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http://eprints.jcu.edu.au/10018/1/adenosine_deaminase_and_A1_receptor_deficiency.pdf
Objective: Adenosine deaminase (ADA) may be multifunctional, regulating adenosine levels and adenosine receptor (AR) agonism, and potentially modifying AR functionality. Herein we assess effects of ADA (and A1AR) deficiency on AR-mediated responses and ischaemic tolerance. Methods: Normoxic function and responses to 20 or 25min ischaemia and 45min reperfusion were studied in isolated hearts from wild-type mice and from mice deficient in ADA and/or A1ARs. Results: Neither ADA or A1AR deficiency significantly modified basal contractility, although ADA deficiency reduced resting heart rate (an effect abrogated by A1AR deficiency). Bradycardia and vasodilation in response to AR agonism (2-chloroadenosine) were unaltered by ADA deficiency, while A1AR deficiency eliminated the heart rate response. Adenosine efflux increased 10- to 20-fold with ADA deficiency (at the expense of inosine). Deletion of ADA improved outcome from 25min ischaemia, reducing ventricular diastolic pressure (by 45%; 21±4 vs. 38±3mm Hg) and lactate dehydrogenase (LDH) efflux (by 40%; 0.12±0.01 vs. 0.21±0.02U/g/min ischaemia), and enhancing pressure development (by 35%; 89±6 vs. 66±5mm Hg). Similar protection was evident after 20min ischaemia, and was mimicked by the ADA inhibitor EHNA (5μM). Deletion of ADA also enhanced tolerance in A1AR deficient hearts, though effects on diastolic pressure were eliminated. Conclusions: Deficiency of ADA does not alter sensitivities of cardiovascular A1 or A2ARs (despite markedly elevated [adenosine]), but significantly improves ischaemic tolerance. Conversely, A1AR deficiency impairs ischaemic tolerance. Effects of ADA deficiency on diastolic pressure appear solely A1AR-dependent while other ARs or processes additionally contribute to improved contractile recovery and reduced cell death. Publisher: Elsevier Format: application/pdf Other identifier: Willems, Laura, Reichelt, Melissa E., Molina, Jose G., Sun, Chun-Xiao, Chunn, Janci L., Ashton, Kevin J., Schnermann, Jurgen, Blackburn, Michael R., and Headrick, John P. (2006) Effects of adenosine deaminase and A1 receptor deficiency in normoxic and ischaemic mouse hearts. Cardiovascular Research, 71 (1). pp. 79-87. ISSN 1755-3245
2010-01-01
Abstract We reported that adenosine A1 receptor (A1AR) knockout (KO) mice develop lethal status epilepticus after experimental traumatic brain injury (TBI), which is not seen in wild-type (WT) mice. Studies in epilepsy, multiple sclerosis, and neuro-oncology suggest enhanced neuro-inflammation and/or neuronal death in A1AR KO. We hypothesized that A1AR deficiency exacerbates the microglial response and neuronal damage after TBI. A1AR KO and WT littermates were subjected to mild controlled cortical impact (3âm/sec; 0.5âmm depth) to left parietal cortex, an injury level below the acute seizure threshold in the KO. At 24âh or 7 days, mice were sacrificed and serial sections prepared. Iba-1 immunostaining was used to quantify microglia at 7 days. To assess neuronal injury, sections were ...
Demonstration of distinct agonist and antagonist conformations of the A1 adenosine receptor
1989-08-05
A1 adenosine receptor-binding subunits can be visualized using high affinity antagonist and agonist photoaffinity radioligands. In the present study, we examined whether agonists and antagonists bind to the same receptor-binding subunit and if agonists and antagonists induce different conformational states of the receptor in intact membranes. It was demonstrated that several agonist and antagonist photoaffinity receptor-binding subunit. When the agonist and antagonist photoaffinity labeled peptides were denatured and subjected to partial peptide map analysis using a two-dimensional gel electrophoresis system similar peptide fragments were generated from each specifically labeled protein. This suggests that both classes of ligand label and incorporate into the same binding subunit. Proteolytic digestions of agonist- and antagonist-occupied receptors in native intact membranes revealed distinct and different peptide fragments depending on whether the ligand was an agonist or an antagonist. Manipulation of incubation conditions to perturb ligand-receptor interactions alter the pattern of peptide fragments generated with each specific protease. These data suggest that agonist and antagonist photoaffinity probes interact with an incorporate into the same binding subunit but that agonist binding is associated with a unique and detectable receptor conformation.
Demonstration of distinct agonist and antagonist conformations of the A1 adenosine receptor
1989-01-01
A1 adenosine receptor-binding subunits can be visualized using high affinity antagonist and agonist photoaffinity radioligands. In the present study, we examined whether agonists and antagonists bind to the same receptor-binding subunit and if agonists and antagonists induce different conformational states of the receptor in intact membranes. It was demonstrated that several agonist and antagonist photoaffinity receptor-binding subunit. When the agonist and antagonist photoaffinity labeled peptides were denatured and subjected to partial peptide map analysis using a two-dimensional gel electrophoresis system similar peptide fragments were generated from each specifically labeled protein. This suggests that both classes of ligand label and incorporate into the same binding subunit. Proteolytic digestions of agonist- and antagonist-occupied receptors ... >>
Adenosine A1 receptors contribute to mitochondria vulnerability to pro-oxidant stressors
2010-01-01
A1 adenosine receptors are highly expressed in the central nervous system. Mitochondrial function is a major player in adenosine receptors-mediated effects. Here, by using mice with genetic deletion of the A1 receptor, we addressed the existence of a relationship between mitochondria functions and adenosine A1 receptor. Mitochondrial functions and effects of MPP+ in primary mixed cultures are influenced by the presence of the A1 receptor, demonstrating, for the first time, the mitochondrial localization of the adenosine A1 receptor and suggesting a role for this receptor as a mitochondrial vulnerability factor.
2010-01-01
Fractalkine/CX3CL1 is a neuron-associated chemokine, which modulates microglia-induced neurotoxicity activating the specific and unique receptor CX3CR1. CX3CL1/CX3CR1 interaction modulates the release of cytokines from microglia, reducing the level of tumor necrosis factor-α, interleukin-1-β, and nitric oxide and induces the production of neurotrophic substances, both in vivo and in vitro. We have recently shown that blocking adenosine A1 receptors (A1R) with the specific antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) abolishes CX3CL1-mediated rescue of neuronal excitotoxic death and that CX3CL1 induces the release of adenosine from microglia. In this study, we show that the presence of extracellular adenosine is mandatory for the neurotrophic effect of CX3CL1 as reducing ...
Photoaffinity labeling of A1-adenosine receptors
1985-01-01
The ligand-binding subunit of the A1-adenosine receptor has been identified by photoaffinity labeling. A photolabile derivative of R-N6-phenylisopropyladenosine, R-2-azido-N6-p-hydroxyphenylisopropyladenosine (R-AHPIA), has been synthesized as a covalent specific ligand for A1-adenosine receptors. In adenylate cyclase studies with membranes of rat fat cells and human platelets, R-AHPIA has adenosine receptor agonist activity with a more than 60-fold selectivity for the A1-subtype. It competes for [3H]N6-phenylisopropyladenosine binding to A1-receptors of rat brain membranes with a Ki value of 1.6 nM. After UV irradiation, R-AHPIA binds irreversibly to the receptor, as indicated by a loss of [3H]N6-phenylisopropyladenosine binding after extensive washing. The Ki value for this photoinactivation is 1.3 nM. The p-hydroxyphenyl ... >>
Characterization of agonist radioligand interactions with porcine atrial A1 adenosine receptors
1988-09-01
The agonist radioligand (-)-N6-(125I)-p-hydroxyphenylisopropyl-adenosine (( 125I)HPIA) was used to characterize adenosine recognition sites in porcine atrial membranes. (125I)HPIA showed saturable binding to an apparently homogeneous population of sites with a maximum binding capacity of 35 +/- 3 fmol/mg of protein and an equilibrium dissociation constant of 2.5 +/- 0.4 nM. Kinetic experiments were performed to address the molecular mechanism of (125I)HPIA binding in porcine atrial membranes. (125I)HPIA apparently interacts with the cardiac adenosine receptor in a simple bimolecular reaction. A kinetically derived (125I) HPIA dissociation constant (2.4 nM) was in good agreement with that parameter measured at equilibrium. Guanyl nucleotides negatively modulated (125I)HPIA binding by increasing its rate of dissociation. This finding is consonant with the formation of a ternary complex in porcine atrial membranes, consisting of ligand, receptor, and guanyl nucleotide-binding protein. Prototypic adenosine receptor agonists and antagonists inhibited specific binding in a manner consistent with the labeling of an A1 adenosine receptor. The results of these experiments suggest that the adenosine receptor present in porcine atrial membranes, as labeled by (125I)HPIA, is of the A1 subtype.
Characterization of agonist radioligand interactions with porcine atrial A1 adenosine receptors
1988-01-01
The agonist radioligand (-)-N6-[125I]-p-hydroxyphenylisopropyl-adenosine ([ 125I]HPIA) was used to characterize adenosine recognition sites in porcine atrial membranes. [125I]HPIA showed saturable binding to an apparently homogeneous population of sites with a maximum binding capacity of 35 +/- 3 fmol/mg of protein and an equilibrium dissociation constant of 2.5 +/- 0.4 nM. Kinetic experiments were performed to address the molecular mechanism of [125I]HPIA binding in porcine atrial membranes. [125I]HPIA apparently interacts with the cardiac adenosine receptor in a simple bimolecular reaction. A kinetically derived [125I] HPIA dissociation constant (2.4 nM) was in good agreement with that parameter measured at equilibrium. Guanyl nucleotides negatively modulated [125I]HPIA binding by increasing its rate of dissociation. This finding is consonant with the ... >>
Cloning and expression of an A1 adenosine receptor from rat brain
1991-07-01
The authors have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist (3H)-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5{prime}-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor.
Cloning and expression of an A1 adenosine receptor from rat brain
1991-01-01
The authors have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding ... >>
Adenosine receptor binding of the A1-selective antagonist [3H]PD 116,948
1986-01-01
PD 116,948 (8-cyclopentyl-1,3-dipropylxanthine) is a potent adenosine (ado) antagonist which is highly selective for the A1 subtype of ado receptor, with an IC50 of 0.8 nM in [3H]CHA binding to A1 receptors and 500 nM in [3H]NECA binding to A2 receptors. [3H]PD 116,948 (117 Ci/mmol) was prepared by reduction of the diallyl analog, and binding was performed at 250C in 2 ml of 50 mM Tris pH 7.7 for 60 min using membranes from 10 mg wet weight of rat whole brain, with 100 muM N6-cyclopentylado used to define nonspecific binding. Under these conditions, [3H]PD 116,948 bound to a single site with a K/sub d/ of 0.4 nM and a B/sub max/ of 46 pmol/g wet weight. Under optimal conditions, more than 99% of the binding of [3H]PD 116,948 was specific. Affinities of ado agonists and antagonists versus ... >>
Effects of the adenosine A1 receptor inhibitor FK 838 on proximal tubular fluid output in rats
2004-01-01
Influence of the adenosine A1 receptor on blood pressure regulation and renin release
2006-01-01
The present study was performed to investigate the role of adenosine A1 receptors in regulating blood pressure in conscious mice. Adenosine A1-receptor knockout (A1R-/-) mice and their wild-type (A1R+/+) littermates were placed on standardized normal-salt (NS), high-salt (HS), or salt-deficient (SD) diets for a minimum of 10 days before telemetric blood pressure and urinary excretion measurements in metabolic cages. On the NS diet, daytime and nighttime mean arterial blood pressure (MAP) was 7-10 mmHg higher in A1R-/- than in A1R+/+ mice. HS diet did not affect the MAP in A1R-/- mice, but the daytime and nighttime MAP of the A1R+/+ mice increased by approximately 10 mmHg, to the same level as that in the A1R-/-. On the SD diet, day- and nighttime MAP decreased by approximately 6 mmHg in both A1R-/- and A1R+/+ mice, although the MAP remained higher in A1R-/- than in A1R+/+ mice. Although plasma renin levels decreased with increased salt intake in both genotypes, the A1R-/- mice had an approximately twofold higher plasma renin concentration on all diets compared with A1R+/+ mice. Sodium excretion was elevated in the A1R-/- compared with the A1R+/+ mice on the NS diet. There was no difference in sodium excretion between the two genotypes on the HS diet. Even on the SD diet, A1R-/- mice had an increased sodium excretion compared with A1R+/+ mice. An abolished tubuloglomerular feedback response and reduced tubular reabsorption can account for the elevated salt excretion found in A1R-/- animals. The elevated plasma renin concentrations found in the A1R-/- mice could also result in increased blood pressure. Our results confirm that adenosine, acting through the adenosine A1 receptor, plays an important role in regulating blood pressure, renin release, and sodium excretion.
