binding of 1 nM [3H]MSX-2 with the following order of potency: 5'-N-ethylcarboxamidoadenosine (NECA)>2-[4-(carboxyethyl)phenylethylamino]-5'-N-ethylcarboxami doaden osine (CGS-21680)>2-chloroadenosine (2-CADO)>N(6)-cyclopentyladenosine (CPA). AR antagonists showed the following order of potency: 8-(m-bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX)>1, 3-dipropyl-8-cyclopentylxanthine (DPCPX)>(R)-5, 6-dimethyl-7-(1-phenylethyl)-2-(4-pyridyl)-7H-pyrrolo[2, 3-d]pyrimidine-4-amine (SH-128)>3,7-dimethyl-1-propargylxanthine (DMPX)>caffeine. The K(i) values for antagonists were in accordance with data from binding studies with the agonist radioligand [3H]CGS21680, while agonist affinities were 3-7-fold lower. [3H]MSX-2 is a highly selective A(2A) AR antagonist radioligand exhibiting a selectivity of at least two orders of magnitude versus all other AR subtypes. The new radioligand shows high specific radioactivity (85 Ci/mmol, 3150 GBq/mmol) and acceptable nonspecific binding at rat striatal membranes of 20-30%, at 1 nM.

  10. Kinetic analysis of transport and opioid receptor binding of ( sup 3 H)(-)-cyclofoxy in rat brain in vivo: Implications for human studies

    Energy Technology Data Exchange (ETDEWEB)

    Sawada, Y.; Kawai, R.; McManaway, M.; Otsuki, H.; Rice, K.C.; Patlak, C.S.; Blasberg, R.G. (National Institutes of Health, Bethesda, MD (USA))

    1991-03-01

    (3H)Cyclofoxy (CF: 17-cyclopropylmethyl-3,14-dihydroxy-4,5-alpha-epoxy-6-beta-fluoromorp hinan) is an opioid antagonist with affinity to both mu and kappa subtypes that was synthesized for quantitative evaluation of opioid receptor binding in vivo. Two sets of experiments in rats were analyzed. The first involved determining the metabolite-corrected blood concentration and tissue distribution of CF in brain 1 to 60 min after i.v. bolus injection. The second involved measuring brain washout for 15 to 120 s following intracarotid artery injection of CF. A physiologically based model and a classical compartmental pharmacokinetic model were compared. The models included different assumptions for transport across the blood-brain barrier (BBB); estimates of nonspecific tissue binding and specific binding to a single opiate receptor site were found to be essentially the same with both models. The nonspecific binding equilibrium constant varied modestly in different brain structures (Keq = 3-9), whereas the binding potential (BP) varied over a much broader range (BP = 0.6-32). In vivo estimates of the opioid receptor dissociation constant were similar for different brain structures (KD = 2.1-5.2 nM), whereas the apparent receptor density (Bmax) varied between 1 (cerebellum) and 78 (thalamus) pmol/g of brain. The receptor dissociation rate constants in cerebrum (k4 = 0.08-0.16 min-1; koff = 0.16-0.23 min-1) and brain vascular permeability (PS = 1.3-3.4 ml/min/g) are sufficiently high to achieve equilibrium conditions within a reasonable period of time. Graphical analysis of the data is inappropriate due to the high tissue-loss rate constant for CF in brain. From these findings, CF should be a very useful opioid receptor ligand for the estimation of the receptor binding parameters in human subjects using (18F)CF and positron emission tomography.

  11. The solvent at antigen-binding site regulated C3d-CR2 interactions through the C-terminal tail of C3d at different ion strengths: insights from molecular dynamics simulation.

    Science.gov (United States)

    Zhang, Yan; Guo, Jingjing; Li, Lanlan; Liu, Xuewei; Yao, Xiaojun; Liu, Huanxiang

    2016-10-01

    The interactions of complement receptor 2 (CR2) and the degradation fragment C3d of complement component C3 play important links between the innate and adaptive immune systems. Due to the importance of C3d-CR2 interaction in the design of vaccines and inhibitors, a number of studies have been performed to investigate C3d-CR2 interaction. Many studies have indicated C3d-CR2 interactions are ionic strength-dependent. To investigate the molecular mechanism of C3d-CR2 interaction and the origin of effects of ionic strength, molecular dynamics simulations for C3d-CR2 complex together with the energetic and structural analysis were performed. Our results revealed the increased interactions between charged protein and ions weaken C3d-CR2 association, as ionic strengths increase. Moreover, ion strengths have similar effects on antigen-binding site and CR2 binding site. Meanwhile, Ala17 and Gln20 will transform between the activated and non-activated states mediated by His133 and Glu135 at different ion strengths. Our results reveal the origins of the effects of ionic strengths on C3d-CR2 interactions are due to the changes of water, ion occupancies and distributions. This study uncovers the origin of the effect of ionic strength on C3d-CR2 interaction and deepens the understanding of the molecular mechanism of their interaction, which is valuable for the design of vaccines and small molecule inhibitors. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Differential regulation of dopamine receptors after chronic typical and atypical antipsychotic drug treatment

    International Nuclear Information System (INIS)

    Creese, I.; Florijn, W.J.; Tarazi, F.I.

    1997-01-01

    Changes in dopamine receptor subtype binding in different brain regions were examined after 28 days treatment of rats with haloperidol, raclopride, clozapine or SCH23390 using in vitro receptor autoradiography. [ 3 H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin binding to dopamine D 3 receptors was not changed in any brain region by any of the drug treatments. [ 3 H]SCH23390 was only increased by chronic SCH23390 treatment. Haloperidol significantly increased [ 3 H]nemonapride and [ 3 H]spiperone binding to dopamine D 2 -like receptors in the caudate-putamen. In contrast, haloperidol caused a small, significant increase in [ 3 H]raclopride binding in the lateral caudate-putamen only. Raclopride also elevated, but to a lesser extent [ 3 H]nemonapride and [ 3 H]spiperone binding in caudate-putamen, whereas it did not affect [ 3 H]raclopride binding. Clozapine did not significantly change D 2 -like striatal binding of [ 3 H]nemonapride, [ 3 H]spiperone or [ 3 H]raclopride. The differences in radioligand binding suggest that [ 3 H]nemonapride and [ 3 H]spiperone may be binding to additional subsets of dopamine D 2 -like receptors (including D 4 -like receptors) that are not recognized by [ 3 H]raclopride, which has high affinity for D 2 and D 3 receptors only.Quantification of [ 3 H]nemonapride or [ 3 H]spiperone binding in the presence of 300 nM raclopride (to block D 2 and D 3 receptors) revealed that haloperidol, raclopride and clozapine up-regulated D 4 -like receptors in the caudate-putamen using either radioligand. These results suggest that D 4 -like receptors may be a common site of action of both typical and atypical antipsychotics. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  13. 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.

  14. Independent associations of polymorphisms in vitamin D binding protein (GC) and vitamin D receptor (VDR) genes with obesity and plasma 25OHD3 levels demonstrate sex dimorphism.

    Science.gov (United States)

    Almesri, Norah; Das, Nagalla S; Ali, Muhallab E; Gumaa, Khalid; Giha, Hayder Ahmed

    2016-04-01

    We investigated a possible association between polymorphisms in vitamin D binding protein (GC) and vitamin D receptor (VDR) genes and obesity in Bahraini adults. For this purpose, 406 subjects with varying body mass indexes (BMIs) were selected. Plasma levels of 25-hydroxyvitamin D3 (25OHD3) were measured by chemiluminescence immunoassay. Six single nucleotide polymorphisms, 2 in the VDR gene (rs731236 TC and rs12721377 AG) and 4 in the GC gene (rs2282679 AC, rs4588 CA, rs7041 GT, and rs2298849 TC), were genotyped by real-time polymerase chain reaction. We found that the rs7041 minor allele (G) and rare genotype (GG) were associated with higher BMI (p = 0.007 and p = 0.012, respectively), but they did not influence 25OHD3 levels. However, the minor alleles of rs2282679 (A) and rs4588 (C) were associated with low 25OHD3 plasma levels (p = 0.039 and p = 0.021, respectively), but not with BMI. Having categorized the subjects based on their sex, we found that (i) rs7041 GG associated with high BMI in females (p = 0.003), (ii) rs4588 CC associated with high BMI in females (p = 0.034) and low 25OHD3 levels in males (p = 0.009), and (iii) rs12721377 AA associated with low 25OHD3 levels in females (p = 0.039). Notably, none of the common haplotypes (6 in the GC gene and 3 in the VDR gene) were associated with BMI. Therefore, polymorphisms in the GC (rs2282679, rs4588, rs7041) and VDR (rs12721377) genes were independently associated with obesity and 25OHD3 levels with a clear sex dimorphism.

  15. Extrastriatal dopamine D-2/3 receptors and cortical grey matter volumes in antipsychotic-naive schizophrenia patients before and after initial antipsychotic treatment

    DEFF Research Database (Denmark)

    Nørbak-Emig, Henrik; Pinborg, Lars H.; Raghava, Jayachandra M.

    2017-01-01

    OBJECTIVES: Long-term dopamine D2/3 receptor blockade, common to all antipsychotics, may underlie progressive brain volume changes observed in patients with chronic schizophrenia. In the present study, we examined associations between cortical volume changes and extrastriatal dopamine D2/3 recept...... binding potentials (BPND) in first-episode schizophrenia patents at baseline and after antipsychotic treatment. METHODS: Twenty-two initially antipsychotic-naïve patients underwent magnetic resonance imaging (MRI), [(123)I]epidepride single-photon emission computerised tomography (SPECT......), and psychopathology assessments before and after 3 months of treatment with either risperidone (N = 13) or zuclopenthixol (N = 9). Twenty healthy controls matched on age, gender and parental socioeconomic status underwent baseline MRI and SPECT. RESULTS: Neither extrastriatal D2/3 receptor BPND at baseline, nor...

  16. Detailed mapping of serotonin 5-HT{sub 1B} and 5-HT{sub 1D} receptor messenger RNA and ligand binding sites in guinea-pig brain and trigeminal ganglion: clues for function

    Energy Technology Data Exchange (ETDEWEB)

    Leysen, J.E. [Graduate School Neurosciences, Amsterdam (Netherlands); Schotte, A.; Jurzak, M.; Luyten, W.H.M.L. [Department of Biochemical Pharmacology, Janssen Research Foundation, Beerse (Belgium); Voorn, P.; Bonaventure, P. [Graduate School Neurosciences, Amsterdam (Netherlands)

    1997-10-17

    The similar pharmacology of the 5-HT{sub 1B} and 5-HT{sub 1D} receptors, and the lack of selective compounds sufficiently distinguishing between the two receptor subtypes, have hampered functional studies on these receptors. In order to provide clues for differential functional roles of the two subtypes, we performed a parallel localization study throughout the guinea-pig brain and the trigeminal ganglia by means of quantitative in situ hybridization histochemistry (using [{sup 35}S]-labelled riboprobes probes for receptor messenger RNA) and receptor autoradiography (using a new radioligand [{sup 3}H]alniditan).The anatomical patterns of 5-HT{sub 1B} and 5-HT{sub 1D} receptor messenger RNA were quite different. While 5-HT{sub 1B} receptor messenger RNA was abundant throughout the brain (with highest levels in the striatum, nucleus accumbens, olfactory tubercle, cortex, hypothalamus, hippocampal formation, amygdala, thalamus, dorsal raphe and cerebellum), 5-HT{sub 1D} receptor messenger RNA exhibited a more restricted pattern; it was found mainly in the olfactory tubercle, entorhinal cortex, dorsal raphe, cerebellum, mesencephalic trigeminal nucleus and in the trigeminal ganglion. The density of 5-HT{sub 1B/1D} binding sites (combined) obtained with [{sup 3}H]alniditan autoradiography was high in the substantia nigra, superior colliculus and globus pallidus, whereas lower levels were detected in the caudate-putamen, hypothalamus, hippocampal formation, amygdala, thalamus and central gray. This distribution pattern was indistinguishable from specific 5-HT{sub 1B} receptor labelling in the presence of ketanserin under conditions to occlude 5-HT{sub 1D} receptor labelling; hence the latter were below detection level. Relationships between the regional distributions of the receptor messenger RNAs and binding sites and particular neuroanatomical pathways are discussed with respect to possible functional roles of the 5-HT{sub 1B} and 5-HT{sub 1D} receptors. (Copyright (c

  17. A photoaffinity ligand for dopamine D2 receptors: azidoclebopride.

    Science.gov (United States)

    Niznik, H B; Guan, J H; Neumeyer, J L; Seeman, P

    1985-02-01

    In order to label D2 dopamine receptors selectively and covalently by means of a photosensitive compound, azidoclebopride was synthesized directly from clebopride. The dissociation constant (KD) of clebopride for the D2 dopamine receptor (canine brain striatum) was 1.5 nM, while that for azidoclebopride was 21 nM. The affinities of both clebopride and azidoclebopride were markedly reduced in the absence of sodium chloride. In the presence of ultraviolet light, azidoclebopride inactivated D2 dopamine receptors irreversibly, as indicated by the inability of the receptors to bind [3H]spiperone. Maximal photoinactivation of about 60% of the D2 dopamine receptors occurred at 1 microM azidoclebopride; 30% of the receptors were inactivated at 80 nM azidoclebopride (pseudo-IC50). Dopamine agonists selectively protected the D2 receptors from being inactivated by azidoclebopride, the order of potency being (-)-N-n-propylnorapomorphine greater than apomorphine greater than (+/-)-6,7-dihydroxy-2-aminotetralin greater than (+)-N-n-propylnorapomorphine greater than dopamine greater than noradrenaline greater than serotonin. Similarly, dopaminergic antagonists prevented the photoinactivation of D2 receptors by azidoclebopride with the following order of potency: spiperone greater than (+)-butaclamol greater than haloperidol greater than clebopride greater than (-)-sulpiride greater than (-)-butaclamol. The degree of D2 dopamine receptor photoinduced inactivation by azidoclebopride was not significantly affected by scavengers such as p-aminobenzoic acid and dithiothreitol. Furthermore, irradiation of striatal membranes with a concentration of azidoclebopride sufficient to inactivate dopamine D2 receptors by 60% did not significantly reduce dopamine D1, serotonin (S2), benzodiazepine, alpha 1- or beta-noradrenergic receptors. This study describes the use of a novel and selective photoaffinity ligand for brain dopamine D2 receptors. The molecule, in radiolabeled form, may aid in the

  18. Direct chemical synthesis of 1 alpha,25-dihydroxy[26,27-3H] vitamin D3 with high specific activity: its use in receptor studies

    International Nuclear Information System (INIS)

    Napoli, J.L.; Mellon, W.S.; Fivizzani, M.A.; Schnoes, H.K.; DeLuca, H.F.

    1980-01-01

    The first direct chemical synthesis of radiolabeled 1 alpha, 25-dihydroxyvitamin D3 is reported. Unlike all previous syntheses, the new approach does not rely on enzymatic 1 alpha-hydroxylation of radiolabeled precursors. Rather, isotope is introduced in the last synthetic step by reaction of [3H] -methylmagnesium bromide with methyl 1 alpha-hydroxy-26,27-dinorvitamin D3-25-carboxylate to give 1 alpha,25-dihydroxy-[26,27-3H] vitamin D3 with a specific activity of 160 Ci/mmol. Mass spectroscopy confirmed that the radiohormone consists of a single isomer with six tritium atoms bound to carbons 26 and 27. Synthetically produced 1 alpha,25-dihydroxy [26,27-3H] vitamin D3 is indistinguishable from 1 alpha,25-dihydroxy-[26,27-3H] vitamin D3 obtained from the enzymatic 1 alpha-hydroxylation of 25-hydroxy[26,27-3H] vitamin D3 (160 Ci/mmol) by high-pressure liquid chromatography analysis and in the competitive binding assay using chick intestinal cytosol as the receptor source. Equilibrium dissociation constant measurements with the high specific activity radiohormone indicate a Kd of 8.2 x 10(-11) M for the chick intestinal cytosol 1 alpha,25-dihydroxyvitamin D3 receptor--a value considerably lower than the constants in the range of (1-5) x 10(-9) M previously reported

  19. Arcaine uncovers dual interactions of polyamines with the N-methyl-D-aspartate receptor

    Energy Technology Data Exchange (ETDEWEB)

    Reynolds, I.J. (Univ. of Pittsburgh, PA (USA))

    1990-12-01

    This study investigated the interaction between the polyamines spermine and spermidine and the N-methyl-D-aspartate (NMDA) receptor by using (+)-(3H)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cyclohepten-5,10-im ine maleate ((3H)MK801) binding to well washed rat brain membranes. The actions of arcaine, agmatine, diethylenetriamine and 1,8-octanediamine as polyamine antagonists were compared to use as tools in this study. Arcaine was found to be the antagonist of choice due to its greater potency. Several divalent cations, including Ba++, Ca++ and Sr++, but not Zn++, decreased the apparent potency of arcaine. These cations enhance (3H)MK801 binding in a similar fashion to spermidine and spermine suggesting that they may share a common site and mechanism of action. Moreover, arcaine competitively reduced the enhancement of (3H)MK801 binding produced by Sr++ did not alter the inhibition produced by higher concentrations of this cation, a phenomenon that also occurs with spermidine. The distinct arcaine sensitivity of the two separate phases of the concentration-response curves of both spermidine and Sr++ suggests two separate mechanisms underlying the action of spermidine-like drugs on the NMDA receptor. Further investigation of the increase in (3H)MK801 binding produced by spermidine revealed that spermidine increased the equilibrium affinity of this ligand by 2-fold without significantly altering the density of binding sites. In contrast, polyamine induced increases in the dissociation of (3H)MK801 required higher polyamine concentrations than necessary to increase ligand binding and were relatively insensitive to arcaine. These findings suggest that polyamines do not activate or promote the activation of the NMDA receptor, but instead enhance (3H)MK801 binding by allosterically increasing ligand affinity.

  20. Arcaine uncovers dual interactions of polyamines with the N-methyl-D-aspartate receptor

    International Nuclear Information System (INIS)

    Reynolds, I.J.

    1990-01-01

    This study investigated the interaction between the polyamines spermine and spermidine and the N-methyl-D-aspartate (NMDA) receptor by using (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK801) binding to well washed rat brain membranes. The actions of arcaine, agmatine, diethylenetriamine and 1,8-octanediamine as polyamine antagonists were compared to use as tools in this study. Arcaine was found to be the antagonist of choice due to its greater potency. Several divalent cations, including Ba++, Ca++ and Sr++, but not Zn++, decreased the apparent potency of arcaine. These cations enhance [3H]MK801 binding in a similar fashion to spermidine and spermine suggesting that they may share a common site and mechanism of action. Moreover, arcaine competitively reduced the enhancement of [3H]MK801 binding produced by Sr++ did not alter the inhibition produced by higher concentrations of this cation, a phenomenon that also occurs with spermidine. The distinct arcaine sensitivity of the two separate phases of the concentration-response curves of both spermidine and Sr++ suggests two separate mechanisms underlying the action of spermidine-like drugs on the NMDA receptor. Further investigation of the increase in [3H]MK801 binding produced by spermidine revealed that spermidine increased the equilibrium affinity of this ligand by 2-fold without significantly altering the density of binding sites. In contrast, polyamine induced increases in the dissociation of [3H]MK801 required higher polyamine concentrations than necessary to increase ligand binding and were relatively insensitive to arcaine. These findings suggest that polyamines do not activate or promote the activation of the NMDA receptor, but instead enhance [3H]MK801 binding by allosterically increasing ligand affinity

  1. [3H]opipramol labels a novel binding site and sigma receptors in rat brain membranes

    International Nuclear Information System (INIS)

    Ferris, C.D.; Hirsch, D.J.; Brooks, B.P.; Snowman, A.M.; Snyder, S.H.

    1991-01-01

    Opipramol (OP), a clinically effective antidepressant with a tricyclic structure, is inactive as an inhibitor of biogenic amine uptake. [ 3 H]Opipramol binds saturably to rat brain membranes (apparent KD = 4 nM, Bmax = 3 pmol/mg of protein). [ 3 H]Opipramol binding can be differentiated into haloperidol-sensitive and -resistant components, with Ki values for haloperidol of 1 nM (Bmax = 1 pmol/mg of protein) and 350 nM (Bmax = 1.9 pmol/mg of protein), respectively. The drug specificity of the haloperidol-sensitive component is the same as that of sigma receptors labeled with (+)-[ 3 H]3-(3-hydroxyphenyl)-N-(1-propyl)piperdine. The haloperidol-resistant component does not correspond to any known neurotransmitter receptor or uptake recognition site. It displays high affinity for phenothiazines and related structures such as perphenazine, clopenthixol, and flupenthixol, whose potencies are comparable to that of opipramol. Because certain of these drugs are more potent at the haloperidol-resistant opipramol site than in exerting any other action, it is possible that this opipramol-selective site may mediate their therapeutic effects

  2. Dopamine D2 receptor radiotracers [11C](+)-PHNO and [3H]raclopride are indistinguishably inhibited by D2 agonists and antagonists ex vivo

    International Nuclear Information System (INIS)

    McCormick, Patrick N.; Kapur, Shitij; Seeman, Philip; Wilson, Alan A.

    2008-01-01

    Introduction: In vitro, the dopamine D2 receptor exists in two states, with high and low affinity for agonists. The high-affinity state is the physiologically active state thought to be involved in dopaminergic illnesses such as schizophrenia. The positron emission tomography radiotracer [ 11 C](+)-PHNO ([ 11 C](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4] oxazin-9-o l), being a D2 agonist, should selectively label the high-affinity state at tracer dose and therefore be more susceptible to competition by agonist as compared to the antagonist [ 3 H]raclopride, which binds to both affinity states. Methods: We tested this prediction using ex vivo dual-radiotracer experiments in conscious rats. D2 antagonists (haloperidol or clozapine), a partial agonist (aripiprazole), a full agonist [(-)-NPA] or the dopamine-releasing drug amphetamine (AMPH) were administered to rats prior to an intravenous coinjection of [ 11 C](+)-PHNO and [ 3 H]raclopride. Rats were sacrificed 60 min after radiotracer injection. Striatum, cerebellum and plasma samples were counted for 11 C and 3 H. The specific binding ratio {SBR, i.e., [%ID/g (striatum)-%ID/g (cerebellum)]/(%ID/g (cerebellum)} was used as the outcome measure. Results: In response to D2 antagonists, partial agonist or full agonist, [ 11 C](+)-PHNO and [ 3 H]raclopride SBRs responded indistinguishably in terms of both ED 50 and Hill slope (e.g., (-)-NPA ED 50 values are 0.027 and 0.023 mg/kg for [ 11 C](+)-PHNO and [ 3 H]raclopride, respectively). In response to AMPH challenge, [ 11 C](+)-PHNO and [ 3 H]raclopride SBRs were inhibited to the same degree. Conclusions: We have shown that the SBRs of [ 11 C](+)-PHNO- and [ 3 H]raclopride do not differ in their response to agonist challenge. These results do not support predictions of the in vivo binding behavior of a D2 agonist radiotracer and cast some doubt on the in vivo applicability of the D2 two-state model, as described by in vitro binding experiments

  3. QSAR of adenosine receptor antagonists. Part 3: Exploring physicochemical requirements for selective binding of 1,2,4-triazolo[5,1-i]purine derivatives with human adenosine A3 receptor subtype.

    Science.gov (United States)

    Roy, Kunal; Leonard, J Thomas; Sengupta, Chandana

    2004-07-16

    Considering potential of selective adenosine A3 receptor antagonists in the development of prospective therapeutic agents, an attempt has been made to explore selectivity requirements of 1,2,4-triazolo[5,1-i]purine derivatives for binding with cloned human adenosine A3 receptor subtype. In this study, partition coefficient (logP) values of the molecules (calculated by Crippen's fragmentation method) and Wang-Ford charges of the common atoms of the triazolopurine nucleus (calculated from molecular electrostatic potential surface of energy minimized geometry using AM1 technique) were used as independent variables along with suitable dummy parameters. The best equation describing A3 binding affinity [n=29, Q2=0.796, Ra2=0.853, R2=0.874, R=0.935, s=0.342, F=41.5 (df 4,24), SDEP=0.396] showed parabolic relation with logP (optimum value being 4.134). Further, it was found that an aromatic substituent conjugated with the triazole nucleus should be present at R2 position for A3 binding affinity. Again, high negative charges on N2 and N4 are conducive to the binding affinity. While exploring selectivity requirements of the compounds for binding with A3 receptor over that with A2A receptor, the selectivity relation [n=23, Q2=0.909, Ra2=0.918, R2=0.933, R=0.966, s=0.401, F=62.4 (df 4,18), SDEP=0.412] showed that an aromatic R2 substituent conjugated with the triazole nucleus contributes significantly to the selectivity. Again, presence of a 4-substituted-phenyl ring (except 4-OH-phenyl and 4-CH3-phenyl) at R2 position also increases selectivity. Further, charge difference between N2 and N11 (negative charge on the former should be higher and that on the latter should be less) contributes significantly to the selectivity. In addition, negative charge on N7 is conducive while presence of substituents like propyl, butyl, pentyl or phenyl at R1 position is detrimental for the A3 selectivity.

  4. Piracetam Defines a New Binding Site for Allosteric Modulators of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors§

    Science.gov (United States)

    Ahmed, Ahmed H.; Oswald, Robert E.

    2010-01-01

    Glutamate receptors are the most prevalent excitatory neurotransmitter receptors in the vertebrate central nervous system and are important potential drug targets for cognitive enhancement and the treatment of schizophrenia. Allosteric modulators of AMPA receptors promote dimerization by binding to a dimer interface and reducing desensitization and deactivation. The pyrrolidine allosteric modulators, piracetam and aniracetam, were among the first of this class of drugs to be discovered. We have determined the structure of the ligand binding domain of the AMPA receptor subtypes GluA2 and GluA3 with piracetam and a corresponding structure of GluA3 with aniracetam. Both drugs bind to both GluA2 and GluA3 in a very similar manner, suggesting little subunit specificity. However, the binding sites for piracetam and aniracetam differ considerably. Aniracetam binds to a symmetrical site at the center of the dimer interface. Piracetam binds to multiple sites along the dimer interface with low occupation, one of which is a unique binding site for potential allosteric modulators. This new site may be of importance in the design of new allosteric regulators. PMID:20163115

  5. 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

  6. 3D-QSAR comparative molecular field analysis on opioid receptor antagonists: pooling data from different studies.

    Science.gov (United States)

    Peng, Youyi; Keenan, Susan M; Zhang, Qiang; Kholodovych, Vladyslav; Welsh, William J

    2005-03-10

    Three-dimensional quantitative structure-activity relationship (3D-QSAR) models were constructed using comparative molecular field analysis (CoMFA) on a series of opioid receptor antagonists. To obtain statistically significant and robust CoMFA models, a sizable data set of naltrindole and naltrexone analogues was assembled by pooling biological and structural data from independent studies. A process of "leave one data set out", similar to the traditional "leave one out" cross-validation procedure employed in partial least squares (PLS) analysis, was utilized to study the feasibility of pooling data in the present case. These studies indicate that our approach yields statistically significant and highly predictive CoMFA models from the pooled data set of delta, mu, and kappa opioid receptor antagonists. All models showed excellent internal predictability and self-consistency: q(2) = 0.69/r(2) = 0.91 (delta), q(2) = 0.67/r(2) = 0.92 (mu), and q(2) = 0.60/r(2) = 0.96 (kappa). The CoMFA models were further validated using two separate test sets: one test set was selected randomly from the pooled data set, while the other test set was retrieved from other published sources. The overall excellent agreement between CoMFA-predicted and experimental binding affinities for a structurally diverse array of ligands across all three opioid receptor subtypes gives testimony to the superb predictive power of these models. CoMFA field analysis demonstrated that the variations in binding affinity of opioid antagonists are dominated by steric rather than electrostatic interactions with the three opioid receptor binding sites. The CoMFA steric-electrostatic contour maps corresponding to the delta, mu, and kappa opioid receptor subtypes reflected the characteristic similarities and differences in the familiar "message-address" concept of opioid receptor ligands. Structural modifications to increase selectivity for the delta over mu and kappa opioid receptors have been predicted on the

  7. COMT Val(158) met genotype and striatal D(2/3) receptor binding in adults with 22q11 deletion syndrome.

    LENUS (Irish Health Repository)

    Boot, Erik

    2011-09-01

    Although catechol-O-methyltransferase (COMT) activity evidently affects dopamine function in prefrontal cortex, the contribution is assumed less significant in striatum. We studied whether a functional polymorphism in the COMT gene (Val(158) Met) influences striatal D(2\\/3) R binding ratios (D(2\\/3) R BP(ND) ) in 15 adults with 22q11 deletion syndrome and hemizygous for this gene, using single photon emission computed tomography and the selective D(2\\/3) radioligand [(123) I]IBZM. Met hemizygotes had significantly lower mean D(2\\/3) R BPND than Val hemizygotes. These preliminary data suggest that low COMT activity may affect dopamine levels in striatum in humans and this may have implications for understanding the contribution of COMT activity to psychiatric disorders.

  8. (D-Pen2,4 prime -125I-Phe4,D-Pen5)enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity

    Energy Technology Data Exchange (ETDEWEB)

    Knapp, R.J.; Sharma, S.D.; Toth, G.; Duong, M.T.; Fang, L.; Bogert, C.L.; Weber, S.J.; Hunt, M.; Davis, T.P.; Wamsley, J.K. (Department of Pharmacology, University of Arizona, College of Medicine, Tucson (United States))

    1991-09-01

    (D-Pen2,4{prime}-125I-Phe4,D-Pen5)enkephalin ((125I)DPDPE) is a highly selective radioligand for the delta opioid receptor with a specific activity (2200 Ci/mmol) that is over 50-fold greater than that of tritium-labeled DPDPE analogs. (125I)DPDPE binds to a single site in rat brain membranes with an equilibrium dissociation constant (Kd) value of 421 {plus minus} 67 pM and a receptor density (Bmax) value of 36.4 {plus minus} 2.7 fmol/mg protein. The high affinity of this site for delta opioid receptor ligands and its low affinity for mu or kappa receptor-selective ligands are consistent with its being a delta opioid receptor. The distribution of these sites in rat brain, observed by receptor autoradiography, is also consistent with that of delta opioid receptors. Association and dissociation binding kinetics of 1.0 nM (125I) DPDPE are monophasic at 25 degrees C. The association rate (k + 1 = 5.80 {plus minus} 0.88 {times} 10(7) M-1 min-1) is about 20- and 7-fold greater than that measured for 1.0 nM (3H) DPDPE and 0.8 nM (3H) (D-Pen2,4{prime}-Cl-Phe4, D-Pen5)enkephalin, respectively. The dissociation rate of (125I)DPDPE (0.917 {plus minus} 0.117 {times} 10(-2) min-1) measured at 1.0 nM is about 3-fold faster than is observed for either of the other DPDPE analogs. The rapid binding kinetics of (125I)DPDPE is advantageous because binding equilibrium is achieved with much shorter incubation times than are required for other cyclic enkephalin analogs. This, in addition to its much higher specific activity, makes (125I)DPDPE a valuable new radioligand for studies of delta opioid receptors.

  9. Modulation of [3H]-glutamate binding by serotonin in the rat hippocampus: An autoradiographic study

    International Nuclear Information System (INIS)

    Mennini, T.; Miari, A.

    1991-01-01

    Serotonin (5-HT) added in vitro increased [ 3 H]-glutamate specific binding in the rat hippocampus, reaching statistical significance in layers rich in N-Methyl-D-Aspartate sensitive glutamate receptors. This effect was explained by a significant increase in the apparent affinity of [ 3 H]-glutamate when 5-HT is added in vitro. Two days after lesion of serotonergic afferents to the hippocampus with 5,7- Dihydroxytryptamine [ 3 H]-glutamate binding was significantly decreased in the CA3 region and stratum lacunosum moleculare of the hippocampus, this reduction being reversed by in vitro addition of 10 μM 5-HT. The decrease observed is due to a significant reduction of quisqualate-insensitive (radiatum CA3) and kainate receptors (strata oriens, radiatum, pyramidal of CA3). Five days after lesion [ 3 H]-glutamate binding increased significantly in the CA3 region of the hippocampus but was not different from sham animals in the other hippocampal layers. Two weeks after lesion [ 3 H]-glutamate binding to quisqualate-insensitive receptors was increased in all the hippocampal layers, while kainate and quisqualate-sensitive receptors were not affected. These data are consistent with the possibility that 5-HT is a direct positive modulator of glutamate receptor subtypes

  10. Photoaffinity labeling of the rat plasma vitamin D binding protein with [26,27-3H]-25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate

    International Nuclear Information System (INIS)

    Ray, R.; Holick, S.A.; Hanafin, N.; Holick, M.F.