A PET study of adenosine A1 receptor in anesthetized monkey brain
2000-01-01
We demonstrated the distribution of adenosine A1 receptors in the anesthetized monkey brain with positron emission tomography (PET) using [11C]KF15372 ([1-propyl-11C]8-dicyclopropylmethyl-1,3-dipropyl xanthine). [11C]KF15372 was injected intravenously. The regional standardized uptake values and the distribution volume were calculated. We also investigated the effect of carrier on the uptake and regional brain distribution of [11C]KF15372. The use of [11C]KF15372 with dynamic PET scanning could be an appropriate method to analyze the regional binding potential of adenosine A1 receptors in living brain
Localization of the adenosine A1 receptor subtype gene (ADORA1) to chromosome 1q32.1
1995-03-20
Adenosine, acting through its receptors, exerts effects on almost all organ systems, influencing a diversity of physiological responses, including the inhibition of neurotransmitter release, the modulation of cardiac rhythmicity and contractility, and the potentiation of IgE-dependent mediator release. Adenosine receptors belong to the G protein-coupled receptor superfamily, a class of cell-surface receptors that, when activated, couple to a heterotrimeric G protein complex to effect signal transduction. Molecular cloning and subsequent pharmacological and biochemical analyses have led to the identification of four different subtypes of adenosine receptor. The A3 receptor has been localized to chromosome 3 in the mouse by interspecific backcross analysis, suggesting a human chromosomal localization of 1p13 from known mouse-human linkage homologies. We have previously mapped the A2b adenosine receptor subtype to chromosome 17p11.2-p12 using fluorescence in situ hybridization (FISH) and PCR-based screening of somatic cell hybrid DNAs. A previous report has concluded that the Al and A2a receptor subtypes are localized on chromosome 22q11.2-q13.1 and 11q11-q13, respectively, but conflicts with that of MacCollin et al., who have mapped the A2a gene to chromosome 22. In this report, we show that the human A1 adenosine receptor subtype does not map to chromosome 22q11.2-q13.1, but is instead localized on chromosome 1q32. 13 refs., 1 fig.
1988-01-01
The purpose of the present study was the identification of A1 adenosine receptors in intact rat ventricular myocytes, which are thought to mediate the negative inotropic effects of adenosine. The adenosine receptor antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine was used as radioligand. Binding of the radioligand to intact myocytes was rapid, reversible, and saturable with a binding capacity of 40,000 binding sites per cell. The dissociation constant of the radioligand was 0.48 nM. The adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener, and theophylline were competitive inhibitors with affinities in agreement with results obtained for A1 receptors in other tissues. Competition experiments using the adenosine receptor agonists R-N(6)-phenylisopropyladenosine, ... >>
1991-01-01
The triazoloquinazoline CGS 15943 is the first reported nonxanthine adenosine antagonist that has high affinity for brain adenosine receptors. In the present study, the binding of (3H) CGS 15943 to recognition sites in rat cortical membranes was characterized. Saturation experiments revealed that (3H)CGS 15943 labeled a single class of recognition sites with high affinity and limited capacity. Competition studies revealed that the binding of (3H)CGS 15943 was consistent with the labeling of brain adenosine A1 receptors. Adenosine agonists inhibited 1 nM (3H)CGS 15943 binding with the following order of activity N6-cyclopentyladenosine (IC50 = 15 nM) greater than 2-chloroadenosine greater than (R)-N6-phenylisopropyladenosine greater than 5'-N6-ethylcarboxamidoadenosine greater than (S)N6-phenylisopropyladenosine greater than CGS 21680 greater than CV 1808 (IC50 greater than 10,000 nM). The potency order for adenosine antagonists was CGS 15943 (IC50 = 5 nM) greater than 8-phenyltheophylline greater than 1,3-dipropyl-8-(4-amino-2-chloro)phenylxanthine greater than 1,3-diethyl-8-phenylxanthine greater than theophylline = caffeine (IC50 greater than 10,000 nM). Antagonist inhibition curves were steep and best described by a one-site binding model. In contrast, adenosine A1 agonist competition curves were shallow, as indicated by Hill coefficients less than unity. Computer analysis revealed that these inhibition curves were best described by a two-site binding model. Agonist competition curves generated in the presence of 1 mM GTP resulted in a rightward shift and steepening of the inhibition-concentration curves, whereas antagonist binding was not altered in the presence of GTP. The complex binding interactions found with adenosine agonists indicate that (3H)CGS 15943 labels both high and low affinity components of the adenosine A1 receptor in the rat cortex.
1991-01-01
The triazoloquinazoline CGS 15943 is the first reported nonxanthine adenosine antagonist that has high affinity for brain adenosine receptors. In the present study, the binding of [3H] CGS 15943 to recognition sites in rat cortical membranes was characterized. Saturation experiments revealed that [3H]CGS 15943 labeled a single class of recognition sites with high affinity and limited capacity. Competition studies revealed that the binding of [3H]CGS 15943 was consistent with the labeling of brain adenosine A1 receptors. Adenosine agonists inhibited 1 nM [3H]CGS 15943 binding with the following order of activity N6-cyclopentyladenosine (IC50 = 15 nM) greater than 2-chloroadenosine greater than (R)-N6-phenylisopropyladenosine greater than 5'-N6-ethylcarboxamidoadenosine greater than (S)N6-phenylisopropyladenosine greater than CGS 21680 greater than CV ... >>
1988-09-01
The purpose of the present study was the identification of A1 adenosine receptors in intact rat ventricular myocytes, which are thought to mediate the negative inotropic effects of adenosine. The adenosine receptor antagonist (/sup 3/H)-8-cyclopentyl-1,3-dipropylxanthine was used as radioligand. Binding of the radioligand to intact myocytes was rapid, reversible, and saturable with a binding capacity of 40,000 binding sites per cell. The dissociation constant of the radioligand was 0.48 nM. The adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, xanthine amine congener, and theophylline were competitive inhibitors with affinities in agreement with results obtained for A1 receptors in other tissues. Competition experiments using the adenosine receptor agonists R-N(6)-phenylisopropyladenosine, 5'-N-ethylcarboxamidoadenosine, and S-N(6)-phenylisopropyladenosine gave monophasic displacement curves with Ki values of 50 nM, 440 nM, and 4,300 nM, which agreed well with the GTP-inducible low affinity state in cardiac membranes. The low affinity for agonists was not due to agonist-induced desensitization, and correlated well with the corresponding IC50 values for the inhibition of cyclic AMP accumulation by isoprenaline. It is suggested that only a low affinity state of A1 receptors can be detected in intact rat myocytes due to the presence of high concentrations of guanine nucleotides in intact cells.
Circadian rhythm in adenosine A1 receptor of mouse cerebral cortex
1991-01-01
In order to investigate diurnal variation in adenosine A1 receptors binding parameters, Bmax and Kd values of specifically bound N6-cyclohexyl-[3H]adenosine were determined in the cerebral cortex of mice that had been housed under controlled light-dark cycles for 4 weeks. Significant differences were found for Bmax values measured at 3-hr intervals across a 24-h period, with low Bmax values during the light period and high Bmax values during the dark period. The amplitude between 03.00 and 18.00 hr was 33%. No substantial rhythm was found in the Kd values. It is suggested that the changes in the density of A1 receptors could reflect a physiologically-relevant mechanism by which adenosine exerts its modulatory role in the central nervous system
Functional expression of rat adenosine A1 receptor in the dimorphic zygomycete Mucor circinelloides
2003-01-01
Mucor, 7TM expression, biotechnology, membrane protein
1988-04-01
We have derivatized a series of /sup 125/I-labeled 8-phenylxanthines with photoactive aryl azide groups on the 1- or 3-position of the xanthine ring. A 3-azidophenethyl derivative was found to be optimal for use as an antagonist photoaffinity label for adenosine A1 receptors. Following photoactivation, radioactivity was covalently and specifically incorporated into a 34,000-dalton and, to a lesser extent, into a 24,000-dalton polypeptide of rat brain membranes. Photoincorporation into both polypeptides was competitively inhibited by adenosine analogues with a potency order typical of adenosine A1 receptors, but the 24,000-dalton polypeptide bound both agonists and antagonists with lower affinity than the 34,000-dalton polypeptide. Specific photolabeling of receptors in brain membranes of rat, guinea pig, dog, and cow did not show any variation in the 34,000-dalton adenosine receptor binding subunit. The adenosine agonist photoaffinity label (/sup 125/I)N6-(4-azido-3-iodobenzyl)adenosine also specifically photolabeled the 34,000-dalton polypeptide, but photoincorporation of the agonist was less efficient than the antagonist and, unlike the antagonist, was greatly reduced by guanosine 5'-(beta,gamma-imidotriphosphate). The results indicate that the antagonist photoaffinity label may be more useful than agonists particularly for labeling uncoupled receptors.
1988-04-01
We have derivatized a series of /sup 125/I-labeled 8-phenylxanthines with photoactive aryl azide groups on the 1- or 3-position of the xanthine ring. A 3-azidophenethyl derivative was found to be optimal for use as an antagonist photoaffinity label for adenosine A1 receptors. Following photoactivation, radioactivity was covalently and specifically incorporated into a 34,000-dalton and, to a lesser extent, into a 24,000-dalton polypeptide of rat brain membranes. Photoincorporation into both polypeptides was competitively inhibited by adenosine analogues with a potency order typical of adenosine A1 receptors, but the 24,000-dalton polypeptide bound both agonists and antagonists with lower affinity than the 34,000-dalton polypeptide. Specific photolabeling of receptors in brain membranes of rat, guinea pig, dog, and cow did not show any variation in the 34,000-dalton adenosine receptor binding subunit. The adenosine agonist photoaffinity label (/sup 125/I)N6-(4-azido-3-iodobenzyl)adenosine also specifically photolabeled the 34,000-dalton polypeptide, but photoincorporation of the agonist was less efficient than the antagonist and, unlike the antagonist, was greatly reduced by guanosine 5'-(beta,gamma-imidotriphosphate). The results indicate that the antagonist photoaffinity label may be more useful than agonists particularly for labeling uncoupled receptors.
125I-labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors
1988-01-01
A series of 8-phenylxanthine derivatives has been synthesized with oxyacetic acid on the para phenyl position to increase aqueous solubility and minimize nonspecific binding and iodinatable groups on the 1- or 3-position of the xanthine ring. The structure-activity relationship for binding of these compounds to A1 adenosine receptors of bovine and rat brain and A2 receptors of human platelets was examined. The addition of arylamine or photosensitive aryl azide groups to the 3-position of xanthine had little effect on A1 binding affinity with or without iodination, whereas substitutions at the 1-position caused greatly reduced A1 binding affinity. The addition of an aminobenzyl group to the 3-position of the xanthine had little effect on A2 binding affinity, but 3-aminophenethyl substitution decreased A2 binding affinity. Two acidic 3-(arylamino)-8-phenylxanthine ... >>
1988-01-01
We have derivatized a series of 125I-labeled 8-phenylxanthines with photoactive aryl azide groups on the 1- or 3-position of the xanthine ring. A 3-azidophenethyl derivative was found to be optimal for use as an antagonist photoaffinity label for adenosine A1 receptors. Following photoactivation, radioactivity was covalently and specifically incorporated into a 34,000-dalton and, to a lesser extent, into a 24,000-dalton polypeptide of rat brain membranes. Photoincorporation into both polypeptides was competitively inhibited by adenosine analogues with a potency order typical of adenosine A1 receptors, but the 24,000-dalton polypeptide bound both agonists and antagonists with lower affinity than the 34,000-dalton polypeptide. Specific photolabeling of receptors in brain membranes of rat, guinea pig, dog, and cow did not show any variation in the ... >>
Abolished tubuloglomerular feedback and increased plasma renin in adenosine A1 receptor deficient mice
2001-01-01
2010-01-01
Clinical and preclinical data concur that sleep disruption causes hyperalgesia, but the brain mechanisms through which sleep and pain interact remain poorly understood. Evidence that pontine components of the ascending reticular activating system modulate sleep and nociception encouraged the present study testing the hypothesis that hypocretin-1 (orexin-A) and an adenosine receptor agonist administered into the pontine reticular nucleus, oral part (PnO) each alter thermal nociception. Adult male rats (n = 23) were implanted with microinjection guide tubes aimed for the PnO. The PnO was microinjected with saline (control), hypocretin-1, the adenosine A1 receptor agonist N6-p-sulfophenyladenosine (SPA), the hypocretin receptor-1 antagonist N-(2-Methyl-6-benzoxazolyl)-N-1,5-naphthyridin-4-yl-...