    1986-01-01

    It is well recognized that the vitamin D binding protein (DBP) is important for the transport of vitamin D, 25-hydroxyvitamin D (25-OH-D), and its metabolites. In an attempt to better understand the molecular-binding properties of this ubiquitous protein, we designed and synthesized a photoaffinity analogue of 25-OH-D3 and its radiolabeled counterpart. This analogue, 25-hydroxyvitamin D3 3 beta-[N-(4-azido-2-nitrophenyl)glycinate] (25-OH-D3-ANG), was recognized by the rat DBP and was about 10 times less active than 25-OH-D3 in terms of binding. Incubation of [ 3 H]25-OH-D3 or [ 3 H]25-OH-D3-ANG with rat DBP revealed that both compounds were specifically bound to a protein with a sedimentation coefficient of 4.1 S. Each was displaced with a 500-fold excess of 25-OH-D3. When [ 3 H]25-OH-D3-ANG was exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3, there was no displacement of tritium from the 4.1S peak. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographic analysis of [ 3 H]25-OH-D3-ANG exposed to UV radiation in the presence of rat DBP followed by the addition of a 500-fold excess of 25-OH-D3 revealed one major band with a molecular weight of 52 000. These data provide strong evidence that [ 3 H]25-OH-D3-ANG was covalently linked to the rat DBP. This photoaffinity probe should provide a valuable tool for the analysis of the binding site on this transport protein

  11. Dopamine D2L receptor-interacting proteins regulate dopaminergic signaling

    Directory of Open Access Journals (Sweden)

    Norifumi Shioda

    2017-10-01

    Full Text Available Dopamine receptor family proteins include seven transmembrane and trimeric GTP-binding protein-coupled receptors (GPCRs. Among them, the dopamine D2 receptor (D2R is most extensively studied. All clinically used antipsychotic drugs serve as D2R antagonists in the mesolimbic dopamine system, and their ability to block D2R signaling is positively correlated with antipsychotic efficiency. Human genetic studies also show a significant association of DRD2 polymorphisms with disorders including schizophrenia and Parkinson's disease. D2R exists as two alternatively spliced isoforms, the long isoform (D2LR and the short isoform (D2SR, which differ in a 29-amino acid (AA insert in the third cytoplasmic loop. Importantly, previous reports demonstrate functional diversity between the two isoforms in humans. In this review, we focus on binding proteins that specifically interact with the D2LR 29AA insert. We discuss how D2R activities are mediated not only by heterotrimeric G proteins but by D2LR-interacting proteins, which in part regulate diverse D2R activities. Keywords: Dopamine D2L receptor, Antipsychotic drugs, DRD2 polymorphisms, Alternatively spliced isoforms, D2LR-interacting proteins

  12. Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions.

    Science.gov (United States)

    Reedijk, M; Liu, X; van der Geer, P; Letwin, K; Waterfield, M D; Hunter, T; Pawson, T

    1992-01-01

    Efficient binding of active phosphatidylinositol (PI) 3'-kinase to the autophosphorylated macrophage colony stimulating factor receptor (CSF-1R) requires the noncatalytic kinase insert (KI) region of the receptor. To test whether this region could function independently to bind PI 3'-kinase, the isolated CSF-1R KI was expressed in Escherichia coli, and was inducibly phosphorylated on tyrosine. The tyrosine phosphorylated form of the CSF-1R KI bound PI 3'-kinase in vitro, whereas the unphosphorylated form had no binding activity. The p85 alpha subunit of PI 3'-kinase contains two Src homology (SH)2 domains, which are implicated in the interactions of signalling proteins with activated receptors. Bacterially expressed p85 alpha SH2 domains complexed in vitro with the tyrosine phosphorylated CSF-1R KI. Binding of the CSF-1R KI to PI 3'-kinase activity, and to the p85 alpha SH2 domains, required phosphorylation of Tyr721 within the KI domain, but was independent of phosphorylation at Tyr697 and Tyr706. Tyr721 was also critical for the association of activated CSF-1R with PI 3'-kinase in mammalian cells. Complex formation between the CSF-1R and PI 3'-kinase can therefore be reconstructed in vitro in a specific interaction involving the phosphorylated receptor KI and the SH2 domains of p85 alpha. Images PMID:1314163

  13. 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.

  14. 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.

  15. [3]tetrahydrotrazodone binding. Association with serotonin binding sites

    International Nuclear Information System (INIS)

    Kendall, D.A.; Taylor, D.P.; Enna, S.J.

    1983-01-01

    High (17 nM) and low (603 nM) affinity binding sites for [ 3 ]tetrahydrotrazodone ([ 3 ] THT), a biologically active analogue of trazodone, have been identified in rat brain membranes. The substrate specificity, concentration, and subcellular and regional distributions of these sites suggest that they may represent a component of the serotonin transmitter system. Pharmacological analysis of [ 3 ]THT binding, coupled with brain lesion and drug treatment experiments, revealed that, unlike other antidepressants, [ 3 ] THT does not attach to either a biogenic amine transporter or serotonin binding sites. Rather, it would appear that [ 3 ]THT may be an antagonist ligand for the serotonin binding site. This probe may prove of value in defining the mechanism of action of trazodone and in further characterizing serotonin receptors

  16. Improving binding mode and binding affinity predictions of docking by ligand-based search of protein conformations: evaluation in D3R grand challenge 2015

    Science.gov (United States)

    Xu, Xianjin; Yan, Chengfei; Zou, Xiaoqin

    2017-08-01

    The growing number of protein-ligand complex structures, particularly the structures of proteins co-bound with different ligands, in the Protein Data Bank helps us tackle two major challenges in molecular docking studies: the protein flexibility and the scoring function. Here, we introduced a systematic strategy by using the information embedded in the known protein-ligand complex structures to improve both binding mode and binding affinity predictions. Specifically, a ligand similarity calculation method was employed to search a receptor structure with a bound ligand sharing high similarity with the query ligand for the docking use. The strategy was applied to the two datasets (HSP90 and MAP4K4) in recent D3R Grand Challenge 2015. In addition, for the HSP90 dataset, a system-specific scoring function (ITScore2_hsp90) was generated by recalibrating our statistical potential-based scoring function (ITScore2) using the known protein-ligand complex structures and the statistical mechanics-based iterative method. For the HSP90 dataset, better performances were achieved for both binding mode and binding affinity predictions comparing with the original ITScore2 and with ensemble docking. For the MAP4K4 dataset, although there were only eight known protein-ligand complex structures, our docking strategy achieved a comparable performance with ensemble docking. Our method for receptor conformational selection and iterative method for the development of system-specific statistical potential-based scoring functions can be easily applied to other protein targets that have a number of protein-ligand complex structures available to improve predictions on binding.

  17. [125I]2-iodo-3,7,8-trichlorodibenzo-p-dioxin-binding species in mouse liver induced by agonists for the Ah receptor: Characterization and identification

    International Nuclear Information System (INIS)

    Poland, A.; Teitelbaum, P.; Glover, E.

    1989-01-01

    The admininistration of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to C57BL/6J mice produces a dose-related increase in the hepatic uptake of [ 125 I]2-iodo-3,7,8-trichlorodibenzo-p-dioxin ([ 125 I]Cl3DpD) in vivo and the binding of the radioligand to liver homogenate in vitro. The TCDD-induced hepatic binding species was found to be predominantly in the microsomal fraction and was inactivated by heating at 60 degree, trypsin, and mercurials. The TCDD-induced binding species was found to have an apparent equilibrium dissociation constant, KD, ([ 125 I]Cl3DpD) of 56 +/- 16 nM and a pool size, Bmax, of 22 +/- 5 nmol/g of liver. A number of halogenated dibenzo-p-dioxins, biphenyls, and polycyclic aromatic hydrocarbons compete with [ 125 I]Cl3DpD binding to this species; all are aromatic and planar. The distinctive profile of this binding species, a protein of large pool size induced in the microsomal fraction of liver but not other tissues and induced by agonists for the Ah receptor, suggested that this moiety might be cytochrome P3-450. The coincidence of the major microsomal species covalently labeled with the photoaffinity ligand [ 125 I]2-iodo-3-azido-7,8-dibromodibenzo-p-dioxin and that immunochemically stained with polyclonal antiserum that binds to cytochrome P3-450 confirms this hypothesis. This is a novel role for a cytochrome P-450 isozyme, as an induced sequestration site that enhances the hepatic localization of the agonist drug

  18. Synthesis of 25-hydroxyvitamin D3 3β-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether, a second-generation photoaffinity analogue of 25-hydroxyvitamin D3: Photoaffinity labeling of rat serum vitamin D binding protein

    International Nuclear Information System (INIS)

    Ray, R.; Holick, M.F.; Bouillon, R.; Van Baelen, H.

    1991-01-01

    Vulnerability of 25-hydroxy-[26,27- 3 H]vitamin D 3 3β-N-(4-azido-2-nitrophenyl)glycinate, a photoaffinity analogue of 25-hydroxyvitamin D 3 (25-OH-D 3 ) toward standard conditions of carboxymethylationin promoted the authors to synthesize 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitrophenyl)amino]propyl ether (25-ANE), a hydrolytically stable photoaffinity analogue of 25-OH-D 3 , and 25-hydroxyvitamin D 3 3β-3'-[N-(4-azido-2-nitro-[3,5- 3 H]phenyl)amino]propyl ether ( 3 H-25-ANE), the radiolabeled counterpart of 25-ANE competes for the 25-OH-D 3 binding site in rat serum vitamin D binding protein (rDBP). On the other hand, UV exposure of a sample of purified rat DBP (rDBP), preincubated in the dark with 3 H-25-ANE, covalently labeled the protein. However, very little covalent labeling was observed in the absence of UV light or in the presence of a large excess of 25-OH-D 3 . These results provide strong evidence for the covalent labeling of the 25-OH-D 3 binding site in rDPB by 3 H-25-ANE

  19. Vitamin D Receptor Signaling and Cancer.

    Science.gov (United States)

    Campbell, Moray J; Trump, Donald L

    2017-12-01

    The vitamin D receptor (VDR) binds the secosteroid hormone 1,25(OH) 2 D 3 with high affinity and regulates gene programs that control a serum calcium levels, as well as cell proliferation and differentiation. A significant focus has been to exploit the VDR in cancer settings. Although preclinical studies have been strongly encouraging, to date clinical trials have delivered equivocal findings that have paused the clinical translation of these compounds. However, it is entirely possible that mining of genomic data will help to refine precisely what are the key anticancer actions of vitamin D compounds and where these can be used most effectively. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  20. Nuclear triiodothyronine receptor binding characteristics and occupancy in obese (ob/ob) mice

    International Nuclear Information System (INIS)

    Hillgartner, F.B.; Romsos, D.R.

    1987-01-01

    Obese (ob/ob) mice exhibit reduced adaptive thermogenesis associated with an impairment of thyroid hormone action. The mechanism underlying the latter defect was investigated by comparing the binding characteristics and occupancy of solubilized nuclear 3,5,3'-triiodothyronine (T 3 ) receptors from livers of lean and obese mice. T 3 concentration was measured by radioimmunoassay. Scatchard analysis showed minimal differences in B/sub max/ and K/sub d/ between phenotypes at both 4 and 8-10 wk of age, indicating that reduced hepatic thyroid hormone expression in obese mice is not caused by alterations in nuclear receptor concentration or affinity. In contrast, nuclear T 3 receptor occupancy (endogenous T 3 associated with the specific receptor divided by B/sub max/) was 14 and 23% lower in 4- and 8- to 10-wk old obese mice, respectively. Together with reported changes in hepatic thyroid hormone-sensitive enzymes, these data are consistent with a diminished nuclear T 3 signal initiating thyroid hormone action in obese mice. Decreased nuclear T 3 receptor occupancy may be secondary to a low transport of plasma T 3 to the nuclear pool. In conclusion, impaired hepatic thyroid hormone action in obese mice is mediated in part at least by a reduction in nuclear T 3 receptor occupancy

  1. Studies on the characterization and regulation of alpha-1 adrenergic receptors and [3H]WB4101 binding sites in the central nervous system

    International Nuclear Information System (INIS)

    Morrow, A.L.

    1985-01-01

    The purpose of these studies has been to resolve the anomalous binding characteristics of two alpha adrenergic receptor ligands, [ 3 H]WB4101 and [ 3 H]prazosin and to study the regulation of the receptors labeled by these compounds after surgical denervation and chronic drug treatments. Preliminary studies indicated that [ 3 H]WB4101 binding sites, which were believed to represent alpha-1 adrenergic receptors, were increased in number following removal of the fimbrial afferents to the hippocampus. This increase was not due to removal of the adrenergic input into this structure since destruction of the locus coeruleus or the dorsal noradrenergic bundle did not produce the up-regulation. Characterization of alpha-1 adrenergic receptors using [ 3 H]prazosin and [ 3 H]WB4101 revealed evidence for subtypes of alpha-1 receptors designated alpha-1A and alpha-1B. The nanomolar affinity component of [ 3 H]WB4101 binding is not adrenergic but serotonergic. The serotonergic agonists, serotonin and 8-hydroxy-dipropylaminotetraline have affinities of 1.5 and 3.0 nM for this site, when studied in the presence of a 30 nM prazosin mask of the alpha-1 component of [ 3 H]WB4101 binding. Fimbria transection or 5,7 dihydroxytryptamine injections produced increases in the Bmax of the nanomolar affinity component of [ 3 H]WB4101 binding in the presence of a prazosin mask. The up-regulated site showed identical serotonergic pharmacology compared to control tissue. Thus, the author concluded that serotonergic denervation of the hippocampus produces the increase in serotonergic binding sites labeled by [ 3 H]WB4101

  2. 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.

  3. 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

  4. SH3 domain-mediated binding of the Drk protein to Dos is an important step in signaling of Drosophila receptor tyrosine kinases.

    Science.gov (United States)

    Feller, Stephan M; Wecklein, Heike; Lewitzky, Marc; Kibler, Eike; Raabe, Thomas

    2002-08-01

    Activation of the Sevenless (Sev) receptor tyrosine kinase (RTK) in the developing Drosophila eye is required for the specification of the R7 photoreceptor cell fate. Daughter of Sevenless (Dos), a putative multi-site adaptor protein, is a substrate of the Sev kinase and is known to associate with the tyrosine phosphatase Corkscrew (Csw). Binding of Csw to Dos depends on the Csw Src homology 2 (SH2) domains and is an essential step for signaling by the Sev RTK. Dos, however, lacks a recognizable phosphotyrosine interaction domain and it was previously unclear how it is recruited to the Sev receptor. Here it is shown that the SH2/SH3 domain adaptor protein Drk can provide this link. Drk binds with its SH2 domain to the autophosphorylated Sev receptor while the C-terminal SH3 domain is able to associate with Dos. The Drk SH3 domain binding motifs on Dos were mapped to two sites which do not conform the known Drk SH3 domain binding motif (PxxPxR) but instead have the consensus PxxxRxxKP. Mutational analysis in vitro and in vivo provided evidence that both Drk binding sites fulfil an important function in the context of Sev and Drosophila epidermal growth factor receptor mediated signaling processes.

  5. Differential regulation of dopamine receptors after chronic typical and atypical antipsychotic drug treatment

    Energy Technology Data Exchange (ETDEWEB)

    Creese, I; Florijn, W J; Tarazi, F I [Center for Molecular and Behavioral Neuroscience, Rutgers University, 197 University Avenue, Newark, NJ (United States)

    1997-04-14

    Changes in dopamine receptor subtype binding in different brain regions were examined after 28 days treatment of rats with haloperidol, raclopride, clozapine or SCH23390 using in vitro receptor autoradiography. [{sup 3}H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin binding to dopamine D{sub 3} receptors was not changed in any brain region by any of the drug treatments. [{sup 3}H]SCH23390 was only increased by chronic SCH23390 treatment. Haloperidol significantly increased [{sup 3}H]nemonapride and [{sup 3}H]spiperone binding to dopamine D{sub 2}-like receptors in the caudate-putamen. In contrast, haloperidol caused a small, significant increase in [{sup 3}H]raclopride binding in the lateral caudate-putamen only. Raclopride also elevated, but to a lesser extent [{sup 3}H]nemonapride and [{sup 3}H]spiperone binding in caudate-putamen, whereas it did not affect [{sup 3}H]raclopride binding. Clozapine did not significantly change D{sub 2}-like striatal binding of [{sup 3}H]nemonapride, [{sup 3}H]spiperone or [{sup 3}H]raclopride. The differences in radioligand binding suggest that [{sup 3}H]nemonapride and [{sup 3}H]spiperone may be binding to additional subsets of dopamine D{sub 2}-like receptors (including D{sub 4}-like receptors) that are not recognized by [{sup 3}H]raclopride, which has high affinity for D{sub 2} and D{sub 3} receptors only.Quantification of [{sup 3}H]nemonapride or [{sup 3}H]spiperone binding in the presence of 300 nM raclopride (to block D{sub 2} and D{sub 3} receptors) revealed that haloperidol, raclopride and clozapine up-regulated D{sub 4}-like receptors in the caudate-putamen using either radioligand. These results suggest that D{sub 4}-like receptors may be a common site of action of both typical and atypical antipsychotics. (Copyright (c) 1997 Elsevier Science B.V., Amsterdam. All rights reserved.)