1988-01-01
3-(4-Amino)phenethyl-1-propyl-8-cyclopentylxanthine (BW-A844U) has been synthesized and shown to bind with high affinity to adenosine A1 receptors of bovine brain membranes (KD = 0.23 nM). This compound is highly selective for A1 receptors. The KI for binding to A2 receptors of human platelet membranes is 2.0 microM (A2/A1 ratio = 8700). Radioiodination of the 3-aminophenethyl group resulted in 125I-BW-A844U, a radioligand that retains high affinity for A1 receptors in bovine brain membranes (KD = 0.14 nM) and to 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate-solubilized receptors (KD = 0.34 nM). Specific binding of 125I-BW-A844U represented greater than 90% of the total binding at the KD. From the association constant (K1 = 5.0 X 10(8) M-1min-1) and the dissociation constant (K-1 = 0.064 min-1), the kinetic KD (K-1/K1) in membranes was calculated to ... >>
1990-04-01
The ubiquitous adenosine A2-like binding protein obscures the binding properties of adenosine receptors assayed with 5'-N-({sup 3}H)ethylcarboxamidoadenosine (({sup 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 ({sup 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.
Novel adenosine receptors in rat hippocampus identification and characterization
1985-01-01
2-chloro[3H]adenosine, a stable analog of adenosine, was used to investigate the presence of adenosine receptors in rat hippocampal membranes that may mediate the depressant effects of adenosine on synaptic transmission in this tissue. Equilibrium binding studies reveal the presence of a previously undescribed class of receptors with a K/sub D/ of 4.7 muM and a Bmax of 130 pmol/mg of protein. Binding is sensitive to alkylxanthines and to a number of adenosine-related compounds. The pharmacological properties of this binding site are distinct from those of the A1 and A2 adenosine receptors associated with adenylate cyclase. The results suggest that this adenosine binding site is a novel central purinergic receptor through which adenosine may regulate hippocampal excitability. 50 references, 2 figures, 1 table
Novel adenosine receptors in rat hippocampus identification and characterization
1985-05-06
2-chloro(/sup 3/H)adenosine, a stable analog of adenosine, was used to investigate the presence of adenosine receptors in rat hippocampal membranes that may mediate the depressant effects of adenosine on synaptic transmission in this tissue. Equilibrium binding studies reveal the presence of a previously undescribed class of receptors with a K/sub D/ of 4.7 ..mu..M and a Bmax of 130 pmol/mg of protein. Binding is sensitive to alkylxanthines and to a number of adenosine-related compounds. The pharmacological properties of this binding site are distinct from those of the A1 and A2 adenosine receptors associated with adenylate cyclase. The results suggest that this adenosine binding site is a novel central purinergic receptor through which adenosine may regulate hippocampal excitability. 50 references, 2 figures, 1 table.
2010-01-01
Exogenous adenosine produces potent synaptic inhibition in spinal substantia gelatinosa (SG), a region involved in nociceptive and thermoreceptive mechanisms. To examine the possibility that endogenous adenosine tonically modulates excitatory synaptic transmission in spinal SG, whole-cell, voltage-clamp recordings were made from SG neurons in adult rat spinal cord slices. In all SG neurons sensitive to exogenous adenosine, the adenosine uptake inhibitor, NBTI, mimics adenosine's inhibitory actions on dorsal root evoked EPSCs (eEPSCs) and miniature spontaneous EPSCs (mEPSCs). These inhibitory effects were antagonized by A1 adenosine receptor antagonist, DPCPX. DPCPX also potentates eEPSCs in those SG neurons in which adenosine or adenosine A1 receptor agonists (CHA, CCPA) suppressed eEPSCs....
Effect of adenosine and adenosine analogs on [14C]aminopyrine accumulation by rabbit parietal cells
1989-01-01
Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on [14C]aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. [14C]Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated [14C]aminopyrine accumulation was studied. The effects of N-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on [14C]aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined ... >>
Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina
1987-06-01
Using specific sensitive antisera against adenosine, we have immunocytochemically localized endogenous adenosine to specific layers of rat, guinea pig, monkey, and human retina. Highest adenosine immunoreactivity was observed in ganglion cells and their processes in the optic nerve fiber layer. Substantial staining was also found throughout the inner plexiform layer and in select cells in the inner nuclear layer. Adenosine A1 receptors, labeled with the agonists L-(/sup 3/H)phenylisopropyladenosine and /sup 125/I-labeled hydroxy-phenylisopropyladenosine, were autoradiographically localized. The highest levels of binding sites occurred in the nerve fiber, ganglion cell, and inner plexiform layers of the retina in all the species examined. The distribution of adenosine A1 receptor sites closely parallels that of retinal neurons and fibers containing immunoreactive adenosine. These results suggest a role for endogenous adenosine as a coneurotransmitter in ganglion cells and their fibers in the optic nerve.
Central adenosinergic system involvement in ethanol-induced motor incoordination in mice
1990-12-01
To clarify if the behavioral interaction between ethanol and adenosine reported previously occur centrally or due to a peripheral hemodynamic change, the effect of i.c.v. adenosine agonists, N6-(R-phenylisopropyl)adenosine (R-PIA), N6-(S-phenylisopropyl)adenosine, 5'-(N-cyclopropyl)-carboxamidoadenosine, antagonists, theophylline and 8-p-(sulfophenyl)theophylline as well as enprofylline on ethanol-(i.p.)-induced motor incoordination was evaluated by rotorod. Adenosine agonists and antagonists dose dependently accentuated and attenuated, respectively, ethanol-induced motor incoordination, thereby suggesting a central mechanism of adenosine modulation of this effect of ethanol and confirmed our previous reports in which adenosine agonists and antagonists were given i.p. Enprofylline, a weak adenosine antagonist but potent inhibitor of cyclic AMP phosphodiesterase, did not alter ethanol's motor incoordination, further supporting involvement of brain adenosine receptor mechanism(s) in ethanol-adenosine interactions. Results from R-PIA and N6-(S-phenylisopropyl)adenosine experiments showed nearly a 40-fold greater potency of R-vs. S-diastereoisomer, suggesting predominance of adenosine A1 subtype. However, 5'-(N-cyclopropyl)-carboxamidoadenosine data indicate complexity of the mechanism(s) and point toward an additional involvement of a yet unknown subtype of adenosine A2. No effect of ethanol on blood or brain levels of (3H)R-PIA was noted and sufficient amount of the latter entered the brain to suggest adenosine receptor activation adequate to produce behavioral interaction with ethanol. There was no escape of i.c.v.-administered (3H)R-PIA from brain to the peripheral circulation ruling out a peripheral and supporting a central mechanism of ethanol-adenosine interaction.
Endogenous adenosine and adenosine receptors localized to ganglion cells of the retina
1987-01-01
Using specific sensitive antisera against adenosine, we have immunocytochemically localized endogenous adenosine to specific layers of rat, guinea pig, monkey, and human retina. Highest adenosine immunoreactivity was observed in ganglion cells and their processes in the optic nerve fiber layer. Substantial staining was also found throughout the inner plexiform layer and in select cells in the inner nuclear layer. Adenosine A1 receptors, labeled with the agonists L-[3H]phenylisopropyladenosine and 125I-labeled hydroxy-phenylisopropyladenosine, were autoradiographically localized. The highest levels of binding sites occurred in the nerve fiber, ganglion cell, and inner plexiform layers of the retina in all the species examined. The distribution of adenosine A1 receptor sites closely parallels that of retinal neurons and fibers ... >>
1989-12-01
Adenosine receptors that modulate adenylate cyclase activity have been identified recently in a number of tissues. Adenosine A2 receptor is stimulatory to adenylate cyclase, whereas adenosine A1 receptor is inhibitory to adenylate cyclase. We investigated the effect of adenosine and its analogs on (14C)aminopyrine accumulation by rabbit parietal cells. Rabbit gastric mucosal cells were isolated by enzyme digestion. Parietal cells were enriched by nonlinear percoll gradients. (14C)Aminopyrine accumulation was used as an indicator of acid secretion. The effect of 2-chloroadenosine on histamine-stimulated (14C)aminopyrine accumulation was studied. The effects of N-ethylcarboxamideadenosine, 2-chloroadenosine, stable analogs of adenosine, and adenosine on (14C)aminopyrine accumulation were assessed. Cyclic AMP content of parietal cells was determined by radioimmunoassay. Histamine and carbachol, known secretagogues, stimulated (14C)aminopyrine accumulation. 2-Chloroadenosine did not suppress histamine-stimulated (14C)aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine dose dependently increased (14C)aminopyrine accumulation. The order of potency was N-ethylcarboxamideadenosine greater than 2-chloroadenosine greater than adenosine. 8-Phenyltheophylline and theophylline, adenosine-receptor antagonists, or cimetidine did not have significant effects on the increase of AP uptake induced by 2-chloroadenosine. Coadministration of dipyridamole, and adenosine uptake inhibitor, augmented the effect of adenosine on (14C)aminopyrine accumulation. 2-Chloroadenosine, N-ethylcarboxamideadenosine, and adenosine each induced a significant increase in cellular cyclic AMP. We conclude that there may be adenosine A2 receptors on rabbit parietal cells which modulate gastric acid secretion.
1991-01-01
It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine [(R)-AHPIA] into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the ... >>
2003-01-01
Elevated extracellular adenosine has been found to stimulate hematopoiesis in experimental mice exposed to radiotherapy (gamma-rays), chemotherapy (5-fluorouracil), or combined action of both these modalities (gamma-rays + carboplatin). These findings have been obtained after treatment of the animals with the combination of dipyridamole (DP), preventing the cellular uptake of adenosine, and adenosine monophosphate (AMP), acting as adenosine prodrug. Increased cycling of hematopoietic progenitor cells following the administration of DP + AMP has been shown to represent an important mechanism of acceleration of regeneration of suppressed hematopoiesis. In recent experiments, non-degradable synthetic adenosine receptor agonists, more or less specific for individual subtypes of adenosine receptors (A1, A2A, A2B, and A3 subtypes) have been studied. These ... >>
Auto-anti-idiotypic approach to adenosine receptors
1986-03-01
If an antibody (Abl) binds to and recognizes the same epitopes on a ligand that are recognized by a receptor, then among anti-idiotypic antibodies (Ab2) to Ab1, there may be some that have combining sites that mimic the ligand (internal image). In the case of the acetylcholine receptor and the glucocorticoid receptor these Ab2 antibodies could be raised through a naturally occurring idiotypic network by immunizing mice with the ligands of the receptors. To test the generality of this procedure and to raise antibodies to adenosine receptors, BALB/c mice were immunized with adenosine 6-aminocaproyl-BSA. Hyberidoma cell lines were raised. Cell lines which secreted antibodies that bound to rabbit anti-adenosine antibody were obtained. Two such mAbs, AA18 and AA21, inhibited the binding of (/sup 3/H)adenosine to rabbit anti-adenosine antibody. Furthermore, they bound to rat cerebral cortical membrane and binding could be inhibited by L-phenylisopropyladenosine (L-PIA), an adenosine receptor agonist. Rat cerebral membrane proteins were solubilized with 1% cholic acid, and analyzed by SDS-PAGE and Western blotting. Both AA18 and AA21 recognized a 62 kD band under non-reducing conditions, and 52 kD and 36 kD bands under reducing conditions. The 36 kD band is consistent with the A1 adenosine receptor binding subunit reported by Stiles, et al. The authors results extend the utility of the autoimmune approach for raising antibody to receptors without the necessity of using isolated receptor as immunogen.
1991-04-01
It has previously been shown that covalent incorporation of the photoreactive adenosine derivative (R)-2-azido-N6-p-hydroxy-phenylisopropyladenosine ((R)-AHPIA) into the A1 adenosine receptor of intact fat cells leads to a persistent activation of this receptor, resulting in a reduction of cellular cAMP levels. In contrast, covalent incorporation of (R)-AHPIA into human platelet membranes, which contain only stimulatory A2 adenosine receptors, reduces adenylate cyclase stimulation via these receptors. This effect of (R)-AHPIA is specific for the A2 receptor and can be prevented by the adenosine receptor antagonist theophylline. Binding studies indicate that up to 90% of A2 receptors can be blocked by photoincorporation of (R)-AHPIA. However, the remaining 10-20% of A2 receptors are sufficient to mediate an adenylate cyclase stimulation of up to 50% of the control value. Similarly, the activation via these 10-20% of receptors occurs with a half-life that is only 2 times longer than that in control membranes. This indicates the presence of a receptor reserve, with respect to both the extent and the rate of adenylate cyclase stimulation. These observations require a modification of the models of receptor-adenylate cyclase coupling.
Correlation of adenosine receptor affinities and cardiovascular activity
1987-01-01
Binding affinities of 28 adenosine analogs at A1 adenosine receptors (rat whole brain membranes, [3H]N6-cyclohexyladenosine, CHA), and at A2 adenosine receptors (rat striatal membranes, [3H]NECA) were compared to their EC25 values for decreasing heart rate and increasing coronary flow in the isolated rat heart. Heart rate (an A1 response) correlated with A1 binding affinity but not with A2 binding affinity. Conversely, coronary flow (an A2 response) correlated with A2 binding affinity but now with A1 binding affinity. These results confirm that the brain A1 and A2 receptors, studied by binding methods, bear close similarities to their respective counterparts in the heart, studied by means of functional responses. 12 references, 4 figures, 2 tables
2010-01-01
Adenosine is a neuromodulator which acts through adenosine receptors regulating functions such as inhibition of glutamate release. Adenosine A1 and A2A receptor activations most often regulate opposing actions. Primary rat cortical neurons and rat C6 cells, an astrocytic derived cell line, were exposed to 100mM l-glutamate, and cell viability and transduction pathways mediated by both A1 and A2A receptors were analyzed. Glutamate-induced excitotoxic damage was found only in cortical neurons, with C6 cells preserved. In C6 cells, adenosine A1 and A2A receptors were increased and decreased, respectively. Consequently, A1-mediated adenylyl cyclase inhibition and A2A-mediated adenylyl cyclase stimulation were, respectively, increased and decreased after glutamate exposure. In cortical neurons,...
2010-01-01
Background: Ethanol intake has significant impact on sleep. However, the cellular substrates responsible for sleep promotion following ethanol intake are unknown. The purine nucleoside, adenosine, is responsible for mediating many neuronal and behavioral responses to ethanol. Studies performed in cell cultures suggest that ethanol inhibits equilibrative nucleoside transporter 1 to block the reuptake of adenosine resulting in increased extracellular adenosine. Adenosine also has a pivotal role in sleep regulation. Adenosine acts via A1 receptor to inhibit the wake-promoting neurons of the basal forebrain (BF) resulting in the promotion of sleep. Is ethanol-induced sleep associated with the inhibition of the BF wake-promoting neurons? Do adenosinergic mechanisms in the BF have a role in slee...
Correlation of adenosine receptor affinities and cardiovascular activity
1987-11-16
Binding affinities of 28 adenosine analogs at A1 adenosine receptors (rat whole brain membranes, (/sup 3/H)N6-cyclohexyladenosine, CHA), and at A2 adenosine receptors (rat striatal membranes, (/sup 3/H)NECA) were compared to their EC/sub 25/ values for decreasing heart rate and increasing coronary flow in the isolated rat heart. Heart rate (an A1 response) correlated with A1 binding affinity but not with A2 binding affinity; conversely, coronary flow (an A2 response) correlated with A2 binding affinity but now with A1 binding affinity. These results confirm that the brain A1 and A2 receptors, studied by binding methods, bear close similarities to their respective counterparts in the heart, studied by means of functional responses. 12 references, 4 figures, 2 tables.
http://eprints.jcu.edu.au/10037/1/Ischaemic_tolerance_and_A1_receptor_overexpression.pdf
None Available Publisher: Physiological Society Format: application/pdf Other identifier: Headrick, John P., Willems, Laura, Ashton, Kevin J., Holmgren, Kirsten, Peart, Jason, and Matherne, G. Paul (2003) Ischaemic tolerance in aged mouse myocardium: the role of adenosine and effects of A1 adenosine receptor overexpression. Journal of Physiology, 549 (3). pp. 823-833. ISSN 1469-7793
http://eprints.qut.edu.au/15127/
Herein we report the synthesis and biological evaluation of some potent and selective A1 adenosine receptor agonists, which incorporate a functionalised linker attached to an antioxidant moiety. N6-(2,2,5,5-Tetramethylpyrrolidin-1-yloxyl-3-ylmethyl)adenosine (VCP28, 2e) proved to be an agonist with high affinity (Ki = 50 nM) and good selectivity (A3/A1 400) for the A1 adenosine receptor. N6-[4-[2-[1,1,3,3-Tetramethylisoindolin-2-yloxyl-5-amido]ethyl]phenyl]adenosine (VCP102, 5a) has higher binding affinity (Ki = 7 nM), but lower selectivity (A3/A1 = 3). All compounds bind weakly (Ki > 1 μM) to A2A and A2B receptors. The combination of A1 agonist activity and antioxidant activity has the potential to produce cardioprotective effects. Publisher: Elsevier Relation: DOI:10.1016/j.bmcl.2007.07.035; Gregg, Alison D. and Bottle, Steven E. and Devine, Shane M. and Figler, Heidi and Linden, Joel and White, Paul and Pouton, Colin W. and Urmaliya, Vijay and Scammells, Peter J. (2007) Dual acting antioxidant A1 adenosine receptor agonists. Bioorganic & Medicinal Chemistry Letters, 17(9). pp. 5437-5441. Format: application/pdf Rights: Copyright 2007 Elsevier; Reproduced in accordance with the copyright policy of the publisher.
1985-03-01
Nucleoside transport sites in rat brain membrane preparations were labeled with (/sup 3/H)nitrobenzylthioinosine ((/sup 3/H) NBI), a potent inhibitor of nucleoside transport systems. The membranes contained a single class of very high affinity binding sites with K/sub D/ and B/sub max/ values of 0.06 nM and 147 fmol/mg of protein, respectively. The displacement of (/sup 3/H)NBI binding by various nucleosides, adenosine receptor agonists and antagonists, and known nucleoside transport inhibitors was examined. The K/sub i/ values (micromolar concentration) of (/sup 3/H)NBI displacement by the nucleosides tested were: adenosine, 3.0; inosine, 160; thymidine, 240; uridine, 390; guanosine, 460; and cytidine, 1000. These nucleosides displayed parallel displacement curves indicating their interaction with a common site labeled by (/sup 3/H)NBI. The nucleobases, hypoxanthine and adenine, exhibited K/sub i/ values of 220 and 3640 microM, respectively. Adenosine receptor agonists exhibited moderate affinities for the (/sup 3/H)NBI site, whereas the adenosine receptor antagonists, caffeine, theophylline, and enprofylline, were ineffective displacers. The K/sub i/ values for cyclohexyladenosine, (+)- and (-)-phenylisopropyladenosine, 2-chloroadenosine, and adenosine 5'-ethylcarboxamide were 0.8, 0.9, 2.6, 12, and 54 microM, respectively. These affinities and the rank order of potencies indicate that (/sup 3/H)NBI does not label any known class of adenosine receptors (i.e., A1, A2, and P). The K/sub i/ values of other nucleoside transport inhibitors were: nitrobenzylthioguanosine, 0.05 nM; dipyridamole, 16 nM; papaverine, 3 microM; and 2'-deoxyadenosine, 22 microM. These results indicate that (/sup 3/H)NBI binds to a nucleoside transporter in brain which specifically recognizes adenosine as its preferred endogenous substrate. This ligand may aid in the identification of CNS neural systems that selectively accumulate adenosine and thereby control adenosinergic function.
1986-01-01
Analysis of (-) [125]iodo-N6-(4-hydroxyphenylisopropyl)-adenosine [( 125I]HPIA) binding to purified sarcolemmal preparations of guinea pig and bovine hearts revealed two classes of binding sites when unlabeled iodo-HPIA (100 mumol/l) was used as non-specific binding marker. In the presence of 1 mmol/l theophylline, however, only the high affinity component was detected. Adenosine receptor agonists caused biphasic displacement of [125I]HPIA binding, with a high affinity potency rank order typical of interaction with A1-adenosine receptors. Biphasic competition curves were also observed with 8-phenyltheophylline and isobutylmethylxanthine, whereas the theophylline curve was monophasic up to 1 mmol/l. In brain membranes, specific binding of [125I]HPIA as well as of [3H]PIA was further reduced when ... >>
Effects of Adenosine Receptor Antagonists on Survival in Amitriptyline-poisoned Mice
2010-01-01
Objective: We investigated the effects of adenosine receptor antagonists on survival rates in a mouse model of amitriptyline poisoning. Materials and Methods: In the preliminary study, amitriptyline was given at doses of 75, 100, and 125 mg/ kg to mice intraperitoneally (i.p.; n = 20 for each dose) to determine the lethal dose at 50% (LD50). Different doses (1, 3, and 5 mg/kg) of DPCPX (selective adenosine A1 antagonists, n = 10 for each dose, total n = 30) or CSC (selective adenosine A2a antagonists, n = 10 for each dose, total n = 30) were given i.p. to find the nonlethal dose. After the administration of the LD50 dose of amitriptyline (125 mg/kg), mice were treated with DPCPX (3 mg/kg), CSC (3 mg/kg), saline, or DMSO (dimethyl sulfoxide) (n = 25 for each group). Mice were observed durin...
2010-01-01
Exogenous zinc can protect cardiac cells from reperfusion injury, but the exact roles of endogenous zinc in the pathogenesis of reperfusion injury and in adenosine A2 receptor activation-induced cardioprotection against reperfusion injury remain unknown. Adenosine A1/A2 receptor agonist 5'-(N-ethylcarboxamido) adenosine (NECA) given at reperfusion reduced infarct size in isolated rat hearts subjected to 30min ischemia followed by 2h of reperfusion. This effect of NECA was partially but significantly blocked by the zinc chelator N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN), and ZnCl2 given at reperfusion mimicked the effect of NECA by reducing infarct size. Total tissue zinc concentrations measured with inductively coupled plasma optical emission spectroscopy (ICPOES) were de...
1982-09-01
Adenosine (A1) receptor binding sites have been localized in rat brain by an in vitro light microscopic autoradiographic method. The binding of (/sup 3/H)N6-cyclohexyladenosine to slide-mounted rat brain tissue sections has the characteristics of A1 receptors. It is saturable with high affinity and has appropriate pharmacology and stereospecificity. The highest densities of adenosine receptors occur in the molecular layer of the cerebellum, the molecular and polymorphic layers of the hippocampus and dentate gyrus, the medial geniculate body, certain thalamic nuclei, and the lateral septum. High densities also are observed in certain layers of the cerebral cortex, the piriform cortex, the caudate-putamen, the nucleus accumbens, and the granule cell layer of the cerebellum. Most white matter areas, as well as certain gray matter areas, such as the hypothalamus, have negligible receptor concentrations. These localizations suggest possible central nervous system sites of action of adenosine.