  6. Modeling and protein engineering studies of active and inactive states of human dopamine D2 receptor (D2R) and investigation of drug/receptor interactions.

    Science.gov (United States)

    Salmas, Ramin Ekhteiari; Yurtsever, Mine; Stein, Matthias; Durdagi, Serdar

    2015-05-01

    Homology model structures of the dopamine D2 receptor (D2R) were generated starting from the active and inactive states of β2-adrenergic crystal structure templates. To the best of our knowledge, the active conformation of D2R was modeled for the first time in this study. The homology models are built and refined using MODELLER and ROSETTA programs. Top-ranked models have been validated with ligand docking simulations and in silico Alanine-scanning mutagenesis studies. The derived extra-cellular loop region of the protein models is directed toward the binding site cavity which is often involved in ligand binding. The binding sites of protein models were refined using induced fit docking to enable the side-chain refinement during ligand docking simulations. The derived models were then tested using molecular modeling techniques on several marketed drugs for schizophrenia. Alanine-scanning mutagenesis and molecular docking studies gave similar results for marketed drugs tested. We believe that these new D2 receptor models will be very useful for a better understanding of the mechanisms of action of drugs to be targeted to the binding sites of D2Rs and they will contribute significantly to drug design studies involving G-protein-coupled receptors in the future.

  7. 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)

  8. Striatal Dopamine D2/D3 Receptor Availability Is Associated with Executive Function in Healthy Controls but Not Methamphetamine Users.

    Directory of Open Access Journals (Sweden)

    Michael E Ballard

    Full Text Available Dopamine D2/D3 receptor availability in the striatum has been linked with executive function in healthy individuals, and is below control levels among drug addicts, possibly contributing to diminished executive function in the latter group. This study tested for an association of striatal D2/D3 receptor availability with a measure of executive function among research participants who met DSM-IV criteria for methamphetamine dependence.Methamphetamine users and non-user controls (n = 18 per group completed the Wisconsin Card Sorting Test and positron emission tomography with [18F]fallypride.The methamphetamine users displayed significantly lower striatal D2/D3 receptor availability on average than controls after controlling for age and education (p = 0.008, but they did not register greater proportions of either perseverative or non-perseverative errors when controlling for education (both ps ≥ 0.622. The proportion of non-perseverative, but not perseverative, errors was negatively correlated with striatal D2/D3 receptor availability among controls (r = -0.588, p = 0.010, but not methamphetamine users (r = 0.281, p = 0.258, and the group-wise interaction was significant (p = 0.030.These results suggest that cognitive flexibility, as measured by perseverative errors on the Wisconsin Card Sorting Test, is not determined by signaling through striatal D2/D3 receptors in healthy controls, and that in stimulant abusers, who have lower D2/D3 receptor availability, compensation can effectively maintain other executive functions, which are associated with D2/D3 receptor signaling in controls.

  9. 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.

  10. 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

  11. Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1

    NARCIS (Netherlands)

    Zwijsen, R.M.L.; Buckle, R.S.; Hijmans, E.M.; Loomans, C.J.M.; Bernards, R.A.

    1998-01-01

    The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast

  12. 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

  13. Kappa-receptor selective binding of opioid ligands with a heterocyclic bicyclo[3.3.1]nonan-9-one structure.

    Science.gov (United States)

    Benyhe, S; Márki, A; Nachtsheim, Corina; Holzgrabe, Ulrike; Borsodi, Anna

    2003-01-01

    Previous pharmacological results have suggested that members of the heterocyclic bicyclo[3.3.1]nonan-9-one-like compounds are potent kappa-opioid receptor specific agonists. One lead molecule of this series. called compound 1 (dimethyl 7-methyl-2,4-di-2-pyridyl-3.7-diazabicyclo[3.3.1]nonan-9-one-1,5-dicarboxylate) exhibited high affinity for [3H]ethylketocyclazocine and [3H]U-69.593 binding sites in guinea pig cerebellar membranes which known to be a good source for kappa1 receptors. It was shown by molecular modelling that heterocyclic bicyclo[3.3.1]nonan-9-ones fit very well with the structure of ketazocine, a prototypic kappa-selective benzomorphan compound; when compared to the arylacetamide structure of U-69.593, a specific kappa1-receptor agonist, a similar geometry was found with a slightly different distribution of the charges. It is postulated, that the essential structural skeleton involved in the opioid activity is an aryl-propyl-amine element distributed along the N7-C6-C5-C4-aryl bonds.

  14. Binding free energy predictions of farnesoid X receptor (FXR) agonists using a linear interaction energy (LIE) approach with reliability estimation: application to the D3R Grand Challenge 2

    Science.gov (United States)

    Rifai, Eko Aditya; van Dijk, Marc; Vermeulen, Nico P. E.; Geerke, Daan P.

    2018-01-01

    Computational protein binding affinity prediction can play an important role in drug research but performing efficient and accurate binding free energy calculations is still challenging. In the context of phase 2 of the Drug Design Data Resource (D3R) Grand Challenge 2 we used our automated eTOX ALLIES approach to apply the (iterative) linear interaction energy (LIE) method and we evaluated its performance in predicting binding affinities for farnesoid X receptor (FXR) agonists. Efficiency was obtained by our pre-calibrated LIE models and molecular dynamics (MD) simulations at the nanosecond scale, while predictive accuracy was obtained for a small subset of compounds. Using our recently introduced reliability estimation metrics, we could classify predictions with higher confidence by featuring an applicability domain (AD) analysis in combination with protein-ligand interaction profiling. The outcomes of and agreement between our AD and interaction-profile analyses to distinguish and rationalize the performance of our predictions highlighted the relevance of sufficiently exploring protein-ligand interactions during training and it demonstrated the possibility to quantitatively and efficiently evaluate if this is achieved by using simulation data only.

  15. Positron emission tomography with ( sup 18 F)methylspiperone demonstrates D sub 2 dopamine receptor binding differences of clozapine and haloperidol

    Energy Technology Data Exchange (ETDEWEB)

    Karbe, H; Wienhard, K; Huber, M; Herholz, K; Heiss, W D [Klinik fuer Neurologie, Univ. of Koeln, Koeln (Germany); Hamacher, K; Coenen, H H; Stoecklin, G [Inst. fuer Chemie 1, Forschungszentrum Juelich Gmbh (Germany); Loevenich, A [Klinik fuer Psychiatrie, Univ. of Koeln, Koeln (Germany)

    1991-01-01

    Four schizophrenic patients were investigated with dynamic positron emission tomography (PET) using ({sup 18}F)fluorodeoxyglucose (FDG) and ({sup 18}F)methylspiperone (MSP) as tracers. Two schizophrenics were on haloperidol therapy at the time of MSP PET. The other two schizophrenics were treated with clozapine, in one of them MSP PET was carried out twice with different daily doses (100 mg and 450mg respectively). Neuroleptic serum levels were measured in all patients. Results were compared with MSP PET of two drugfree male control subjects and with a previous fluoroethylspiperone (FESP) study of normals. Three hours after tracer injection specific binding of MSP was observed in the striatum in all cases. The striatum to cerebellum ratio was used to estimate the degree of neuroleptic-caused striatal D{sub 2} dopamine receptor occupancy. In the haloperidol treated patients MSP binding was significantly decreased, whereas in the clozapine treated patients striatum to cerebellum ratio was normal. Even the increase of clozapine dose in the same patient had no influence on this ratio. Despite the smaller number of patients the study shows for the first time in humans that striatal MSP binding reflects the different D{sub 2} dopamine receptor affinities of clozapine and haloperidol. (authors).

  16. 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.

  17. (3H)leukotriene B4 binding to the guinea pig spleen membranes: a rich tissue source for a high affinity leukotriene B4 receptor site

    International Nuclear Information System (INIS)

    Cheng, J.B.; Kohi, F.; Townley, R.G.

    1986-01-01

    To select a tissue rich for the high affinity leukotriene (LT)B 4 receptor site, they compared binding of 1 nM ( 3 H)LTB 4 (180 Ci/mmol) to the crude membrane preparations of guinea pig spleen, thymus, lung, uterus, bladder, brain, adrenal gland, small intestine, liver, kidney and heart. They found that the membrane preparations from spleen contained the highest binding activity per mg protein. They characterized the LTB 4 binding to the spleen preparation in detail. LTB 4 binding was rapid, reversible, stereoselective and saturable. The data from equilibrium experiments showed a linear Scatchard plot with a K/sub d/ of 1.6 nM and a binding site density of 259 fmol/mg prot. The rank order of agents competing for spleen ( 3 H)LTB 4 binding at 25 0 C was: LTB 4 (K/sub i/ = 2.8 nM) > 20-OH-LTB 4 (23 nM) > LTA 4 (48 nM) > LTA 4 methyl ester (0.13 μM) > 20-COOH-LTB 4 (> 6.6 μM) ≥ arachidonic acid (0.15 mM) similarly ordered FPL-55,712 (0.11 mM). At 4 0 C, LTB 4 (2.3 nM) competed at least 10x more effectively than 20-OH-LTB 4 (29 nM) and 20-COOH-LTB 4 (> 6.6 μM). HPLC analysis indicated that incubation of 84 ng LTB 4 with the spleen membrane at 25 0 C did not result in the formation of 20-OH-LTB 4 ( 3 H)LTB 4 receptor binding sites

  18. Characterisation of the binding of [3H]methyllycaconitine: a new radioligand for labelling α7-type neuronal nicotinic acetylcholine receptors

    International Nuclear Information System (INIS)

    Davies, A.R.L.; Wolstenholme, A.J.; Wonnacott, S.; Hardick, D.J.; Blagbrough, I.S.; Potter, B.V.L.

    1999-01-01

    Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for αbungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [ 3 H]MLA bound to rat brain membranes with high affinity (K d =1.86±0.31 nM) with a good ratio of specific to non-specific binding. The binding of [ 3 H]MLA was characterised by rapid association (t 1/2 =2.3 min) and dissociation (t 1/2 =12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with αbungarotoxin-sensitive nAChR. The snake α-toxins, αbungarotoxin and αcobratoxin, displaced [ 3 H]MLA with high affinity (K i =1.8±0.5 and 5.5±0.9 nM, respectively), whereas nicotine was less potent (K i =6.1±1.1 μM). The distribution of [ 3 H]MLA binding sites in crudely dissected rat brain regions was identical to that of [ 125 I]αbungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [ 3 H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake αtoxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [ 3 H]MLA, therefore, is a novel radiolabel for characterising α7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled αbungarotoxin for rapid and accurate equilibrium binding assays. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  19. Functional Selectivity of Allosteric Interactions within G Protein–Coupled Receptor Oligomers: The Dopamine D1-D3 Receptor Heterotetramer

    Science.gov (United States)

    Guitart, Xavier; Navarro, Gemma; Moreno, Estefania; Yano, Hideaki; Cai, Ning-Sheng; Sánchez-Soto, Marta; Kumar-Barodia, Sandeep; Naidu, Yamini T.; Mallol, Josefa; Cortés, Antoni; Lluís, Carme; Canela, Enric I.; Casadó, Vicent; McCormick, Peter J.

    2014-01-01

    The dopamine D1 receptor–D3 receptor (D1R-D3R) heteromer is being considered as a potential therapeutic target for neuropsychiatric disorders. Previous studies suggested that this heteromer could be involved in the ability of D3R agonists to potentiate locomotor activation induced by D1R agonists. It has also been postulated that its overexpression plays a role in L-dopa–induced dyskinesia and in drug addiction. However, little is known about its biochemical properties. By combining bioluminescence resonance energy transfer, bimolecular complementation techniques, and cell-signaling experiments in transfected cells, evidence was obtained for a tetrameric stoichiometry of the D1R–D3R heteromer, constituted by two interacting D1R and D3R homodimers coupled to Gs and Gi proteins, respectively. Coactivation of both receptors led to the canonical negative interaction at the level of adenylyl cyclase signaling, to a strong recruitment of β-arrestin-1, and to a positive cross talk of D1R and D3R agonists at the level of mitogen-activated protein kinase (MAPK) signaling. Furthermore, D1R or D3R antagonists counteracted β-arrestin-1 recruitment and MAPK activation induced by D3R and D1R agonists, respectively (cross-antagonism). Positive cross talk and cross-antagonism at the MAPK level were counteracted by specific synthetic peptides with amino acid sequences corresponding to D1R transmembrane (TM) domains TM5 and TM6, which also selectively modified the quaternary structure of the D1R-D3R heteromer, as demonstrated by complementation of hemiproteins of yellow fluorescence protein fused to D1R and D3R. These results demonstrate functional selectivity of allosteric modulations within the D1R-D3R heteromer, which can be involved with the reported behavioral synergism of D1R and D3R agonists. PMID:25097189

  20. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor-binding domain of botulinum neurotoxin serotype D

    International Nuclear Information System (INIS)

    Zhang, Yanfeng; Gao, Xiaoli; Qin, Ling; Buchko, Garry W.; Robinson, Howard; Varnum, Susan M.

    2010-01-01

    The receptor-binding domain of botulinum neurotoxin serotype D was expressed in E. coli using a codon-optimized cDNA. The highly purified protein crystallized in space group P2 1 2 1 2 1 , with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 Å, and the crystals diffracted to 1.65 Å resolution. Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D-HCR was expressed at a high level (150–200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D-HCR was obtained. The recombinant BoNT/D-HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belonged to space group P2 1 2 1 2 1 , with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 Å. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit

  1. Photoaffinity labelling of high affinity dopamine binding proteins

    International Nuclear Information System (INIS)

    Ross, G.M.; McCarry, B.E.; Mishra, R.K.

    1986-01-01

    A photoactive analogue of the dopamine agonist 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronapthalene (ADTN) has been synthesized and used to photoaffinity label dopamine binding proteins prepared from bovine caudate nucleus. N-(3-]N'-4-azidobenzamidol]-aminopropyl)-aminopropyl)-ADTN (AzB-AP-ADTN) was incubated with caudate membranes and irradiated with UV light. Membranes were then repeatedly washed by centrifugation to remove excess photolabel. A binding assay, using ( 3 H)-SCH 23390 (a D 1 specific antagonist), was then performed to evaluate the loss of receptor density in the photolyzed preparation. AzB-AP-ADTN irreversibly blocked ( 3 H)-SCH 23390 binding in a dose-dependent manner. Scatchard analysis revealed a decrease in the B/sub max/, with no significant change in the K/sub d/, of ( 3 H)-SCH 23390 binding. Compounds which compete for D 1 receptor binding (such as dopamine, SKF 38393 or apomorphine), proteted the SCH 23390 binding site from inactivation. This data would suggest that the novel photoaffinity ligand, AzB-AP-ADTN, can covalently label the D 1 (adenylate cyclase linked) dopamine receptor

  2. ( sup 3 H)opipramol labels a novel binding site and sigma receptors in rat brain membranes

    Energy Technology Data Exchange (ETDEWEB)

    Ferris, C.D.; Hirsch, D.J.; Brooks, B.P.; Snowman, A.M.; Snyder, S.H. (Johns Hopkins Univ. School of Medicine, Baltimore, MD (USA))

    1991-02-01

    Opipramol (OP), a clinically effective antidepressant with a tricyclic structure, is inactive as an inhibitor of biogenic amine uptake. ({sup 3}H)Opipramol binds saturably to rat brain membranes (apparent KD = 4 nM, Bmax = 3 pmol/mg of protein). ({sup 3}H)Opipramol binding can be differentiated into haloperidol-sensitive and -resistant components, with Ki values for haloperidol of 1 nM (Bmax = 1 pmol/mg of protein) and 350 nM (Bmax = 1.9 pmol/mg of protein), respectively. The drug specificity of the haloperidol-sensitive component is the same as that of sigma receptors labeled with (+)-({sup 3}H)3-(3-hydroxyphenyl)-N-(1-propyl)piperdine. The haloperidol-resistant component does not correspond to any known neurotransmitter receptor or uptake recognition site. It displays high affinity for phenothiazines and related structures such as perphenazine, clopenthixol, and flupenthixol, whose potencies are comparable to that of opipramol. Because certain of these drugs are more potent at the haloperidol-resistant opipramol site than in exerting any other action, it is possible that this opipramol-selective site may mediate their therapeutic effects.

  3. 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

  4. 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

  5. Cloning retinoid and peroxisome proliferator-activated nuclear receptors of the Pacific oyster and in silico binding to environmental chemicals.

    Directory of Open Access Journals (Sweden)

    Susanne Vogeler

    Full Text Available Disruption of nuclear receptors, a transcription factor superfamily regulating gene expression in animals, is one proposed mechanism through which pollution causes effects in aquatic invertebrates. Environmental pollutants have the ability to interfere with the receptor's functions through direct binding and inducing incorrect signals. Limited knowledge of invertebrate endocrinology and molecular regulatory mechanisms, however, impede the understanding of endocrine disruptive effects in many aquatic invertebrate species. Here, we isolated three nuclear receptors of the Pacific oyster, Crassostrea gigas: two isoforms of the retinoid X receptor, CgRXR-1 and CgRXR-2, a retinoic acid receptor ortholog CgRAR, and a peroxisome proliferator-activated receptor ortholog CgPPAR. Computer modelling of the receptors based on 3D crystal structures of human proteins was used to predict each receptor's ability to bind to different ligands in silico. CgRXR showed high potential to bind and be activated by 9-cis retinoic acid and the organotin tributyltin (TBT. Computer modelling of CgRAR revealed six residues in the ligand binding domain, which prevent the successful interaction with natural and synthetic retinoid ligands. This supports an existing theory of loss of retinoid binding in molluscan RARs. Modelling of CgPPAR was less reliable due to high discrepancies in sequence to its human ortholog. Yet, there are suggestions of binding to TBT, but not to rosiglitazone. The effect of potential receptor ligands on early oyster development was assessed after 24h of chemical exposure. TBT oxide (0.2μg/l, all-trans retinoic acid (ATRA (0.06 mg/L and perfluorooctanoic acid (20 mg/L showed high effects on development (>74% abnormal developed D-shelled larvae, while rosiglitazone (40 mg/L showed no effect. The results are discussed in relation to a putative direct (TBT disruption effect on nuclear receptors. The inability of direct binding of ATRA to CgRAR suggests

  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. Agonist and antagonist actions of antipsychotic agents at 5-HT1A receptors: a [35S]GTPgammaS binding study.

    Science.gov (United States)

    Newman-Tancredi, A; Gavaudan, S; Conte, C; Chaput, C; Touzard, M; Verrièle, L; Audinot, V; Millan, M J

    1998-08-21

    Recombinant human (h) 5-HT1A receptor-mediated G-protein activation was characterised in membranes of transfected Chinese hamster ovary (CHO) cells by use of guanosine-5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS binding). The potency and efficacy of 21 5-HT receptor agonists and antagonists was determined. The agonists, 5-CT (carboxamidotryptamine) and flesinoxan displayed high affinity (subnanomolar Ki values) and high efficacy (Emax > 90%, relative to 5-HT = 100%). In contrast, ipsapirone, zalospirone and buspirone displayed partial agonist activity. EC50s for agonist stimulation of [35S]GTPgammaS binding correlated well with Ki values from competition binding (r = +0.99). Among the compounds tested for antagonist activity, methiothepin and (+)butaclamol exhibited 'inverse agonist' behaviour, inhibiting basal [35S]GTPgammaS binding. The actions of 17 antipsychotic agents were investigated. Clozapine and several putatively 'atypical' antipsychotic agents, including ziprasidone, quetiapine and tiospirone, exhibited partial agonist activity and marked affinity at h5-HT1A receptors, similar to their affinity at hD2 dopamine receptors. In contrast, risperidone and sertindole displayed low affinity at h5-HT1A receptors and behaved as 'neutral' antagonists, inhibiting 5-HT-stimulated [35S]GTPgammaS binding. Likewise the 'typical' neuroleptics, haloperidol, pimozide, raclopride and chlorpromazine exhibited relatively low affinity and 'neutral' antagonist activity at h5-HT1A receptors with Ki values which correlated with their respective Kb values. The present data show that (i) [35S]GTPgammaS binding is an effective method to evaluate the efficacy and potency of agonists and antagonists at recombinant human 5-HT1A receptors. (ii) Like clozapine, several putatively 'atypical' antipsychotic drugs display balanced serotonin h5-HT1A/dopamine hD2 receptor affinity and partial agonist activity at h5-HT1A receptors. (iii) Several 'typical' and some putatively 'atypical

  8. Molecular mechanism of AMD3100 antagonism in the CXCR4 receptor: transfer of binding site to the CXCR3 receptor

    DEFF Research Database (Denmark)

    Rosenkilde, Mette M; Gerlach, Lars-Ole; Jakobsen, Janus S

    2004-01-01

    , respectively. Metal ion binding in the cyclam rings of AMD3100 increased its dependence on Asp(262) and provided a tighter molecular map of the binding site, where borderline mutational hits became clear hits for the Zn(II)-loaded analog. The proposed binding site for AMD3100 was confirmed by a gradual build......-up in the rather distinct CXCR3 receptor, for which the compound normally had no effect. Introduction of only a Glu at position VII:06 and the removal of a neutralizing Lys residue at position VII:02 resulted in a 1000-fold increase in affinity of AMD3100 to within 10-fold of its affinity in CXCR4. We conclude...

  9. 3D structure of muscle dihydropyridine receptor

    Directory of Open Access Journals (Sweden)

    Montserrat Samsó

    2015-01-01

    Full Text Available Excitation contraction coupling, the rapid and massive Ca2+ release under control of an action potential that triggers muscle contraction, takes places at specialized regions of the cell called triad junctions. There, a highly ordered supramolecular complex between the dihydropyridine receptor (DHPR and the ryanodine receptor (RyR1 mediates the quasi‐instantaneous conversion from T‐tubule depolarization into Ca2+ release from the sarcoplasmic reticulum (SR. The DHPR has several key modules required for EC coupling: the voltage sensors and II‐III loop in the alpha1s subunit, and the beta subunit. To gain insight into their molecular organization, this review examines the most updated 3D structure of the DHPR as obtained by transmission electron microscopy and image reconstruction. Although structure determination of a heteromeric membrane protein such as the DHPR is challenging, novel technical advances in protein expression and 3D labeling facilitated this task. The 3D structure of the DHPR complex consists of a main body with five irregular corners around its perimeter encompassing the transmembrane alpha 1s subunit besides the intracellular beta subunit, an extended extracellular alpha 2 subunit, and a bulky intracellular II‐III loop. The structural definition attained at 19 Å resolution enabled docking of the atomic coordinates of structural homologs of the alpha1s and beta subunits. These structural features, together with their relative location with respect to the RyR1, are discussed in the context of the functional data.

  10. Capacity for cooperative binding of thyroid hormone (T3) receptor dimers defines wild type T3 response elements.

    Science.gov (United States)

    Brent, G A; Williams, G R; Harney, J W; Forman, B M; Samuels, H H; Moore, D D; Larsen, P R

    1992-04-01

    Thyroid hormone response elements (T3REs) have been identified in a variety of promoters including those directing expression of rat GH (rGH), alpha-myosin heavy chain (rMHC), and malic enzyme (rME). A detailed biochemical and genetic analysis of the rGH element has shown that it consists of three hexamers related to the consensus [(A/G)GGT(C/A)A]. We have extended this analysis to the rMHC and rME elements. Binding of highly purified thyroid hormone receptor (T3R) to T3REs was determined using the gel shift assay, and thyroid hormone (T3) induction was measured in transient tranfections. We show that the wild type version of each of the three elements binds T3R dimers cooperatively. Mutational analysis of the rMHC and rME elements identified domains important for binding T3R dimers and allowed a direct determination of the relationship between T3R binding and function. In each element two hexamers are required for dimer binding, and mutations that interfere with dimer formation significantly reduce T3 induction. Similar to the rGH element, the rMHC T3RE contains three hexameric domains arranged as a direct repeat followed by an inverted copy, although the third domain is weaker than in rGH. All three are required for full function and T3R binding. The rME T3RE is a two-hexamer direct repeat T3RE, which also binds T3R monomer and dimer. Across a series of mutant elements, there was a strong correlation between dimer binding in vitro and function in vivo for rMHC (r = 0.99, P less than 0.01) and rME (r = 0.67, P less than 0.05) T3REs. Our results demonstrate a similar pattern of T3R dimer binding to a diverse array of hexameric sequences and arrangements in three wild type T3REs. Addition of nuclear protein enhanced T3R binding but did not alter the specificity of binding to wild type or mutant elements. Binding of purified T3R to T3REs was highly correlated with function, both with and without the addition of nuclear protein. T3R dimer formation is the common

  11. Binding affinities of the farnesoid X receptor in the D3R Grand Challenge 2 estimated by free-energy perturbation and docking

    Science.gov (United States)

    Olsson, Martin A.; García-Sosa, Alfonso T.; Ryde, Ulf

    2018-01-01

    We have studied the binding of 102 ligands to the farnesoid X receptor within the D3R Grand Challenge 2016 blind-prediction competition. First, we employed docking with five different docking software and scoring functions. The selected docked poses gave an average root-mean-squared deviation of 4.2 Å. Consensus scoring gave decent results with a Kendall's τ of 0.26 ± 0.06 and a Spearman's ρ of 0.41 ± 0.08. For a subset of 33 ligands, we calculated relative binding free energies with free-energy perturbation. Five transformations between the ligands involved a change of the net charge and we implemented and benchmarked a semi-analytic correction (Rocklin et al., J Chem Phys 139:184103, 2013) for artifacts caused by the periodic boundary conditions and Ewald summation. The results gave a mean absolute deviation of 7.5 kJ/mol compared to the experimental estimates and a correlation coefficient of R 2 = 0.1. These results were among the four best in this competition out of 22 submissions. The charge corrections were significant (7-8 kJ/mol) and always improved the results. By employing 23 intermediate states in the free-energy perturbation, there was a proper overlap between all states and the precision was 0.1-0.7 kJ/mol. However, thermodynamic cycles indicate that the sampling was insufficient in some of the perturbations.

  12. The effect of infectious brain edema on NMDA receptor binding in rat's brain

    International Nuclear Information System (INIS)

    Cheng Guansheng; Chen Jianfang; Chen Xiang

    1997-01-01

    PURPOSE: The effect of the infectious brain edema (IBE) induced by Bordetella Pertussis (BP) on the specific binding of 3 H MK-801 in rat's brain in vivo was determined. METHODS: BP was injected via left internal carotid artery in rat model of infectious brain edema. Male SD rats were divided into three groups: 1) Group control (NS, n = 11); 2) Group IBF (BP, n = 12); 3) Group pretreatment of MK-801 + PB (MK-801, n = 4). Normal saline or BP 0.2 ml/kg was injected into left internal carotid artery in NS and BP group respectively. MK-801 0.5 mg/kg per day was injected i.p. two days before injection of BP in group MK-801. Rats were killed by decapitation at 24 hours after injection of BP. The specific binding of N-methyl-D-aspartate (NMDA) receptor were measured with 3 H-MK-801 in the neuronal membrane of cerebral cortex. The Scatchard plots were performed. RESULTS: The B max values were 0.623 +- 0.082 and 0.606 +- 0.087 pmol/mg protein in group NS and BP respectively (t = 0.48, P>0.05). The Kd values were 43.1 +- 4.2 and 30.5 +- 3.0 nmol/L in group NS and BP respectively (t = 7.8, P<0.05). The specific binding of NMDA receptor was decreased by pretreatment of MK-801. CONCLUSIONS: The total number of NMDA receptor had not changed, whereas its affinity increased significantly in the model of brain edema induced by pertussis bacilli in rat. The increase of affinity of NMDA receptor can be blockaded by MK-801 pretreatment in vivo

  13. 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

  14. Association study between schizophrenia and dopamine D3 receptor gene polymorphism

    Energy Technology Data Exchange (ETDEWEB)

    Tanaka, Toshihisa; Takahashi, Makoto; Maeda, Masaya [Niigata Univ. (Japan)] [and others

    1996-07-26

    Crocq et al. reported the existence of an association between schizophrenia and homozygosity of a BalI polymorphism in the first exon of the dopamine D3 receptor (DRD3) gene. In response to this report, further studies were conducted; however, these studies yielded conflicting results. In the present study, we examined 100 unrelated Japanese schizophrenics and 100 normal controls to determine any association between this polymorphism and schizophrenia. Results suggest that neither allele nor genotype frequencies of the DRD3 gene in the schizophrenics as a whole are significantly different from those of the controls. Further, we found no association between any allele or genotype and any clinical subtype based on family history of schizophrenia and age-at-onset. A significantly high frequency of homozygosity of a dopamine D3 receptor gene allele was not observed in the schizophrenics as a whole, or in clinical subtypes. Our results suggest that an association between the dopamine D3 receptor gene and schizophrenia is unlikely to exist. 26 refs., 1 tab.

  15. Predicting the affinity of Farnesoid X Receptor ligands through a hierarchical ranking protocol: a D3R Grand Challenge 2 case study

    Science.gov (United States)

    Réau, Manon; Langenfeld, Florent; Zagury, Jean-François; Montes, Matthieu

    2018-01-01

    The Drug Design Data Resource (D3R) Grand Challenges are blind contests organized to assess the state-of-the-art methods accuracy in predicting binding modes and relative binding free energies of experimentally validated ligands for a given target. The second stage of the D3R Grand Challenge 2 (GC2) was focused on ranking 102 compounds according to their predicted affinity for Farnesoid X Receptor. In this task, our workflow was ranked 5th out of the 77 submissions in the structure-based category. Our strategy consisted in (1) a combination of molecular docking using AutoDock 4.2 and manual edition of available structures for binding poses generation using SeeSAR, (2) the use of HYDE scoring for pose selection, and (3) a hierarchical ranking using HYDE and MM/GBSA. In this report, we detail our pose generation and ligands ranking protocols and provide guidelines to be used in a prospective computer aided drug design program.

  16. 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)

  17. Characterization with 3H-haloperidol of the dopamine receptor in the rat kidney particulate preparation

    International Nuclear Information System (INIS)

    Nakajima, Tohru; Kuruma, Isami

    1980-01-01

    The dopamine receptor of rat kidney particulate preparation was identified and characterized by the use of 3 H-haloperidol binding. Binding of 3 H-haloperidol to the kidney particulate preparation was slow and saturable. The dissociation constants (K sub(D)) were 0.41 nM and 5.88 nM, respectively, according to the model of two classes of independent binding sites. Maximal binding of high affinity site was obtained with 166 fmole/mg protein which was about 40% of the total receptor density. A wide variety of neuroleptics at specifically low concentrations in nanomolar range inhibited the 3 H-haloperidol binding. There was an excellent correlation between the affinity of numerous neuroleptics for the kidney particulate preparation and that for the brain striatum. (author)

  18. 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.

  19. [3H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

    International Nuclear Information System (INIS)

    Benke, D.; Honer, M.; Mohler, H.; Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C.

    1999-01-01

    Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [ 3 H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [ 3 H]CGP 61594 labeled with high affinity (K D =23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [ 3 H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [ 3 H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [ 3 H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  20. Association between the dopamine D3 receptor gene locus (DRD3) and unipolar affective disorder.

    Science.gov (United States)

    Dikeos, D G; Papadimitriou, G N; Avramopoulos, D; Karadima, G; Daskalopoulou, E G; Souery, D; Mendlewicz, J; Vassilopoulos, D; Stefanis, C N

    1999-12-01

    Dopamine neurotransmission has been implicated in the pathophysiology of schizophrenia and, more recently, affective disorders. Among the dopamine receptors, D3 can be considered as particularly related to affective disorders due to its neuroanatomical localization in the limbic region of the brain and its relation to the serotoninergic activity of the CNS. The possible involvement of dopamine receptor D3 in unipolar (UP) major depression was investigated by a genetic association study of the D3 receptor gene locus (DRD3) on 36 UP patients and 38 ethnically matched controls. An allelic association of DRD3 (Bal I polymorphism) and UP illness was observed, with the Gly-9 allele (allele '2', 206/98 base-pairs long) being more frequent in patients than in controls (49% vs 29%, P < 0.02). The genotypes containing this allele (1-2 and 2-2) were found in 75% of patients vs 50% of controls (P < 0.03, odds ratio = 3.00, 95% CI = 1.12-8.05). The effect of the genotype remained significant (P < 0.02) after sex and family history were controlled by a multiple linear regression analysis. These results further support the hypothesis that dopaminergic mechanisms may be implicated in the pathogenesis of affective disorder. More specifically, the '2' allele of the dopamine receptor D3 gene seems to be associated with unipolar depression and can be considered as a 'phenotypic modifier' for major psychiatric disorders.