1989-06-01
Adenosine receptors in a spontaneously contracting atrial myocyte culture from 14-day chick embryos were characterized by radioligand binding studies and by examining the involvement of G-protein in coupling these receptors to a high-affinity state and to the adenylate cyclase and the myocyte contractility. Binding of the antagonist radioligand (3H)-8-cyclopentyl-1,3-diproylxanthine ((3H)CPX) was rapid, reversible and saturable and was to a homogeneous population of sites with a Kd value of 2.1 +/- 0.2 nM and an apparent maximum binding of 26.2 +/- 3 fmol/mg of protein (n = 10, +/- S.E.). Guanyl-5-yl-(beta, gamma-imido)diphosphate had no effect on either the Kd or the maximum binding and CPX reversed the N6-R-phenyl-2-propyladenosine-induced inhibition of adenylate cyclase activity and contractility, indicating that (3H) CPX is an antagonist radioligand. Competition curves for (3H) CPX binding by a series of reference adenosine agonists were consistent with labeling of an A1 adenosine receptor and were better fit by a two-site model than by a one-site model. ADP-ribosylation of the G-protein by the endogenous NAD+ in the presence of pertussis toxin shifted the competition curves from bi to monophasic with Ki values similar to those of the KL observed in the absence of prior pertussis intoxication. The adenosine agonists were capable of inhibiting both the adenylate cyclase activity and myocyte contractility in either the absence or the presence of isoproterenol. The A1 adenosine receptor-selective antagonist CPX reversed these agonist effects. The order of ability of the reference adenosine receptor agonists in causing these inhibitory effects was similar to the order of potency of the same agonists in inhibiting the specific (3H)CPX binding (N6-R-phenyl-2-propyladenosine greater than N6-S-phenyl-2-propyladenosine or N-ethyladenosine-5'-uronic acid).
http://hdl.handle.net/10072/6136
The genesis of the ischaemia intolerant phenotype in aged myocardium is poorly understood. We tested the hypothesis that impaired adenosine-mediated protection contributes to ischaemic intolerance, and examined whether this is countered by A1 adenosine receptor (A1AR) overexpression. Responses to 20 min ischaemia and 45 min reperfusion were assessed in perfused hearts from young (2 4 months) and moderately aged (16 18 months) mice. Post-ischaemic contractility was impaired by ageing with elevated ventricular diastolic (32 ± 2 vs. 18 ± 2 mmHg in young) and reduced developed (37 ± 3 vs. 83 ± 6 mmHg in young) pressures. Lactate dehydrogenase (LDH) loss was exaggerated (27 ± 2 vs. 16 ± 2 IU g1in young) whereas the incidence of tachyarrhythmias was similar in young (15 ± 1 %) and aged hearts (16 ± 1 %). Functional analysis confirmed equipotent effects of 50 ¼m adenosine at A1 and A2 receptors in young and aged hearts. Nonetheless, while 50 ¼m adenosine improved diastolic (5 ± 1 mmHg) and developed pressures (134 ± 7 mmHg) and LDH loss (6 ± 2 IU g1) in young hearts, it did not alter these variables in the aged group. Adenosine did attenuate arrhythmogenesis for both ages (to 10 %). In contrast to adenosine, 50 ¼m diazoxide reduced ischaemic damage and arrhythmogenesis for both ages. Contractile and anti-necrotic effects of adenosine were limited by 100 ¼m 5-hydroxydecanoate (5-HD) and 3 ¼m chelerythrine. Anti-arrhythmic effects were limited by 5-HD but not chelerythrine. Non-selective (100 ¼m 8-sulfophenyltheophylline) and A1-selective (150 nm 8-cyclopentyl-1,3-dipropylxanthine) adenosine receptor antagonism impaired ischaemic tolerance in young but not aged hearts. Quantitative real-time PCR and radioligand analysis indicated that impaired protection is unrelated to changes in A1AR mRNA transcription, or receptor density (8 fmol mg1 protein in both age groups). However, A1AR overexpression improved tolerance for both ages, restoring adenosine-mediated protection. These data reveal impaired protection via exogenous and endogenous adenosine contributes to ischaemic intolerance with ageing. This is independent of A1AR expression, and involves ineffective activation of a 5-HD-/diazoxide-sensitive process. The effects of A1AR overexpression indicate that the age-related failure in signalling can be overcome. There is increasing evidence of a decline in myocardial tolerance to injury with ageing. A reduction in the 'intrinsic' tolerance to ischaemic insult is supported by data from animal models (Pahor et al. 1985; Frolkis et al. 1991; Misare et al. 1992; Lesnefsky et al. 1994; Tani et al. 1997; Headrick, 1998; Abete et al. 1999; Rosenfeldt et al. 2002) and humans (Mariani et al. 2000; Rosenfeldt et al. 2002). The molecular basis of this intolerant phenotype is unclear, but it may involve multiple alterations including mitochondrial abnormalities (Lesnefsky et al. 2001), impaired anti-oxidant responses (Boucher et al. 1998; Coombes et al. 2000; Lesnefsky et al. 2001), loss of proteasome function (Bulteau et al. 2002) and modified Ca2+ handling (Cain et al. 1998). One possibility receiving increased attention is impairment of intrinsic cardioprotective responses (Abete et al. 1996; Gao et al. 2000; Schulman et al. 2001; Lee et al. 2002). This may be a particularly important factor since it may impact directly on the therapeutic approach to ischaemic injury in aged hearts. It is increasingly evident that conventional therapeutic strategies developed through findings in young tissues and subjects may not be relevant in aged subjects (Rosenfeldt et al. 2002). Adenosine is an important determinant of ischaemic (Zhao et al. 1993, 1994; Peart & Headrick, 2000) or hypoxic tolerance (Matherne et al. 1996). We previously acquired evidence that altered adenosine handling might contribute to impaired ischaemic tolerance with age (Headrick, 1998), and more recent evidence supports an age-related decline in adenosine-mediated protec Publisher: Blackwell Publishing Ltd.; UK; http://www.blackwell-synergy.com/doi/full/10.1113/jphysiol.2003.041541 Relation: The Journal of Physiology; 823; 833; 549 Other identifier: 0022-3751 Language: en_AU Rights: Copyright 2003 Blackwell Publishing. The definitive version is available at [www.blackwell-synergy.com.]
http://hdl.handle.net/10072/29784
Extracellular adenosine concentrations increase within the heart during ischemia, and any exogenous adenosine receptor agonists therefore work in the context of significant local agonist concentrations. We evaluated the interactions between A1, A2A, A2B, and A3 receptors in the presence and absence of adenosine deaminase (ADA, which is used to remove endogenous adenosine) in a cardiac cell ischemia model. Simulated ischemia (SI) was induced by incubating H9c2(2-1) cells in SI medium for 12 hours in 100% N2 gas before assessment of necrosis using propidium iodide (5 μM) or apoptosis using AnnexinV-PE flow cytometry. N6-Cyclopentyladenosine (CPA; 10-7M) and N6-(3-iodobenzyl) adenosine-5′-N-methyluronamide (IB-MECA; 10-7M) reduced the proportion of nonviable cells to 30.87 ± 2.49% and 35.18 ± 10.30%, respectively (% of SI group). In the presence of ADA, the protective effect of CPA was reduced (62.82 ± 3.52% nonviable), whereas the efficacy of IB-MECA was unchanged (35.81 ± 3.84% nonviable; P < 0.05, n = 3-5, SI vs. SI + ADA). The protective effects of CPA and IB-MECA were abrogated in the presence of their respective antagonists DPCPX (8-cyclopentyl-1,3-dipropylxanthine) and MRS1191 [3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate], whereas A2A and A2B agonists had no significant effect. CPA-mediated protection was abrogated in the presence of both A2A (ZM241385, 4-(2-[7-amino-2-(2-furyl)[1,2,4]triazolo[2,3-a][1,3,5]triazin-5-lamino]ethyl)phenol; 50 nM) and A2B (MRS1754, 8-[4-[((4-cyanophenyl)carbamoylmethyl)oxy]phenyl]-1,3-di(n-propyl)xanthine; 200 nM) antagonists (n = 3-5, P < 0.05). In the absence of endogenous adenosine, significant protection was observed with CPA in presence of CGS21680 (4-[2-[[6-amino-9-(N-ethyl-b-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid) or LUF5834 [2-amino-4-(4-hydroxyphenyl)-6-(1H-imidazol-2-ylmethylsulfanyl)pyridine-3,5-dicarbonitrile] (P < 0.05 vs. SI + ADA + CPA). Apoptosis (14.35 ± 0.15% of cells in SI + ADA group; P < 0.05 vs. control) was not significantly reduced by CPA or IB-MECA. In conclusion, endogenous adenosine makes a significant contribution to A1 agonist-mediated prevention of necrosis in this SI model by cooperative interactions with both A2A and A2B receptors but does not play a role in A3 agonist-mediated protection. Publisher: http://dx.doi.org/10.1097/FJC.0b013e3181a443e2; Lippincott Williams & Wilkins; United States; http://journals.lww.com/cardiovascularpharm/pages/default.aspx Contributor: Michael R Rosen Relation: 5; Journal of Cardiovascular Pharmacology; 424; 433; Y; 53 Other identifier: 0160-2446 Language: en_AU Rights: Y
http://eprints.jcu.edu.au/10034/1/genetic_deletion_of_A1_receptor.pdf
Adenosine receptors may be important determinants of intrinsic ischemic tolerance. Genetically modified mice were used to examine effects of global A1 adenosine receptor (A1AR) knockout (KO) on function and ischemic tolerance in perfused mouse hearts. Baseline contractile function and heart rate were unaltered by A1AR KO, which was shown to abolish the negative chronotropic effects of 2-chloroadenosine (A1AR-mediated) without altering A2 adenosine receptor–mediated coronary dilation. Tolerance to 25 minutes global normothermic ischemia (followed by 45 minutes reperfusion) was significantly limited by A1AR KO, with impaired contractile recovery (reduced by {approx}25%) and enhanced lactate dehydrogenase (LDH) efflux (increased by {approx}100%). Functional effects of A1AR KO involved worsened systolic pressure development with little to no change in diastolic dysfunction. In contrast, cardiac specific A1AR overexpression enhanced ischemic tolerance with a primary action on diastolic dysfunction. Nonselective receptor agonism (10 µmol/L 2-chloroadenosine) protected wild-type and also A1AR KO hearts (albeit to a lesser extent), implicating protection via subtypes additional to A1ARs. However, A1AR KO abrogated effects of 2-chloroadenosine on ischemic contracture and diastolic dysfunction. These data are the first demonstrating global deletion of the A1AR limits intrinsic myocardial resistance to ischemia. Data indicate the function of intrinsically activated A1ARs appears primarily to be enhancement of postischemic contractility and limitation of cell death. Publisher: Lippincott Williams & Wilkins Format: application/pdf Other identifier: Reichelt, Melissa E., Willems, Laura, Molina, Jose G., Sun, Chun-Xiao, Noble, Janci C., Ashton, Kevin J., Schnermann, Jurgen, Blackburn, Michael R., and Headrick, John P. (2005) Genetic deletion of the A1 adenosine receptor limits myocardial ischemic tolerance. Circulation Research, 96 . pp. 363-367. ISSN 1524-4571
2010-01-01
Fluorescence polarization (FP) assay has many advantages over the traditional radioreceptor binding studies. We developed an A2A adenosine receptor (AR) FP assay using a newly synthesized fluorescent antagonist of the A2AAR (MRS5346), a pyrazolo[4,3-e][1,2,4]triazolo[1,5-c]pyrimidin-5-amine derivative conjugated to the fluorescent dye Alexa Fluor-488. MRS5346 displayed a Ki value of 111+/-16nM in radioligand binding using [^3H]CGS21680 and membranes prepared from HEK293 cells stably expressing the human A2AAR. In a cyclic AMP functional assay, MRS5346 was shown to be an A2AAR antagonist. MRS5346 did not show any effect on A1 and A3 ARs in binding or the A2BAR in a cyclic AMP assay at 10mM. Its suitability as a fluorescent tracer was indicated in an initial observation of an FP signal follo...