  1. Binding of 3H-actinomycin D with polytene chromosomes of Drosophila melanogaster

    International Nuclear Information System (INIS)

    Lakhotia, S.C.

    1976-01-01

    Binding of 3 H-AMD (actinomycin D) in different regions of polytene nuclei of late third instar larvae of Drosophila melanogaster has been examined by EM autoradiography. It is observed that the binding capacity of 3 H-AMD is not related to the transcribing activity of a given region of nuclei, but it may be related to the DNA content. (M.G.B.)

  2. Myocardial slice: a physiological approach to beta-adrenergic ([3H] CGP-12177) receptor binding in hamster and guinea pig heart.

    Science.gov (United States)

    Watson-Wright, W M; Armour, J A; Johnstone, D E; Wilkinson, M

    1989-08-01

    A new technique is described for the characterization and quantification of beta-adrenergic receptors in biologically viable slices of myocardium from the hamster right ventricle using the hydrophilic radioligand, [3H]CGP-12177 (CGP). Binding was stereospecific, saturable, of high affinity, reversible, displaceable by appropriate drugs, and highly positively correlated with increasing tissue concentrations. Bmax for CGP binding to myocardial slices from 50-day-old male Golden Syrian hamsters was 3.28 +/- 0.15 fmol/mg wet weight, while Kd was 0.21 +/- 0.02 nM. Freezing resulted in a close to 50% loss of receptor number with no apparent change in affinity. The slice preparation may be utilized to detect in vivo changes in myocardial cell surface receptors, as evidenced by the fact that the number of receptors in slices from ischemic guinea pigs was increased (Bmax = 15.5 +/- 1.25 fmol/mg wet wt) compared with sham-operated controls (Bmax = 10.4 +/- 0.38 fmol/mg wet wt). The minimal tissue disruption associated with this procedure, as well as its speed, simplicity, and relatively low cost, suggest that the myocardial slice preparation provides an important methodology for the study of beta-adrenergic receptor binding in the semiintact myocardium.

  3. Elevated dopamine D1 receptor availability in striatum of Göttingen minipigs after electroconvulsive therapy

    DEFF Research Database (Denmark)

    Landau, Anne M; Alstrup, Aage Ko; Audrain, Helene

    2018-01-01

    Electroconvulsive therapy (ECT), a direct form of brain stimulation, is an effective antidepressant. We hypothesized that the beneficial effects of ECT are mediated by increased dopaminergic neurotransmission, in which the baseline activity of D1 receptors may predict the response to ECT. We esta......, the baseline binding capacity of D1 receptors predicts the magnitude of increased binding, up to a maximum binding capacity....

  4. Characterisation of the binding of [{sup 3}H]methyllycaconitine: a new radioligand for labelling {alpha}7-type neuronal nicotinic acetylcholine receptors

    Energy Technology Data Exchange (ETDEWEB)

    Davies, A.R.L.; Wolstenholme, A.J.; Wonnacott, S. [Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY (United Kingdom); Hardick, D.J.; Blagbrough, I.S.; Potter, B.V.L. [Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY (United Kingdom)

    1999-05-15

    Methyllycaconitine (MLA), a norditerpenoid alkaloid isolated from Delphinium seeds, is one of the most potent non-proteinacious ligands that is selective for {alpha}bungarotoxin-sensitive neuronal nicotinic acetylcholine receptors (nAChR). [{sup 3}H]MLA bound to rat brain membranes with high affinity (K{sub d}=1.86{+-}0.31 nM) with a good ratio of specific to non-specific binding. The binding of [{sup 3}H]MLA was characterised by rapid association (t{sub 1/2}=2.3 min) and dissociation (t{sub 1/2}=12.6 min) kinetics. The radioligand binding displayed nicotinic pharmacology, consistent with an interaction with {alpha}bungarotoxin-sensitive nAChR. The snake {alpha}-toxins, {alpha}bungarotoxin and {alpha}cobratoxin, displaced [{sup 3}H]MLA with high affinity (K{sub i}=1.8{+-}0.5 and 5.5{+-}0.9 nM, respectively), whereas nicotine was less potent (K{sub i}=6.1{+-}1.1 {mu}M). The distribution of [{sup 3}H]MLA binding sites in crudely dissected rat brain regions was identical to that of [{sup 125}I]{alpha}bungarotoxin binding sites, with a high binding site density in hippocampus and hypothalamus, but low density in striatum and cerebellum. [{sup 3}H]MLA also labelled a sub-population of binding sites which are not sensitive to the snake {alpha}toxins, but which did not differ significantly from the major population with respect to their other pharmacological properties or regional distribution. [{sup 3}H]MLA, therefore, is a novel radiolabel for characterising {alpha}7-type nAChR. A good signal to noise ratio and rapid binding kinetics provide advantages over the use of radiolabelled {alpha}bungarotoxin for rapid and accurate equilibrium binding assays. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  5. Examining the role of dopamine D2 and D3 receptors in Pavlovian conditioned approach behaviors.

    Science.gov (United States)

    Fraser, Kurt M; Haight, Joshua L; Gardner, Eliot L; Flagel, Shelly B

    2016-05-15

    Elucidating the neurobiological mechanisms underlying individual differences in the extent to which reward cues acquire the ability to act as incentive stimuli may contribute to the development of successful treatments for addiction and related disorders. We used the sign-tracker/goal-tracker animal model to examine the role of dopamine D2 and D3 receptors in the propensity to attribute incentive salience to reward cues. Following Pavlovian training, wherein a discrete lever-cue was paired with food reward, rats were classified as sign- or goal-trackers based on the resultant conditioned response. We examined the effects of D2/D3 agonists, 7-OH-DPAT (0.01-0.32mg/kg) or pramipexole (0.032-0.32mg/kg), the D2/D3 antagonist raclopride (0.1mg/kg), and the selective D3 antagonist, SB-277011A (6 or 24mg/kg), on the expression of sign- and goal-tracking conditioned responses. The lever-cue acquired predictive value and elicited a conditioned response for sign- and goal-trackers, but only for sign-trackers did it also acquire incentive value. Following administration of either 7-OH-DPAT, pramipexole, or raclopride, the performance of the previously acquired conditioned response was attenuated for both sign- and goal-trackers. For sign-trackers, the D2/D3 agonist, 7-OH-DPAT, also attenuated the conditioned reinforcing properties of the lever-cue. The selective D3 antagonist did not affect either conditioned response. Alterations in D2/D3 receptor signaling, but not D3 signaling alone, transiently attenuate a previously acquired Pavlovian conditioned response, regardless of whether the response is a result of incentive motivational processes. These findings suggest activity at the dopamine D2 receptor is critical for a reward cue to maintain either its incentive or predictive qualities. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Examining the Role of Dopamine D2 and D3 Receptors in Pavlovian Conditioned Approach Behaviors

    Science.gov (United States)

    Fraser, Kurt M.; Haight, Joshua L.; Gardner, Eliot L.; Flagel, Shelly B.

    2016-01-01

    Elucidating the neurobiological mechanisms underlying individual differences in the extent to which reward cues acquire the ability to act as incentive stimuli may contribute to the development of successful treatments for addiction and related disorders. We used the sign-tracker/goal-tracker animal model to examine the role of dopamine D2 and D3 receptors in the propensity to attribute incentive salience to reward cues. Following Pavlovian training, wherein a discrete lever-cue was paired with food reward, rats were classified as sign- or goal-trackers based on the resultant conditioned response. We examined the effects of D2/D3 agonists, 7-OH-DPAT (0.01–0.32 mg/kg) or pramipexole (0.032–0.32 mg/kg), the D2/D3 antagonist raclopride (0.1 mg/kg), and the selective D3 antagonist, SB-277011A (6 or 24 mg/kg), on the expression of sign- and goal-tracking conditioned responses. The lever-cue acquired predictive value and elicited a conditioned response for sign- and goal-trackers, but only for sign-trackers did it also acquire incentive value. Following administration of either 7-OH-DPAT, pramipexole, or raclopride, the performance of the previously acquired conditioned response was attenuated for both sign- and goal-trackers. For sign-trackers, the D2/D3 agonist, 7-OH-DPAT, also attenuated the conditioned reinforcing properties of the lever-cue. The selective D3 antagonist did not affect either conditioned response. Alterations in D2/D3 receptor signaling, but not D3 signaling alone, transiently attenuate a previously acquired Pavlovian conditioned response, regardless of whether the response is a result of incentive motivational processes. These findings suggest activity at the dopamine D2 receptor is critical for a reward cue to maintain either its incentive or predictive qualities. PMID:26909847

  7. Dopamine D3 receptor ligands for drug addiction treatment: update on recent findings.

    Science.gov (United States)

    Le Foll, Bernard; Collo, Ginetta; Rabiner, Eugenii A; Boileau, Isabelle; Merlo Pich, Emilio; Sokoloff, Pierre

    2014-01-01

    The dopamine D3 receptor is located in the limbic area and apparently mediates selective effects on motivation to take drugs and drug-seeking behaviors, so that there has been considerable interest on the possible use of D3 receptor ligands to treat drug addiction. However, only recently selective tools allowing studying this receptor have been developed. This chapter presents an overview of findings that were presented at a symposium on the conference Dopamine 2013 in Sardinia in May 2013. Novel neurobiological findings indicate that drugs of abuse can lead to significant structural plasticity in rodent brain and that this is dependent on the availability of functional dopamine D3 autoreceptor, whose activation increased phosphorylation in the ERK pathway and in the Akt/mTORC1 pathway indicating the parallel engagement of a series of intracellular signaling pathways all involved in cell growth and survival. Preclinical findings using animal models of drug-seeking behaviors confirm that D3 antagonists have a promising profile to treat drug addiction across drugs of abuse type. Imaging the D3 is now feasible in human subjects. Notably, the development of (+)-4-propyl-9-hydroxynaphthoxazine ligand used in positron emission tomography (PET) studies in humans allows to measure D3 and D2 receptors based on the area of the brain under study. This PET ligand has been used to confirm up-regulation of D3 sites in psychostimulant users and to reveal that tobacco smoking produces elevation of dopamine at the level of D3 sites. There are now novel antagonists being developed, but also old drugs such as buspirone, that are available to test the D3 hypothesis in humans. The first results of clinical investigations are now being provided. Overall, those recent findings support further exploration of D3 ligands to treat drug addiction. © 2014 Elsevier B.V. All rights reserved.

  8. Dopamine D3 receptors mediate the discriminative stimulus effects of quinpirole in free-feeding rats.

    Science.gov (United States)

    Baladi, Michelle G; Newman, Amy H; France, Charles P

    2010-01-01

    The discriminative stimulus effects of dopamine (DA) D3/D2 receptor agonists are thought to be mediated by D2 receptors. To maintain responding, access to food is often restricted, which can alter neurochemical and behavioral effects of drugs acting on DA systems. This study established stimulus control with quinpirole in free-feeding rats and tested the ability of agonists to mimic and antagonists to attenuate the effects of quinpirole. The same antagonists were studied for their ability to attenuate quinpirole-induced yawning and hypothermia. DA receptor agonists apomorphine and lisuride, but not amphetamine and morphine, occasioned responding on the quinpirole lever. The discriminative stimulus effects of quinpirole were attenuated by the D3 receptor-selective antagonist N-{4-[4-(2,3-dichlorophenyl)-piperazin-1-yl]-trans-but-2-enyl}-4-pyridine-2-yl-benzamide HCl (PG01037) and the nonselective D3/D2 receptor antagonist raclopride, but not by the D2 receptor-selective antagonist 3-[4-(4-chlorophenyl)-4-hydroxypiperidin-1-yl]methyl-1H-indole (L-741,626); the potencies of PG01037 and raclopride to antagonize this effect of quinpirole paralleled their potencies to antagonize the ascending limb of the quinpirole yawning dose-response curve (thought to be mediated by D3 receptors). L-741,626 selectively antagonized the descending limb of the quinpirole yawning dose-response curve, and both L-741,626 and raclopride, but not PG01037, antagonized the hypothermic effects of quinpirole (thought to be mediated by D2 receptors). Food restriction (10 g/day/7 days) significantly decreased quinpirole-induced yawning without affecting the quinpirole discrimination. Many discrimination studies on DA receptor agonists use food-restricted rats; together with those studies, the current experiment using free-feeding rats suggests that feeding conditions affecting the behavioral effects of direct-acting DA receptor agonists might also have an impact on the effects of indirect

  9. Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

    Science.gov (United States)

    Mazor, Ohad; Hillairet de Boisferon, Marc; Lombet, Alain; Gruaz-Guyon, Anne; Gayer, Batya; Skrzydelsky, Delphine; Kohen, Fortune; Forgez, Patricia; Scherz, Avigdor; Rostene, William; Salomon, Yoram

    2002-02-01

    We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.

  10. GABAA receptors, but not dopamine, serotonin or NMDA receptors, are increased in the frontal cortex from schizophrenic subjects

    International Nuclear Information System (INIS)

    Daen, B.; Hussain, T.; Scarr, E.; Tomaskovic, E.; Kitsoulis, S.; Pavey, G.; Hill, C.; Keks, N.; Opeskin, K.; Copolov, D.L.

    1998-01-01

    Full text: Having shown changed 5HT 2A receptor density in the frontal cortex (FC) from schizophrenic subjects (1) we now report on further studies of the molecular neuroanatomy of the FC in schizophrenia. We used in situ radioligand binding and autoradiography to measure the density of [ 3 H]8OH-DPAT (1 nM) binding (5HT 1A receptors) and [ 3 H]GR113808 (2.4nM) binding (5HT 4 receptors) in Brodmann's areas (BA) 8, 9 and 10 from 10 schizophrenic and 10 controls subjects. In addition, [ 3 H]muscimol (100 nM) binding (GABA A receptors), [ 3 H]TCP (20nM) binding (NMDA receptors), [ 3 H]SCH 23390 (3nM) binding (DA D 1 like receptors) and [ 3 H]YM-09151-2 (4nM) binding (DA D 2 -like receptors) was measured in BA 9 from 17 schizophrenic and 17 control subjects. Subjects were matched for age and sex and the post-mortem interval for tissue collection did not differ. There was a significant increase (18%) in the density of GABA A receptors in BA 9 from subjects with schizophrenia (p<0.05) with no change in NMDA, dopamine or serotonin receptors. These data support the hypothesis that there are selective changes in neurotransmitter receptors in the FC of subjects with schizophrenia. It is not yet clear if such changes contribute to the pathology of the illness. Copyright (1998) Australian Neuroscience Society

  11. 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

  12. The binding site for neohesperidin dihydrochalcone at the human sweet taste receptor

    Directory of Open Access Journals (Sweden)

    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

  13. Characterization of the binding of [3H]-(+/-)-L-364,718: a new potent, nonpeptide cholecystokinin antagonist radioligand selective for peripheral receptors

    International Nuclear Information System (INIS)

    Chang, R.S.; Lotti, V.J.; Chen, T.B.; Kunkel, K.A.

    1986-01-01

    [3H]-(+/-)-L-364,718 a new, potent and selective nonpeptide peripheral cholecystokinin (CCK) antagonist bound saturably and reversibly to rat pancreatic membranes. The radioligand recognized a single class of binding sites with a high affinity (Kd = 0.23 nM). The binding of [ 3 H]-(+/-)-L-364,718 was stereospecific in that the more biologically active (-)-enantiomer demonstrated greater potency than the (+)-enantiomer. The rank order of potency of various CCK agonists and antagonists in displacing [ 3 H]-(+/-)-L-364,718 correlated with their ability to displace [ 125 I]CCK-8 and their known pharmacological activities in peripheral tissues. However, the absolute potencies of agonists were greater in displacing [ 125 I]CCK-8 than [ 3 H]-(+/-)-L-364,718. As described for other physiologically relevant receptor systems, the potency for displacement of [ 3 H]-(+/-)-L-364,718 binding by CCK agonists, but not antagonists, was reduced by guanosine 5'-(beta, gamma-imido)triphosphate and NaCl and enhanced by MgCl 2 . [ 3 H]-(+/-)-L-364,718 also demonstrated specific binding to bovine gall bladder tissue but not guinea pig brain or gastric glands, consistent with its selectivity as a peripheral CCK antagonist. [ 3 H]-(+/-)-L-364,718 binding to pancreatic membranes was not affected by various pharmacological agents known to interact with other common peptide and nonpeptide receptor systems. These data indicate that [ 3 H]-(+/-)-L-364,718 represents a new potent nonpeptide antagonist radioligand for the study of peripheral CCK receptors which may allow differentiation of agonist and antagonist interactions

  14. 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.

  15. Binding of [125I]-N-(p-aminophenethyl)spiroperidol to the D-2 dopamine receptor in the neurointermediate lobe of the rat pituitary gland: a thermodynamic study

    International Nuclear Information System (INIS)

    Agui, T.; Amlaiky, N.; Caron, M.G.; Kebabian, J.W.

    1988-01-01

    The novel iodinated ligand [ 125 I]-N-(p-aminophenethyl)spiroperidol ([ 125 I]NAPS) was used to identify the D-2 dopamine receptor in the intermediate lobe of the rat pituitary gland. The binding of [ 125 I]NAPS was of high affinity and saturable, given that the dissociation constant and the maximal binding were 34.7 +/- 4.8 pM and 21.1 +/- 2.5 fmol/mg of protein, respectively. The ability of dopaminergic agonists and antagonists to compete with [ 125 I]NAPS varied markedly with incubation temperature. The marked decrease of the molar potency associated with increasing incubation temperature in the competitive displacement curve suggested that the binding of five agonists, dopamine, (-)-apomorphine, (-)-n-propylnorapomorphine, N-0434, and LY-171555, to the D-2 dopamine receptor was enthalpy-driven, with a negative change in entropy. In contrast, the binding of three antagonists, fluphenazine, (+)-butaclamol, and domperidone, was entropy-driven, with positive change in entropy, suggesting less temperature-sensitive change in the molar potency. Several molecules gave unanticipated results; the molar potency of two dopamine agonists, bromocriptine and lisuride, was much less temperature-sensitive than the other agonists used in this study. The thermodynamic parameters for the atypical agonists indicated entropy-driven binding. Conversely, the molar potency of (+)-apomorphine, a dopamine receptor antagonist, was markedly affected by incubation temperature, indicating enthalpy-driven binding. Another antagonist, YM-09151-2, was affected by the inclusion of sodium chloride in the assay system: in the absence of sodium chloride, the drug was relatively weak and displayed enthalpy-driven binding; in the presence of sodium chloride, its molar potency was increased and its binding manner turned into entropy-driven

  16. 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

  17. 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.

  18. Comparison of nicotinic receptor binding and biotransformation of coniine in the rat and chick.

    Science.gov (United States)

    Forsyth, C S; Speth, R C; Wecker, L; Galey, F D; Frank, A A

    1996-12-31

    Coniine, an alkaloid from Conium maculatum (poison hemlock), is a known teratogen in many domestic species with maternal ingestion resulting in arthrogryposis of the offspring. We have previously shown that rats are not susceptible and rabbits only weakly susceptible to coniine-induced arthrogryposis. However, the chick embryo does provide a reproducible laboratory animal model of coniine-induced teratogenesis. The reason for this cross-species variation is unknown. The purpose of this study was to evaluate coniine binding to nicotinic receptors and to measure coniine metabolism in vitro between susceptible and non-susceptible species. Using the chick model, neither the peripheral nicotinic receptor antagonist d-tubocurarine chloride nor the central nicotinic receptor antagonist trimethaphan camsylate blocked the teratogenesis or lethality of 1.5% coniine (50 microliters/egg). Trimethaphan camsylate enhanced coniine-induced lethality in a dose-dependent manner. Neither nicotinic receptor blocker prevented nicotine sulfate-induced malformations but d-tubocurarine chloride did block lethality in a dose-dependent manner. Competition by coniine for [125I]-alpha-bungarotoxin to nicotinic receptors isolated from adult rat diaphragm and chick thigh muscle and competition by coniine for [3H]-cytisine to receptors from rat and chick brain were used to assess coniine binding to nicotinic receptors. The IC50 for coniine in rat diaphragm was 314 microM while that for chick leg muscle was 70 microM. For neuronal nicotinic receptors, the IC50s of coniine for maternal rat brain, fetal rat brain, and chick brain were 1100 microM, 820 microM, and 270 microM, respectively. There were no differences in coniine biotransformation in vitro by microsomes from rat or chick livers. Differences in apparent affinity of coniine for nicotinic receptors or differences in the quantity of the nicotinic receptor between the rat and chick may explain, in part, the differences in susceptibility of

  19. Effects of vitamin D(3)-binding protein-derived macrophage activating factor (GcMAF) on angiogenesis.

    Science.gov (United States)

    Kanda, Shigeru; Mochizuki, Yasushi; Miyata, Yasuyoshi; Kanetake, Hiroshi; Yamamoto, Nobuto

    2002-09-04

    The vitamin D(3)-binding protein (Gc protein)-derived macrophage activating factor (GcMAF) activates tumoricidal macrophages against a variety of cancers indiscriminately. We investigated whether GcMAF also acts as an antiangiogenic factor on endothelial cells. The effects of GcMAF on angiogenic growth factor-induced cell proliferation, chemotaxis, and tube formation were examined in vitro by using cultured endothelial cells (murine IBE cells, porcine PAE cells, and human umbilical vein endothelial cells [HUVECs]) and in vivo by using a mouse cornea micropocket assay. Blocking monoclonal antibodies to CD36, a receptor for the antiangiogenic factor thrombospondin-1, which is also a possible receptor for GcMAF, were used to investigate the mechanism of GcMAF action. GcMAF inhibited the endothelial cell proliferation, chemotaxis, and tube formation that were all stimulated by fibroblast growth factor-2 (FGF-2), vascular endothelial growth factor-A, or angiopoietin 2. FGF-2-induced neovascularization in murine cornea was also inhibited by GcMAF. Monoclonal antibodies against murine and human CD36 receptor blocked the antiangiogenic action of GcMAF on the angiogenic factor stimulation of endothelial cell chemotaxis. In addition to its ability to activate tumoricidal macrophages, GcMAF has direct antiangiogenic effects on endothelial cells independent of tissue origin. The antiangiogenic effects of GcMAF may be mediated through the CD36 receptor.

  20. Striatal dopamine D2/3 receptor regulation by stress inoculation in squirrel monkeys

    Directory of Open Access Journals (Sweden)

    Alex G. Lee

    2016-06-01

    Full Text Available Intermittent mildly stressful situations provide opportunities to learn, practice, and improve coping in a process called stress inoculation. Stress inoculation also enhances cognitive control and response inhibition of impulsive motivated behavior. Cognitive control and motivation have been linked to striatal dopamine D2 and/or D3 receptors (DRD2/3 in rodents, monkeys, and humans. Here, we study squirrel monkeys randomized early in life to stress inoculation with or without maternal companionship and a no-stress control treatment condition. Striatal DRD2/3 availability in adulthood was measured in vivo by [11C]raclopride binding using positron emission tomography (PET. DRD2/3 availability was greater in caudate and putamen compared to ventral striatum as reported in PET studies of humans and other non-human primates. DRD2/3 availability in ventral striatum was also consistently greater in stress inoculated squirrel monkeys compared to no-stress controls. Squirrel monkeys exposed to stress inoculation in the presence of their mother did not differ from squirrel monkeys exposed to stress inoculation without maternal companionship. Similar effects in different social contexts extend the generality of our findings and together suggest that stress inoculation increases striatal DRD2/3 availability as a correlate of cognitive control in squirrel monkeys.

  1. Heteroreceptor Complexes Formed by Dopamine D1, Histamine H3, and N-Methyl-D-Aspartate Glutamate Receptors as Targets to Prevent Neuronal Death in Alzheimer's Disease.

    Science.gov (United States)

    Rodríguez-Ruiz, Mar; Moreno, Estefanía; Moreno-Delgado, David; Navarro, Gemma; Mallol, Josefa; Cortés, Antonio; Lluís, Carme; Canela, Enric I; Casadó, Vicent; McCormick, Peter J; Franco, Rafael

    2017-08-01

    Alzheimer's disease (AD) is a neurodegenerative disorder causing progressive memory loss and cognitive dysfunction. Anti-AD strategies targeting cell receptors consider them as isolated units. However, many cell surface receptors cooperate and physically contact each other forming complexes having different biochemical properties than individual receptors. We here report the discovery of dopamine D 1 , histamine H 3 , and N-methyl-D-aspartate (NMDA) glutamate receptor heteromers in heterologous systems and in rodent brain cortex. Heteromers were detected by co-immunoprecipitation and in situ proximity ligation assays (PLA) in the rat cortex where H 3 receptor agonists, via negative cross-talk, and H 3 receptor antagonists, via cross-antagonism, decreased D 1 receptor agonist signaling determined by ERK1/2 or Akt phosphorylation, and counteracted D 1 receptor-mediated excitotoxic cell death. Both D 1 and H 3 receptor antagonists also counteracted NMDA toxicity suggesting a complex interaction between NMDA receptors and D 1 -H 3 receptor heteromer function. Likely due to heteromerization, H 3 receptors act as allosteric regulator for D 1 and NMDA receptors. By bioluminescence resonance energy transfer (BRET), we demonstrated that D 1 or H 3 receptors form heteromers with NR1A/NR2B NMDA receptor subunits. D 1 -H 3 -NMDA receptor complexes were confirmed by BRET combined with fluorescence complementation. The endogenous expression of complexes in mouse cortex was determined by PLA and similar expression was observed in wild-type and APP/PS1 mice. Consistent with allosteric receptor-receptor interactions within the complex, H 3 receptor antagonists reduced NMDA or D 1 receptor-mediated excitotoxic cell death in cortical organotypic cultures. Moreover, H 3 receptor antagonists reverted the toxicity induced by ß 1-42 -amyloid peptide. Thus, histamine H 3 receptors in D 1 -H 3 -NMDA heteroreceptor complexes arise as promising targets to prevent neurodegeneration.

  2. Alteration of CNS dopamine transporter and D2 receptor in aged and scopolamine induced amnestic rats

    International Nuclear Information System (INIS)

    Lin Yansong; Ding Shiyu; Chen Zhengping; Zhou Xiang; Fang Ping; Wang Bocheng; Zhang Manda

    2002-01-01

    Objective: To evaluate the effect of aging and scopolamine (Sco) induced amnesia on central dopamine transporter (DAT), D 2 receptor in rats. Methods: The 3 month old amnestic rat models were made by peritoneal injection of the muscarinic receptor antagonist Sco (5 mg/kg) for 10 d. Passive avoidance task was carried out to evaluate the recent learning and memory of rats. The biodistribution of 125 I-2-β-carbomethoxy-3-β(4-iodophenyl)-tropan ( 125 I-β-CIT) and 125 I-s-3-iodo-N-(1-ethyl-2-pyrolidinyl) methyl-2-hydroxy-6-methoxybenzamide (IBZM) in the brain was used to evaluate the DAT and D 2 receptor. Results: During 10 d passive avoidance task testing, no difference was found for the first day among 3 month control, 26 month old and Sco group rats, on the 10th day the entry number of aged and Sco group rats was (1.33 +- 0.82)/10 min, (3.00 +- 0.63)/10 min, respectively, higher than that of the control rats (t was 5.682 and 6.372, respectively, P 125 I-β-CIT binding were found in the striatum (ST), hippocampus (HIP) and frontal cortex (FC) of the aged and Sco group rats (t was 4.151, 5.416, 4.871, 6.922, 7.331 and 3.990, respectively, P 125 I-IBZM binding in ST was found in both Sco and old rats (t was 6.021 and 3.227, respectively, P 2 receptor, was found in ST, HIP and cortex of the aged and Sco group suggesting a gradual degeneration of dopaminergic neurons in aged rats. The decreased levels of 125 I-β-CIT and 125 I-IBZM binding in cortex area might be responsible for the amnesia in he Sco group through the dopaminergic pathway of midbrain-frontal cortex

  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. Characterization with /sup 3/H-haloperidol of the dopamine receptor in the rat kidney particulate preparation

    Energy Technology Data Exchange (ETDEWEB)

    Nakajima, T; Kuruma, I [Nippon Roche Research Center, Kanagawa (Japan)

    1980-12-01

    The dopamine receptor of rat kidney particulate preparation was identified and characterized by the use of /sup 3/H-haloperidol binding. Binding of /sup 3/H-haloperidol to the kidney particulate preparation was slow and saturable. The dissociation constants (K sub(D)) were 0.41 nM and 5.88 nM, respectively, according to the model of two classes of independent binding sites. Maximal binding of high affinity site was obtained with 166 fmole/mg protein which was about 40% of the total receptor density. A wide variety of neuroleptics at specifically low concentrations in nanomolar range inhibited the /sup 3/H-haloperidol binding. There was an excellent correlation between the affinity of numerous neuroleptics for the kidney particulate preparation and that for the brain striatum.

  5. Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

    Science.gov (United States)

    André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

    1999-11-01

    Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential

  6. 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.

  7. Estrogenic activity and estrogen receptor β binding of the UV filter 3-benzylidene camphor Comparison with 4-methylbenzylidene camphor

    International Nuclear Information System (INIS)

    Schlumpf, Margret; Jarry, Hubert; Wuttke, Wolfgang; Ma, Risheng; Lichtensteiger, Walter

    2004-01-01

    UV filters represent new classes of estrogenic [Environ. Health Perspect. 109 (2001) 239] or antiandrogenic [Toxicol. Sci. 74 (2003) 43] chemicals. We tested 3-benzylidene camphor (3-BC), reported as estrogenic in fish [Pharmacol. Toxicol. 91 (2002) 204], and mammalian systems in comparison to 4-methylbenzylidene camphor (4-MBC), shown to be active in rats, and analyzed binding to estrogen receptor subtypes. 3-BC and 4-MBC stimulated MCF-7 cell proliferation (EC 50 : 0.68 and 3.9 μM). The uterotrophic assay of 3-BC (oral gavage) in immature rats showed unexpected potency with ED50 45.3 mg/kg per day; lowest effective dose 2 mg/kg per day, and maximum effect with 70% of ethinylestradiol. After comparing with literature data, we found that the oral 3-BC was considerably more potent than oral bisphenol A and almost as active as subcutaneous genistein. 3-BC and 4-MBC displaced 16α 125 I-estradiol from porcine uterine cytosolic receptors (IC 50 : 14.5 and 112 μM), and from recombinant human estrogen receptor β (hERβ) (IC 50 : 3-BC, 11.8 μM; 4-MBC, 35.3 μM), whereas no displacement was detected at human estrogen receptor α (hERα) up to 3 mM. This subtype selectivity makes the two camphor derivatives interesting model compounds. Their activity on immature rat uterus is not easily explained by ERβ activation. It cannot be excluded that active metabolites with possibly different receptor binding characteristics are formed in vivo

  8. 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

  9. Identifying polymorphisms in the Rattus norvegicus D3 dopamine receptor gene and regulatory region

    NARCIS (Netherlands)

    Smits, B.M.; D'Souza, U.M.; Berezikov, E.; Cuppen, E.; Sluyter, F.

    2004-01-01

    The D(3) dopamine receptor has been implicated in several neuropsychiatric disorders, including schizophrenia, Parkinson's disease and addiction. Sequence variation in the D(3) gene can lead to subtle alteration in receptor structure or gene expression and thus to a different phenotype. In this

  10. [{sup 3}H]CGP 61594, the first photoaffinity ligand for the glycine site of NMDA receptors

    Energy Technology Data Exchange (ETDEWEB)

    Benke, D.; Honer, M.; Mohler, H. [Institute of Pharmacology, ETH and University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich (Switzerland); Heckendorn, R.; Pozza, M.F.; Allgeier, H.; Angst, C. [NS Research, Novartis Pharma AG, CH-4002 Basle (Switzerland)

    1999-02-01

    Activation of NMDA receptors requires the presence of glycine as a coagonist which binds to a site that is allosterically linked to the glutamate binding site. To identify the protein constituents of the glycine binding site in situ the photoaffinity label [{sup 3}H]CGP 61594 was synthesized. In reversible binding assays using crude rat brain membranes, [{sup 3}H]CGP 61594 labeled with high affinity (K{sub D}=23 nM) the glycine site of the NMDA receptor. This was evident from the Scatchard analysis, the displacing potencies of various glycine site ligands and the allosteric modulation of [{sup 3}H]CGP 61594 binding by ligands of the glutamate and polyamine sites. Electrophysiological experiments in a neocortical slice preparation identified CGP 61594 as a glycine antagonist. Upon UV-irradiation, a protein band of 115 kDa was specifically photolabeled by [{sup 3}H]CGP 61594 in brain membrane preparations. The photolabeled protein was identified as the NR1 subunit of the NMDA receptor by NR1 subunit-specific immunoaffinity chromatography. Thus, [{sup 3}H]CGP 61594 is the first photoaffinity label for the glycine site of NMDA receptors. It will serve as a tool for the identification of structural elements that are involved in the formation of the glycine binding domain of NMDA receptors in situ and will thereby complement the mutational analysis of recombinant receptors. (Copyright (c) 1999 Elsevier Science B.V., Amsterdam. All rights reserved.)