http://hdl.handle.net/10072/3472
1. Chronotropic and vasodilatory effects of adenosine receptor activation with 2-chloroadenosine (2-ClAdo) and ²-adrenoceptor activation with isoproterenol were studied in wild-type murine hearts and transgenic hearts overexpressing the A1 adenosine receptor. 2. Treatment of wild-type hearts with 2-ClAdo induced bradycardia (pEC50 6.4±0.2) and vasodilatation (pEC50 7.9±0.1; minimal resistance 2.2±0.2 mmHg/mL per min per g). The A1 receptor-mediated bradycardia was 20-fold more sensitive in transgenic hearts (pEC50 7.7±0.2), whereas coronary vasoactivity of 2-ClAdo was unaltered (pEC50 7.6±0.1). 3. ²-Adrenoceptor stimulation with isoproterenol increased heart rate (pEC50 8.5±0.2; maximal rate 594±23 b.p.m.) and produced vasodilation (pEC50 8.7±0.1; minimal resistance 1.7±0.2 mmHg/mL per min per g) in wild-type hearts. Treatment with 10 IU/mL adenosine deaminase increased the magnitude of the tachycardia (maximal rate 653±27 b.p.m.) without altering potency (pEC50 8.5±0.1). Antagonism of A1 receptors with 10 nmol/L 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) produced a comparable increase in the magnitude of the chronotropic response (maximal rate 695±26 b.p.m.) without altering potency (pEC50 8.3±0.1). 4. Isoproterenol-mediated vasodilatation was unaltered by transgenic A1 receptor overexpression. Overexpression of A1 receptors significantly reduced the maximal heart rate during ²-adrenoceptor stimulation by 35% (to 381±28 b.p.m.) without altering potency (pEC50 8.4±0.2). At 10 nmol/L, DPCPX increased the magnitude of the chronotropic response to isoproterenol in transgenic hearts (maximal heart rate 484±36 b.p.m.) without altering potency (pEC50 8.3±0.2). 5. The data show that transgenic A1 receptor overexpression selectively sensitizes the cardiovascular A1 receptor response and that A1 receptor activation by endogenous adenosine depresses the magnitude, but not potency, of the ²-adrenoceptor-mediated chronotropic response in mouse heart. The A1 receptor-mediated depression of ²-adrenoceptor responsiveness is non-competitive (reduced response magnitude with no change in sensitivity). This indicates that A1 receptor activation non-competitively inhibits effector mechanisms activated by ²-adrenoceptors (e.g. adenylate cyclase) and/or A1 receptors activate unrelated but opposing mechanisms. This inhibitory response may have physiological importance during periods of sympathetic stimulation of cardiac work. Publisher: http://www.blackwell-synergy.com/links/doi/10.1046%2Fj.1440-1681.2000.03218.x; Blackwell Publishing; Australia; http://www.blackwell-synergy.com/doi/full/10.1046/j.1440-1681.2000.03218.x Relation: Clinical and Experimental Pharmacology & Physiology; 185; 190; 27 Other identifier: 0305-1870 Language: en_AU Rights: Copyright 2000 Blackwell Publishing. The definitive version is available at [www.blackwell-synergy.com.]
2010-01-01
Adenosine receptors (ARs) belong to the G-protein-coupled receptor (GPCR) superfamily and consist of four subtypes referred to as A1, A2A, A2B, and A3. It is important to develop potent and selective modulators of ARs for therapeutic applications. In order to develop reliable in silico models that can effectively classify antagonists of each AR, we carried out three machine learning methods: Laplacian-modified naive Bayesian, recursive partitioning, and support vector machine. The results for each classification model showed values high in accuracy, sensitivity, specificity, area under the receiver operating characteristic curve and Matthews correlation coefficient. By highlighting representative antagonists, the models demonstrated their power and usefulness, and these models could be uti...
Attenuated renovascular constrictor responses to angiotensin II in adenosine 1 receptor knockout mice
2003-01-01
In the present experiments we examined the renovascular constrictor effects of ANG II in the chronic and complete absence of A1 adenosine receptors (A1AR) using mice with targeted deletion of the A1AR gene. Glomerular filtration rate (GFR) was not different between A1AR +/+ and A1AR -/- mice under control conditions (450.5 +/- 60 vs. 475.2 +/- 62.5 microl/min) but fell significantly less in A1AR -/- mice during infusion of ANG II at 1.5 ng/min (A1AR +/+: 242 +/- 32.5 microl/min, A1AR -/-: 371 +/- 42 microl/min; P = 0.03). Bolus injection of 1, 10, and 100 ng of ANG II reduced renal blood flow and increased renal vascular resistance significantly more in A1AR +/+ than in A1AR -/- mice. Perfused afferent arterioles isolated from A1AR +/+ mice constricted in response to bath ANG II with an EC50 of 1.5 +/- 0.4 x 10(-10) mol/l, whereas a right shift in the dose-response relationship with an EC50 of 7.3 +/- 1.2 x 10(-10) mol/l (P
2010-01-01
GPR41 is reportedly expressed in murine adipose tissue and mediates short chain fatty acid (SCFA)-stimulated leptin secretion by activating Gai. Here, we agree with a contradictory report in finding no expression of GPR41 in murine adipose tissue. Nevertheless, in the presence of adenosine deaminase to minimise Gai signalling via the adenosine A1 receptor, SCFA stimulated leptin secretion by adipocytes from wild-type but not GPR41 knockout mice. Expression of GPR43 was reduced in GPR41 knockout mice. Acetate but not butyrate stimulated leptin secretion in wild-type mesenteric adipocytes, consistent with mediation of the response by GPR43 rather than GPR41. Pertussis toxin prevented stimulation of leptin secretion by propionate in epididymal adipocytes, implicating Gai signalling mediated b...
2010-01-01
Multivalent dendrimeric conjugates of GPCR ligands may have increased potency or selectivity in comparison to monomeric ligands, a phenomenon that was tested in a model of cytoprotection in mouse HL-1 cardiomyocytes. Quantitative RT-PCR indicated high expression levels of endogenous A1 and A2A adenosine receptors (ARs), but not of A2B and A3ARs. Activation of the heterologously expressed human A3AR in HL-1 cells by AR agonists significantly attenuated cell damage following 4h exposure to H2O2 (750mM) but not in untransfected cells. The A3 agonist IB-MECA (EC50 3.8mM) and the non-selective agonist NECA (EC50 3.9mM) protected A3 AR-transfected cells against H2O2 in a concentration-dependent manner, as determined by lactate dehydrogenase release. A generation 5.5 PAMAM (polyamidoamine) dendri...
Role of neutrophil purinergic receptors in organ dysfunction
2009-01-01
Neutrophils [polymorphonuclear leukocytes (PMNs)] express several purinergic receptors, including the nucleotide receptors P2Y2 and P2X7 and the adenosine receptor subtypes A1, A2A and A3. Activation of these receptors modulates PMN function and ultimately, in concert with other cells, affects the host's innate inflammatory response. PMN activities that can be altered by purinergic receptor stimulation include adhesion, aggregation, migration, phagocytosis, microbicidal function, release of tissue-damaging products and apoptosis. Interventions that alter PMN purinergic receptor stimulation are being developed to reduce organ dysfunction in conditions such as sepsis, ischemiaâreperfusion injury and the acute respiratory distress syndrome.
http://eprints.jcu.edu.au/6718/1/6718_Canyon_%26_Dobson_2005.pdf
Objective: The heart possesses an extraordinary ability to remember short episodes of sublethal ischemia and reperfusion (angina), which protects the myocardium and coronary vasculature from a subsequent lethal insult, a phenomenon known as ischemic preconditioning. A therapeutic goal for more than 2 decades has been to develop a pharmacologic mimetic comparable with ischemic preconditioning. Our aim was to investigate the preconditioning effect of a new combinatorial therapy targeting adenosine A1 receptors and voltage-dependent sodium fast channels in the in vivo rat model of regional ischemia. Methods: Ischemia-reperfusion was achieved by placing a reversible tie around the left coronary artery in anesthetized and ventilated Sprague-Dawley rats (n = 37). Rats were randomly assigned to 1 of 5 groups: (1) saline control (n = 13); (2) ischemic preconditioning (n = 6); (3) lidocaine only (608 μg · kg−1·min−1, n = 5); (4) adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA; 5 μg/kg, n = 7); and (5) CCPA plus lidocaine (n = 6). Ischemic preconditioning was achieved by using 3 cycles of ischemia and reperfusion lasting 3 minutes each. Lidocaine was infused continuously 5 minutes before and throughout 30 minutes of ischemia and ceased at reperfusion. A bolus of CCPA was infused 5 minutes before ligation along with a constant infusion of lidocaine (as above). All animals were reperfused for 120 minutes for infarct size measurement. Results: Fifty-four percent of saline control rats, 17% of ischemic preconditioning-treated rats, and 29% of CCPA-treated rats died during ischemia from ventricular fibrillation. Infarct size of saline control animals was 61% ± 5%. Pretreating with CCPA and lidocaine infusion resulted in no deaths, no severe arrhythmias, and significant infarct size reduction compared with that seen in saline control animals (P < .05). Remarkably, infarct size reduction in CCPA plus lidocaine-treated rats (12% ± 4%) was equivalent to that achieved with ischemic preconditioning (11% ± 3%), whereas infarct size in rats undergoing CCPA-only and lidocaine-only treatments was 42% ± 7% and 60% ± 6%, respectively. Although CCPA plus lidocaine treatment reduced heart rate, mean arterial pressure, and systolic pressure during ischemia, no correlation was found between these variables and infarct size reduction. Conclusion: We conclude that activating adenosine A1 receptor subtype with CCPA and concomitantly modulating sodium fast channels with lidocaine was comparable with ischemic preconditioning and might offer a new therapeutic window to minimize myocardial damage during surgical ischemia and reperfusion. Publisher: Elsevier Format: application/pdf Other identifier: Canyon, Sarah J., and Dobson, Geoffrey P. (2005) Pretreatment with an adenosine A1 receptor agonist and lidocaine: a possible alternative to myocardial ischemic preconditioning. Journal of Thoracic and Cardiovascular Surgery, 130 (2). pp. 371-377. ISSN 1097-685X
2010-01-01
A new series of 9-deazaxanthine derivatives with various substituents at the heterocyclic system were synthesized and evaluated for their binding affinities for the four human recombinant adenosine receptors, A1–A3 subtypes. A number of the 9-deazaxanthines derivatives 3a–m showed moderate-to-high affinity for hA2B receptors, with compound 3f showing a 32-fold selectivity for A2B over A1 and a 2750-fold selectivity for A2B over A2A.
http://hdl.handle.net/10072/6132
Objectives: To identify potential molecular genetic determinants of cardiovascular ischemic tolerance in wild-type and transgenic hearts overexpressing A1 adenosine receptors (A1ARs). Methods: cDNA microarrays were used to explore expression of 1824 genes in wild-type hearts and ischemia-tolerant mouse hearts overexpressing A1ARs. Results: Overexpression of A1ARs reduced post-ischemic contractile dysfunction, limited arrhythmogenesis, and reduced necrosis by 80% in hearts subjected to 30 min global ischemia 60 min reperfusion. Cardioprotection was abrogated by acute A1AR antagonism, and only a small number (19) of genes were modified by A1AR overexpression in normoxic hearts. Ischemia-reperfusion significantly altered expression of 75 genes in wild-type hearts (14 induced, 61 down-regulated), including genes for metabolic enzymes, structural/motility proteins, cell signaling proteins, defense/growth proteins, and regulators of transcription and translation. A1AR overexpression reversed the majority of gene down-regulation whereas gene induction was generally unaltered. Additionally, genes involved in cell defence, signaling and gene expression were selectively modified by ischemia in transgenic hearts (33 induced, 10 down-regulated), possibly contributing to the protected phenotype. Real-time PCR verified changes in nine selected genes, revealing concordance with array data. Transcription of the A1AR gene was also modestly reduced post-ischemia, consistent with impaired functional sensitivity to A1AR stimulation Conclusions: Data are presented regarding the early post-ischemic gene profile of intact heart. Reduced A1AR transcription is observed which may contribute to poor outcome from ischemia. A1AR overexpression selectively modifies post-ischemic gene expression, potentially contributing to ischemic-tolerance. Publisher: Elsevier Science; Netherlands; http://www.elsevier.com/wps/find/journaldescription.cws_home/525398/description#description Relation: Cardiovascular Research; 715; 726; 57 Other identifier: 0008-6363 Language: en_AU Rights: Copyright 2003 Elsevier : Reproduced in accordance with the copyright policy of the publisher : This journal is available online - use hypertext links.