  11. Comparison between dopamine-stimulated adenylate cyclase and 3H-SCH 23390 binding in rat striatum

    International Nuclear Information System (INIS)

    Andersen, P.H.; Groenvald, F.C.; Jansen, J.A.

    1985-01-01

    Methods for measuring 3 H-SCH 23990 binding and dopamine (DA) stimulated adenylate cyclase (AC) were established in identical tissue preparations and under similar experimental conditions. Pharmacological characterization revealed that both assays involved interaction with the D1 receptor or closely associated sites. In order to investigate whether the binding sites for 3 H-SCH 23390 and DA in fact are identical, the antagonistic effects of a variety of pharmacologically active compounds were examined. Surprisingly, the K/sub i/-values obtained from Schild-plot analysis of the antagonism of DA-stimulated AC, were 80-240 times higher than the K/sub i/-values obtained from competition curves of 3 H-SCH 23390 binding. Since both assays were performed under identical conditions, the differences in K/sub i/-values indicate the possibility of different binding sites for DA and 3 H-SCH 23390 or, that DA and 3 -SCH 23390 label different states of the same receptor. 19 references, 7 figures, 2 tables

  12. Glucocorticoid receptors on leukemic cells as evidenced by dexamethasone-induced cytolysis and /sup 3/H-dexamethasone binding

    Energy Technology Data Exchange (ETDEWEB)

    Thraenhardt, H; Haefer, R; Zintl, F

    1987-01-01

    The presence of glucocorticoid receptors on the leukemic cells of 33 patients affected with acute lymphatic leukemia (ALL) and 6 patients affected with acute myeloic leukemia (AML) was investigated by dexamethasone-induced cytolysis and (/sup 3/H)-dexamethasone binding. The tests undertaken proved that after 20 hours of incubation 9 of 26 non-T-non-B-ALL (c-ALL and unclassified ALL) and 2 of AML were lysed with dexamethasone; blood lymphocytes and bone marrow leukocytes of healthy donors, however, were not affected. Non-T-non-B-ALL and AML were able to bind essentially more (/sup 3/H)-dexamethasone than T-ALL. There existed no correlation between dexamethasone binding and dexamethasone-induced cytolysis.

  13. Positive versus negative modulation of different endogenous chemokines for CC-chemokine receptor 1 by small molecule agonists through allosteric versus orthosteric binding

    DEFF Research Database (Denmark)

    Jensen, Pia C; Thiele, Stefanie; Ulven, Trond

    2008-01-01

    7 transmembrane-spanning (7TM) chemokine receptors having multiple endogenous ligands offer special opportunities to understand the molecular basis for allosteric mechanisms. Thus, CC-chemokine receptor 1 (CCR1) binds CC-chemokine 3 and 5 (CCL3 and CCL5) with K(d) values of 7.3 and 0.16 nm......5 and not CCL3 activation is affected by substitutions in the main ligand binding pocket including the conserved GluVII:06 anchor point. A series of metal ion chelator complexes were found to act as full agonists on CCR1 and to be critically affected by the same substitutions in the main ligand...... binding pocket as CCL5 but not by mutations in the extracellular domain. In agreement with the overlapping binding sites, the small non-peptide agonists displaced radiolabeled CCL5 with high affinity. Interestingly, the same compounds acted as allosteric enhancers of the binding of CCL3, with which...

  14. 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.

  15. Characteristics of central binding sites for [3H] DAMGO in spontaneously hypertensive rats

    International Nuclear Information System (INIS)

    Gulati, A.; Bhargava, H.N.

    1990-01-01

    The binding of [ 3 H] DAMGO, a highly selective ligand for μ-opiate receptors, to membranes of discrete brain regions and spinal cord of 10 week old spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats were determined. The brain regions examined were hypothalamus, amygdala, hippocampus, corpus striatum, pons and medulla, midbrain and cortex. [ 3 H] DAMGO bound to membranes of brain regions and spinal cord at a single high affinity site. The receptor density (B max value) and apparent dissociation constant (K d value) of [ 3 H] DAMGO to bind to membranes of hippocampus, corpus striatum, pons and medulla, cortex and spinal cord of WKY and SHR rats did not differ. The B max value of [ 3 H] DAMGO in membranes of hypothalamus and midbrain of SHR rats was significantly higher than in WKY rats but the K d values in the two strains did not differ. On the other hand, the B max value of [ 3 H] DAMGO in membranes of amygdala of SHR rats was lower than that of WKY rats but the K d values in the two strains were similar

  16. 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.

  17. Naloxone-sensitive, haloperidol-sensitive, [3H](+)SKF-10047-binding protein partially purified from rat liver and rat brain membranes: an opioid/sigma receptor?

    Science.gov (United States)

    Tsao, L I; Su, T P

    1997-02-01

    A naloxone-sensitive, haloperidol-sensitive, [3H](+)SKF-10047-binding protein was partially purified from rat liver and rat brain membranes in an affinity chromatography originally designed to purify sigma receptors. Detergent-solubilized extracts from membranes were adsorbed to Sephadex G-25 resin containing an affinity ligand for sigma receptors: N-(2- 3,4-dichlorophenyl]ethyl)-N-(6-aminohexyl)-(2-[1-pyrrolidinyl]) ethylamine (DAPE). After eluting the resin with haloperidol, a protein that bound [3H](+)SKF-10047 was detected in the eluates. However, the protein was not the sigma receptor. [3H](+)SKF-10047 binding to the protein was inhibited by the following compounds in the order of decreasing potency: (+)pentazocine > (-) pentazocine > (+/-)cyclazocine > (-)morphine > (-)naloxone > haloperidol > (+)SKF-10047 > DADLE > (-)SKF-10047. Further, the prototypic sigma receptor ligands, such as 1,3-di-o-tolylguanidine (DTG), (+)3-PPP, and progesterone, bound poorly to the protein. Tryptic digestion and heat treatment of the affinity-purified protein abolished radioligand binding. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) of the partially-purified protein from the liver revealed a major diffuse band with a molecular mass of 31 kDa, a polypeptide of 65 kDa, and another polypeptide of > 97 kDa. This study demonstrates the existence of a novel protein in the rat liver and rat brain which binds opioids, benzomorphans, and haloperidol with namomolar affinity. The protein resembles the opioid/sigma receptor originally proposed by Martin et al. [(1976): J. Pharmacol. Exp. Ther., 197:517-532.]. A high degree of purification of this protein has been achieved in the present study.

  18. 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

  19. Radioiodination of central nerves system dopamine D2 receptor imaging agent. IBZM preparation and preclinical study

    International Nuclear Information System (INIS)

    Lin Yansong; Lin Xiangtong; Hu Mingyang; Pan Shangren; Wang Bocheng

    1996-01-01

    To study preparation of central nerves system dopamine D2 imaging agent 131 I-IBZM and its preclinical investigation, peracetic acid was used as the oxidant for preparing radioiodinated 125 I-IBZM and 131 I-IBZM, D2 binding properties of IBZM were examined by in vitro binding saturation analysis, rat whole body and regional brain biodistribution, rat brain autoradiography and rabbit SPECT static imaging, etc. The results are: 1. The radiolabelling yields of 125 I-IBZM and 131 I-IBZM were 84.18% +- 3.06% and 78.50% +- 3.47%. The radiochemical purity were over 95% after being isolated by HPLC; and were over 90% after being isolated by organic extraction. 2. Scatchard plot of D2 receptor saturation binding analysis showed: K d = 0.53 +- 0.06 nmol/L, B max = 466.45 +- 45.88 fmol/mg protein. 3. The rat brain autoradiography and analysis showed that there was high 125 I-IBZM uptake in striatal area 2 hr after injection, the striatal/cerebellum ratio was 6.22 +- 0.48; the high 125 -IBZM uptake can be blocked by haloperidol--a special dopamine D2 receptor antagonist. 4. 131 I-IBZM rat biodistribution and rabbit SPECT planar imaging showed good initial brain uptake and retention, the initial uptake of rat brain was 1.893 +- 0.147% ID/g at 2 min and 1.044 +- 0.135% ID/g at 60 min. The results showed that the radioiodinated IBZM had high affinity, saturation and specificity to rat's and rabbit's central nerves system dopamine D2 receptors

  20. Cross-talk between an activator of nuclear receptors-mediated transcription and the D1 dopamine receptor signaling pathway.

    Science.gov (United States)

    Schmidt, Azriel; Vogel, Robert; Rutledge, Su Jane; Opas, Evan E; Rodan, Gideon A; Friedman, Eitan

    2005-03-01

    Nuclear receptors are transcription factors that usually interact, in a ligand-dependent manner, with specific DNA sequences located within promoters of target genes. The nuclear receptors can also be controlled in a ligand-independent manner via the action of membrane receptors and cellular signaling pathways. 5-Tetradecyloxy-2-furancarboxylic acid (TOFA) was shown to stimulate transcription from the MMTV promoter via chimeric receptors that consist of the DNA binding domain of GR and the ligand binding regions of the PPARbeta or LXRbeta nuclear receptors (GR/PPARbeta and GR/LXRbeta). TOFA and hydroxycholesterols also modulate transcription from NF-kappaB- and AP-1-controlled reporter genes and induce neurite differentiation in PC12 cells. In CV-1 cells that express D(1) dopamine receptors, D(1) dopamine receptor stimulation was found to inhibit TOFA-stimulated transcription from the MMTV promoter that is under the control of chimeric GR/PPARbeta and GR/LXRbeta receptors. Treatment with the D(1) dopamine receptor antagonist, SCH23390, prevented dopamine-mediated suppression of transcription, and by itself increased transcription controlled by GR/LXRbeta. Furthermore, combined treatment of CV-1 cells with TOFA and SCH23390 increased transcription controlled by the GR/LXRbeta chimeric receptor synergistically. The significance of this in vitro synergy was demonstrated in vivo, by the observation that SCH23390 (but not haloperidol)-mediated catalepsy in rats was potentiated by TOFA, thus showing that an agent that mimics the in vitro activities of compounds that activate members of the LXR and PPAR receptor families can influence D1 dopamine receptor elicited responses.

  1. 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

    OpenAIRE

    Bolton, Michael J; Garry, Robert F

    2011-01-01

    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 infectio...

  2. Curcumin induces human cathelicidin antimicrobial peptide gene expression through a vitamin D receptor-independent pathway

    DEFF Research Database (Denmark)

    Guo, Chunxiao; Rosoha, Elena; Lowry, Malcolm B

    2013-01-01

    The vitamin D receptor (VDR) mediates the pleiotropic biologic effects of 1α,25 dihydroxy-vitamin D(3). Recent in vitro studies suggested that curcumin and polyunsaturated fatty acids (PUFAs) also bind to VDR with low affinity. As potential ligands for the VDR, we hypothesized that curcumin...... cancer cell line HT-29 and keratinocyte cell line HaCaT. We demonstrated that PUFAs failed to induce CAMP or CYP24A1 mRNA expression in all three cell lines, but curcumin up-regulated CAMP mRNA and protein levels in U937 cells. Curcumin treatment induced CAMP promoter activity from a luciferase reporter...... construct lacking the VDR binding site and did not increase binding of the VDR to the CAMP promoter as determined by chromatin immunoprecipitation assays. These findings indicate that induction of CAMP by curcumin occurs through a vitamin D receptor-independent manner. We conclude that PUFAs and curcumin do...

  3. Dopamine D2 receptor radiotracers [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride are indistinguishably inhibited by D2 agonists and antagonists ex vivo

    Energy Technology Data Exchange (ETDEWEB)

    McCormick, Patrick N. [Institute of Medical Science, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada)], E-mail: patrick.mccormick@camhpet.ca; Kapur, Shitij [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); PET Center, Center for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada); Seeman, Philip [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); Department of Pharmacology, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); Wilson, Alan A. [Department of Psychiatry, University of Toronto, Toronto, Ontario, M5S 1A8 (Canada); PET Center, Center for Addiction and Mental Health, Toronto, Ontario, M5T 1R8 (Canada)

    2008-01-15

    Introduction: In vitro, the dopamine D2 receptor exists in two states, with high and low affinity for agonists. The high-affinity state is the physiologically active state thought to be involved in dopaminergic illnesses such as schizophrenia. The positron emission tomography radiotracer [{sup 11}C](+)-PHNO ([{sup 11}C](+)-4-propyl-3,4,4a,5,6,10b-hexahydro-2H-naphtho[1,2-b][1,4] oxazin-9-o l), being a D2 agonist, should selectively label the high-affinity state at tracer dose and therefore be more susceptible to competition by agonist as compared to the antagonist [{sup 3}H]raclopride, which binds to both affinity states. Methods: We tested this prediction using ex vivo dual-radiotracer experiments in conscious rats. D2 antagonists (haloperidol or clozapine), a partial agonist (aripiprazole), a full agonist [(-)-NPA] or the dopamine-releasing drug amphetamine (AMPH) were administered to rats prior to an intravenous coinjection of [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride. Rats were sacrificed 60 min after radiotracer injection. Striatum, cerebellum and plasma samples were counted for {sup 11}C and {sup 3}H. The specific binding ratio {l_brace}SBR, i.e., [%ID/g (striatum)-%ID/g (cerebellum)]/(%ID/g (cerebellum){r_brace} was used as the outcome measure. Results: In response to D2 antagonists, partial agonist or full agonist, [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride SBRs responded indistinguishably in terms of both ED{sub 50} and Hill slope (e.g., (-)-NPA ED{sub 50} values are 0.027 and 0.023 mg/kg for [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride, respectively). In response to AMPH challenge, [{sup 11}C](+)-PHNO and [{sup 3}H]raclopride SBRs were inhibited to the same degree. Conclusions: We have shown that the SBRs of [{sup 11}C](+)-PHNO- and [{sup 3}H]raclopride do not differ in their response to agonist challenge. These results do not support predictions of the in vivo binding behavior of a D2 agonist radiotracer and cast some doubt on the in vivo

  4. Intrastriatal administration of botulinum neurotoxin A normalizes striatal D2 R binding and reduces striatal D1 R binding in male hemiparkinsonian rats.

    Science.gov (United States)

    Wedekind, Franziska; Oskamp, Angela; Lang, Markus; Hawlitschka, Alexander; Zilles, Karl; Wree, Andreas; Bauer, Andreas

    2018-01-01

    Cerebral administration of botulinum neurotoxin A (BoNT-A) has been shown to improve disease-specific motor behavior in a rat model of Parkinson disease (PD). Since the dopaminergic system of the basal ganglia fundamentally contributes to motor function, we investigated the impact of BoNT-A on striatal dopamine receptor expression using in vitro and in vivo imaging techniques (positron emission tomography and quantitative autoradiography, respectively). Seventeen male Wistar rats were unilaterally lesioned with 6-hydroxydopamine (6-OHDA) and assigned to two treatment groups 7 weeks later: 10 rats were treated ipsilaterally with an intrastriatal injection of 1 ng BoNT-A, while the others received vehicle (n = 7). All animals were tested for asymmetric motor behavior (apomorphine-induced rotations and forelimb usage) and for striatal expression of dopamine receptors and transporters (D 1 R, D 2 R, and DAT). The striatal D 2 R availability was also quantified longitudinally (1.5, 3, and 5 months after intervention) in 5 animals per treatment group. The 6-OHDA lesion alone induced a unilateral PD-like phenotype and a 13% increase of striatal D 2 R. BoNT-A treatment reduced the asymmetry in both apomorphine-induced rotational behavior and D 2 R expression, with the latter returning to normal values 5 months after intervention. D 1 R expression was significantly reduced, while DAT concentrations showed no alteration. Independent of the treatment, higher interhemispheric symmetry in raclopride binding to D 2 R was generally associated with reduced forelimb akinesia. Our findings indicate that striatal BoNT-A treatment diminishes motor impairment and induces changes in D 1 and D 2 binding site density in the 6-OHDA rat model of PD. © 2017 Wiley Periodicals, Inc.

  5. 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