http://hdl.handle.net/10072/6796
None Available Publisher: Steinkopff Verlag; Germany Relation: Basic Research in Cardiology; 232; 238; 97 Other identifier: 0300-8428 Language: en_AU
http://hdl.handle.net/10072/4732
None Available Publisher: Lippincott Williams & Wilkins; United States Relation: 3; Circulation Research: a journal of the American Heart Association; 363; 367; N; 96 Other identifier: 0009-7330 Language: en_AU Rights: Y
http://hdl.handle.net/10072/3452
None Available Publisher: American Physiological Society; USA Relation: American Journal of Physiology: Heart and Circulatory Physiology; H789; H795; 278 Other identifier: 0363-6135 Language: en_AU
http://hdl.handle.net/10072/21913
Ethyl-2-amino-4,5,6,7-tetrahydro-1-benzoselenophene-3-carboxylate (4), has been prepared as a potential dual-acting selenium-containing allosteric enhancer of adenosine A1A receptor binding utilising a modified Gewald reaction. While preliminary testing indicated that 4 is a superior enhancer of A1AR binding than its thiophene counterpart, its instability under mildly acidic conditions is cause for concern. X-Ray crystallography, together with DFT calculations, provide evidence that the decomposition of 4 involves the ring-opening of selenophenium ion (12b) followed by the loss of elemental selenium through a radical chain process. Publisher: http://dx.doi.org/10.1039/b700812k; The Royal Society of Chemistry; Cambridge, UK; http://www.rsc.org/ Relation: 8; Organic and Biomolecular Chemistry; 1276; 1281; Y; 5 Other identifier: 1477-0520 Language: en_AU Rights: Y
http://hdl.handle.net/10072/14103
None Available Publisher: American Physiology Society; Rockville Pike, Bethesda, MD, USA Relation: American Journal of Physiology (Heart and Circulatory Physiology; 1469; 1473; N; 290 Other identifier: 0363-6135 Language: en_AU Rights: Y
http://epublications.bond.edu.au/era_bcr/84
None Available Publisher: ePublications@bond Format: application/pdf Source: ERA - Biomedical and Clinical Health Research
http://epublications.bond.edu.au/era_bcr/2
None Available Publisher: ePublications@bond Format: application/pdf Source: ERA - Biomedical and Clinical Health Research
http://epublications.bond.edu.au/era_bcr/181
None Available Publisher: ePublications@bond Format: application/pdf Source: ERA - Biomedical and Clinical Health Research
http://arrow.monash.edu.au/hdl/1959.1/127519
None Available Publisher: Bentham Science Publishers Ltd Other identifier: monash:19457 Language: eng Source:
http://arrow.monash.edu.au/hdl/1959.1/125016
None Available Publisher: Pergamon Other identifier: monash:18666 Language: eng Source: 0968-0896
http://arrow.monash.edu.au/hdl/1959.1/124857
None Available Publisher: Nature Publishing Group Other identifier: monash:18613 Language: eng Source: 0007-1188
Full Text Available.BackgroundThe Red-headed krait (Bungarus flaviceps, Squamata: Serpentes: Elapidae) is a medically important venomous snake that inhabits South-East Asia. Although the venoms of most species of the snake genus Bungarus have been well characterized, a detailed compositional analysis of B. flaviceps is currently lacking.ResultsHere, we have sequenced 845 expressed sequence tags (ESTs) from the venom gland of a B. flaviceps. Of the transcripts, 74.8% were putative toxins; 20.6% were cellular; and 4.6% were unknown. The main venom protein families identified were three-finger toxins (3FTxs), Kunitz-type serine protease inhibitors (including chain B of β-bungarotoxin), phospholipase A2 (including chain A of β-bungarotoxin), natriuretic peptide (NP), CRISPs, and C-type lectin.ConclusionThe 3FTxs were found to be the major component of the venom (39%). We found eight groups of unique 3FTxs and most of them were different from the well-characterized 3FTxs. We found three groups of Kunitz-type serine protease inhibitors (SPIs); one group was comparable to the classical SPIs and the other two groups to chain B of β-bungarotoxins (with or without the extra cysteine) based on sequence identity. The latter group may be functional equivalents of dendrotoxins in Bungarus venoms. The natriuretic peptide (NP) found is the first NP for any Asian elapid, and distantly related to Australian elapid NPs. Our study identifies several unique toxins in B. flaviceps venom, which may help in understanding the evolution of venom toxins and the pathophysiological symptoms induced after envenomation.
BackgroundThe Red-headed krait (Bungarus flaviceps, Squamata: Serpentes: Elapidae) is a medically important venomous snake that inhabits South-East Asia. Although...Full Text Available
TNF-α and neuropathic pain - a review
Tumor necrosis factor alpha (TNF-α) was discovered more than a century ago, and its known roles have extended from within the immune system to include a neuro-inflammatory domain in the nervous...Full Text Available
TNF-α and neuropathic pain - a review
Full Text Available.Tumor necrosis factor alpha (TNF-α) was discovered more than a century ago, and its known roles have extended from within the immune system to include a neuro-inflammatory domain in the nervous system. Neuropathic pain is a recognized type of pathological pain where nociceptive responses persist beyond the resolution of damage to the nerve or its surrounding tissue. Very often, neuropathic pain is disproportionately enhanced in intensity (hyperalgesia) or altered in modality (hyperpathia or allodynia) in relation to the stimuli. At time of this writing, there is as yet no common consensus about the etiology of neuropathic pain - possible mechanisms can be categorized into peripheral sensitization and central sensitization of the nervous system in response to the nociceptive stimuli. Animal models of neuropathic pain based on various types of nerve injuries (peripheral versus spinal nerve, ligation versus chronic constrictive injury) have persistently implicated a pivotal role for TNF-α at both peripheral and central levels of sensitization. Despite a lack of success in clinical trials of anti-TNF-α therapy in alleviating the sciatic type of neuropathic pain, the intricate link of TNF-α with other neuro-inflammatory signaling systems (e.g., chemokines and p38 MAPK) has indeed inspired a systems approach perspective for future drug development in treating neuropathic pain.
Structure-Based Discovery of A2A Adenosine Receptor Ligands
2010-05-13
The recent determination of X-ray structures of pharmacologically relevant...Full Text Available
Structure-Based Discovery of A2A Adenosine Receptor Ligands
2010-05-13
Full Text Available.The recent determination of X-ray structures of pharmacologically relevant GPCRs has made these targets accessible to structure-based ligand discovery. Here we explore whether novel chemotypes may be discovered for the A2A adenosine receptor, based on complementarity to its recently determined structure. The A2A adenosine receptor signals in the periphery and the CNS, with agonists explored as anti-inflammatory drugs and antagonists explored for neurodegenerative diseases. We used molecular docking to screen a 1.4 million compound database against the X-ray structure computationally and tested 20 high-ranking, previously unknown molecules experimentally. Of these 35% showed substantial activity with affinities between 200 nM and 9 μM. For the most potent of these new inhibitors, over 50-fold specificity was observed for the A2A versus the related A1 and A3 subtypes. These high hit rates and affinities at least partly reflect the bias of commercial libraries toward GPCR-like chemotypes, an issue that we attempt to investigate quantitatively. Despite this bias, many of the most potent new ligands were novel, dissimilar from known ligands, providing new lead structures for modulation of this medically important target.
Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury
2010-05-01
Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation...Full Text Available
Signaling through the A2B Adenosine Receptor Dampens Endotoxin-Induced Acute Lung Injury
2010-05-01
Full Text Available.Sepsis and septic acute lung injury are among the leading causes for morbidity and mortality of critical illness. Extracellular adenosine is a signaling molecule implicated in the cellular adaptation to hypoxia, ischemia or inflammation. Therefore, we pursued the role of the A2B adenosine receptor (A2BAR) as potential therapeutic target in endotoxin-induced acute lung injury. We gained initial insight from in vitro studies of cultured endothelia or epithelia exposed to inflammatory mediators showing time-dependent induction of the A2BAR (up to 12.9±3.4-fold, p<0.05). Similarly, murine studies of endotoxin-induced lung injury identified an almost 4.6-fold induction of A2BAR transcript and corresponding protein induction with LPS-exposure. Studies utilizing A2BAR promoter constructs and RNA-protection assays indicated that A2BAR induction involved mRNA stability. Functional studies of LPS-induced lung injury revealed that pharmacological inhibition or genetic deletion of the A2BAR was associated with dramatic increases in lung inflammation and histologic tissue injury. Studies of A2BAR-bone marrow chimeric mice suggested pulmonary A2BAR signaling in lung protection. Finally, studies with a specific A2BAR agonist (BAY 60-6583) demonstrated attenuation of lung inflammation and pulmonary edema in wild-type but not in gene-targeted mice for the A2BAR. These studies suggest the A2BAR as potential therapeutic target in the treatment of endotoxin-induced forms of acute lung injury.
Signaling from Axon Guidance Receptors
2010-05-01
Full Text Available.Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote axon regeneration after injury or disease. Signaling mechanisms that act downstream of four prominent families of axon guidance cues—netrins, semaphorins, ephrins, and slits—have been extensively studied in both invertebrate and vertebrate model systems. Although details of these signaling mechanisms are still fragmentary and there appears to be considerable diversity in how different guidance receptors regulate the motility of the axonal growth cone, a number of common themes have emerged. Here, we review recent insights into how specific receptors for each of these guidance cues engage downstream regulators of the growth cone cytoskeleton to control axon guidance.
Signaling from Axon Guidance Receptors
2010-05-01
Determining how axon guidance receptors transmit signals to allow precise pathfinding decisions is fundamental to our understanding of nervous system development and may suggest new strategies to promote...Full Text Available
Regulation of hepatic gene expression by saturated fatty acids
2010-04-01
Full Text Available.AbstractDiets rich in saturated fatty acids have long been associated with increased plasma cholesterol concentrations and hence increased risk of cardiovascular disease. More recently, they have also been suggested to promote the development of non-alcoholic fatty liver disease. While there is now considerable evidence to suggest that polyunsaturated fatty acids exert many of their effects through regulating the activity of transcription factors, including peroxisome proliferator activated receptors, sterol regulatory binding proteins (SREBPs) and liver X receptor, our understanding of how saturated fatty acids act is still limited. Here we review the potential mechanisms whereby saturated fatty acids modulate hepatic lipid metabolism thereby impacting on the synthesis, storage and secretion of lipids. Evidence is presented that their effects are, at least partly, mediated through modulation of the activity of the SREBP family of transcription factors.
Regulation of hepatic gene expression by saturated fatty acids
2010-04-01
AbstractDiets rich in saturated fatty acids have long been associated with increased plasma cholesterol concentrations and hence increased risk of cardiovascular disease. More recently,...Full Text Available
Full Text Available.BackgroundEcto-5'-nucleotidase (NT5E, also known as CD73) hydrolyzes extracellular adenosine 5'-monophosphate (AMP) to adenosine in nociceptive circuits. Since adenosine has antinociceptive effects in rodents and humans, we hypothesized that NT5E, an enzyme that generates adenosine, might also have antinociceptive effects in vivo.ResultsTo test this hypothesis, we purified a soluble version of mouse NT5E (mNT5E) using the baculovirus expression system. Recombinant mNT5E hydrolyzed AMP in biochemical assays and was inhibited by α,β-methylene-adenosine 5'-diphosphate (α,β-me-ADP; IC50 = 0.43 μM), a selective inhibitor of NT5E. mNT5E exhibited a dose-dependent thermal antinociceptive effect that lasted for two days when injected intrathecally in wild-type mice. In addition, mNT5E had thermal antihyperalgesic and mechanical antiallodynic effects that lasted for two days in the complete Freund's adjuvant (CFA) model of inflammatory pain and the spared nerve injury (SNI) model of neuropathic pain. In contrast, mNT5E had no antinociceptive effects when injected intrathecally into adenosine A1 receptor (A1R, Adora1) knockout mice.ConclusionOur data indicate that the long lasting antinociceptive effects of mNT5E are due to hydrolysis of AMP followed by activation of A1R. Moreover, our data suggest recombinant NT5E could be used to treat chronic pain and to study many other physiological processes that are regulated by NT5E.
BackgroundEcto-5'-nucleotidase (NT5E, also known as CD73) hydrolyzes extracellular adenosine 5'-monophosphate (AMP) to adenosine in nociceptive circuits. Since adenosine has antinociceptive...Full Text Available
Pulsatility of insulin release - a clinically important phenomenon
2009-01-01
Abstract The mechanisms and clinical importance of pulsatile insulin release are presented against the background of more than half a century of companionship with the islets of Langerhans. The insulin-secreting b-cells are oscillators with intrinsic variations of cytoplasmic ATP and Ca2+. Within the islets the b-cells are mutually entrained into a common rhythm by gap junctions and diffusible factors (ATP). Synchronization of the different islets in the pancreas is supposed to be due to adjustment of the oscillations to the same phase by neural output of acetylcholine and ATP. Studies of hormone secretion from the perfused pancreas of rats and mice revealed that glucose induces pulses of glucagon anti-synchronous with pulses of insulin and somatostatin. The anti-synchrony may result from ...
Proteomics of Trypanosoma evansi Infection in Rodents
Full Text Available.BackgroundTrypanosoma evansi infections, commonly called ‘surra’, cause significant economic losses to livestock industry. While this infection is mainly restricted to large animals such as camels, donkeys and equines, recent reports indicate their ability to infect humans. There are no World Animal Health Organization (WAHO) prescribed diagnostic tests or vaccines available against this disease and the available drugs show significant toxicity. There is an urgent need to develop improved methods of diagnosis and control measures for this disease. Unlike its related human parasites T. brucei and T. cruzi whose genomes have been fully sequenced T. evansi genome sequence remains unavailable and very little efforts are being made to develop improved methods of prevention, diagnosis and treatment. With a view to identify potential diagnostic markers and drug targets we have studied the clinical proteome of T. evansi infection using mass spectrometry (MS).Methodology/Principal FindingsUsing shot-gun proteomic approach involving nano-lc Quadrupole Time Of Flight (QTOF) mass spectrometry we have identified over 160 proteins expressed by T. evansi in mice infected with camel isolate. Homology driven searches for protein identification from MS/MS data led to most of the matches arising from related Trypanosoma species. Proteins identified belonged to various functional categories including metabolic enzymes; DNA metabolism; transcription; translation as well as cell-cell communication and signal transduction. TCA cycle enzymes were strikingly missing, possibly suggesting their low abundances. The clinical proteome revealed the presence of known and potential drug targets such as oligopeptidases, kinases, cysteine proteases and more.Conclusions/SignificancePrevious proteomic studies on Trypanosomal infections, including human parasites T. brucei and T. cruzi, have been carried out from lab grown cultures. For T. evansi infection this is indeed the first ever proteomic study reported thus far. In addition to providing a glimpse into the biology of this neglected disease, our study is the first step towards identification of diagnostic biomarkers, novel drug targets as well as potential vaccine candidates to fight against T. evansi infections.
Proteomics of Trypanosoma evansi Infection in Rodents
BackgroundTrypanosoma evansi infections, commonly called ‘surra’, cause significant economic losses to livestock industry. While this infection is...Full Text Available
2010-05-01
Objective: To determine the effect of steroidogenic acute regulatory protein (StAR) overexpression on the levels of adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) and ATP-binding...Full Text Available
2010-05-01
Full Text Available.Objective: To determine the effect of steroidogenic acute regulatory protein (StAR) overexpression on the levels of adenosine triphosphate (ATP)-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) in an endothelial cell line (bEnd.3). Methods: The StAR gene was induced in bEnd.3 cells with adenovirus infection. The infection efficiency was detected by fluorescence activated cell sorter (FACS) and fluorescence microscopy. The expressions of StAR gene and protein levels were detected by real-time polymerase chain reaction (PCR) and Western blot. The gene and protein levels of ABCA1 and ABCG1 were detected by real-time PCR and Western blot after StAR overexpression. Results: The result shows that StAR was successfully overexpressed in bEnd.3 cells by adenovirus infection. The mRNA and protein expressions of ABCA1 and ABCG1 were greatly increased by StAR overexpression in bEnd.3 cells. Conclusion: Overexpression of StAR increases ABCA1 and ABCG1 expressions in endothelial cells.
Full Text Available.BackgroundQuantum dots (QDs) are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand probes with fluorescent reporter groups are needed for detection and characterization of G protein-coupled receptors (GPCRs).ResultsSynthetic strategies for coupling the A2A adenosine receptor (AR) agonist CGS21680 (2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxamidoadenosine) to functionalized QDs were explored. Conjugates tethered through amide-linked chains and poly(ethyleneglycol) (PEG) displayed low solubility and lacked receptor affinity. The anchor to the dendron was either through two thiol groups of (R)-thioctic acid or through amide formation to a commercial carboxy-derivatized QD. The most effective approach was to use polyamidoamine (PAMAM) D5 dendrons as multivalent spacer groups, grafted on the QD surface through a thioctic acid moiety. In radioligand binding assays, dendron nucleoside conjugate 11 displayed a moderate affinity at the human A2AAR (Kiapp 1.02 ± 0.15 μM). The QD conjugate of increased water solubility 13, resulting from the anchoring of this dendron derivative, interacted with the receptor with Kiapp of 118 ± 54 nM. The fluorescence emission of 13 occurred at 565 nm, and the presence of the pendant nucleoside did not appreciably quench the fluorescence.ConclusionsThis is a feasibility study to demonstrate a means of conjugating to a QD a small molecular pharmacophore of a GPCR that is relatively hydrophobic. Further enhancement of affinity by altering the pharmacophore or the linking structures will be needed to make useful affinity probes.
BackgroundQuantum dots (QDs) are crystalline nanoparticles that are compatible with biological systems to provide a chemically and photochemically stable fluorescent label. New ligand...Full Text Available
2010-04-01
Location analysis for estrogen receptor-α (ERα)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here,...Full Text Available
2010-04-01
Full Text Available.Location analysis for estrogen receptor-α (ERα)-bound cis-regulatory elements was determined in MCF7 cells using chromatin immunoprecipitation (ChIP)-on-chip. Here, we present the estrogen response element (ERE) sequences that were identified at ERα-bound loci and quantify the incidence of ERE sequences under two stringencies of detection: <10% and 10–20% nucleotide deviation from the canonical ERE sequence. We demonstrate that ∼50% of all ERα-bound loci do not have a discernable ERE and show that most ERα-bound EREs are not perfect consensus EREs. Approximately one-third of all ERα-bound ERE sequences reside within repetitive DNA sequences, most commonly of the AluS family. In addition, the 3-bp spacer between the inverted ERE half-sites, rather than being random nucleotides, is C(A/T)G-enriched at bona fide receptor targets. Diverse ERα-bound loci were validated using electrophoretic mobility shift assay and ChIP-polymerase chain reaction (PCR). The functional significance of receptor-bound loci was demonstrated using luciferase reporter assays which proved that repetitive element ERE sequences contribute to enhancer function. ChIP-PCR demonstrated estrogen-dependent recruitment of the coactivator SRC3 to these loci in vivo. Our data demonstrate that ERα binds to widely variant EREs with less sequence specificity than had previously been suspected and that binding at repetitive and nonrepetitive genomic targets is favored by specific trinucleotide spacers.
2010-06-01
Full Text Available.Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1−/−) mice accumulate beta-carotene (BC) similarly to humans, whereas wild-type (Bcmo1+/+) mice efficiently cleave BC. Bcmo1−/− mice are therefore suitable to investigate BC-induced alterations in gene expression in lung, assessed by microarray analysis. Bcmo1−/− mice receiving control diet had increased expression of inflammatory genes as compared to BC-supplemented Bcmo1−/− mice and Bcmo1+/+ mice that received either control or BC-supplemented diets. Differential gene expression in Bcmo1−/− mice was confirmed by real-time quantitative PCR. Histochemical analysis indeed showed an increase in inflammatory cells in lungs of control Bcmo1−/− mice. Supported by metabolite and gene-expression data, we hypothesize that the increased inflammatory response is due to an altered BC metabolism, resulting in an increased vitamin A requirement in Bcmo1−/− mice. This suggests that effects of BC may depend on inter-individual variations in BC-metabolizing enzymes, such as the frequently occurring human polymorphisms in BCMO1.
2010-06-01
Beta-carotene 15,15′-monooxygenase 1 knockout (Bcmo1−/−) mice accumulate beta-carotene (BC) similarly...Full Text Available
2010-01-01
The primary amino acid sequence of the hepatitis B virus (HBV) proteome was searched for identity spots in the human proteome by using the Protein Information Resource database. We find that the HBV...Full Text Available
2010-01-01
Full Text Available.The primary amino acid sequence of the hepatitis B virus (HBV) proteome was searched for identity spots in the human proteome by using the Protein Information Resource database. We find that the HBV polyprotein shares sixty-five heptapeptides, one octapeptide, and one nonapeptide with the human proteins. The viral matches are disseminated among fundamental human proteins such as adhesion molecules, leukocyte differentiation antigens, enzymes, proteins associated with spermatogenesis, and transcription factors. As a datum of special interest, a number of peptide motifs are shared between the virus- and brain-specific antigens involved in neuronal protection. This study may help to evaluate the potential cross reactions and side effects of HBV antigen-based vaccines.
Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma
PurposePreviously we showed that epithelial cell adhesion molecule (Ep-CAM), a cell surface molecule, was highly expressed in primary retinoblastoma tumors. In the present study,...Full Text Available
Genome-wide changes accompanying the knockdown of Ep-CAM in retinoblastoma
Full Text Available.PurposePreviously we showed that epithelial cell adhesion molecule (Ep-CAM), a cell surface molecule, was highly expressed in primary retinoblastoma tumors. In the present study, we studied the genes regulated by Ep-CAM in a retinoblastoma Y79 cell line in vitro using a combination of short interference RNA and microarray technology.MethodsFlow cytometry, quantitative reverse transcriptase PCR (Q-RT–PCR), and immunohistochemistry were performed to confirm the Ep-CAM re-expression in the Y79 cells treated with 5′-azacytidine (AZC). Ep-CAM expression in AZC-treated Y79 cells was silenced using synthetic anti-Ep-CAM short interference RNA, and whole genome microarray was performed to determine the gene expression changes post Ep-CAM knockdown. Ep-CAM inhibition was confirmed by Q-RT–PCR, western blotting, and immunofluorescence.ResultsEp-CAM expression was significantly restored in Y79 cells on day 5 of AZC treatment. Ep-CAM inhibition significantly affected Y79 cell proliferation. We identified 465 upregulated genes (≥1.0 fold) and 205 downregulated genes (≤0.5 fold) in response to knockdown of Ep-CAM. These genes regulate several aspects of tumor function, including cell survival/proliferation, DNA replication/transcription, apoptosis, and angiogenesis. Quantitative pathway analysis using Biointerpreter further revealed that the most pronounced effect of Ep-CAM knockdown was deregulation of pathways that include mitogen-activated protein kinase (MAP) kinase and tumor protein 53 (P53) pathways. Real-time Q-RT–PCR confirmed microarray gene expression changes for selected genes.ConclusionsEp-CAM silencing significantly decreases Y79 cell proliferation and revealed a wide network of deregulated pathways in vitro. Future studies targeting Ep-CAM gene expression in vivo will help to delineate the mechanisms associated with Ep-CAM gene function in neoplastic transformation and define the potential for Ep-CAM-based molecular intervention in retinoblastoma patients.
Fatty Acid- and Cholesterol Transporter Protein Expression along the Human Intestinal Tract
Full Text Available.BackgroundProtein distribution profiles along the human intestinal tract of transporters involved in the absorption of cholesterol and long-chain fatty acids (LCFA) have been scarcely evaluated.Methodology/Principal FindingsIn post-mortem samples from 11 subjects, intestinal transporter distribution profiles were determined via Western Blot. Differences in transporter protein levels were statistically tested using ANOVA and Tukey's Post Hoc comparisons. Levels in all segments were expressed relative to those in duodenum. Except for ABCG5 and FATP4, levels (mean±SEM) were the highest in the ileum. For ABCA1, ileal levels (1.80±0.26) differed significantly from those in duodenum (P = 0.049) and proximal colon (0.92±0.14; P = 0.029). ABCG8 levels in ileum (1.91±0.30) differed from those in duodenum (P = 0.041) and distal colon (0.84±0.22; P = 0.010) and jejunum (1.64±0.26) tended to be higher than distal colon (0.84±0.22; P = 0.087). Ileal NPC1L1 levels (2.56±0.51) differed from duodenum levels (P = 0.019) and from distal colon (1.09±0.22; P = 0.030). There was also a trend (P = 0.098) for higher jejunal (2.23±0.37) than duodenal NPC1L1 levels. The levels of ABCG5 did not correlate with those of ABCG8. FAT/CD36 levels in ileum (2.03±0.42) differed from those in duodenum (P = 0.017), and proximal and distal colon (0.89±0.13 and 0.97±0.15 respectively; P = 0.011 and P = 0.014). FABPpm levels in ileum (1.04±0.13) differed from proximal (0.64±0.07; P = 0.026) and distal colon (0.66±0.09; P = 0.037).Conclusions/SignificanceThe distribution profiles showed a bell-shape pattern along the GI-tract with the highest levels in ileum for ABCA1, ABCG8, NPC1L1, FATCD36 and FABPm, suggesting a prominent role for ileum in transporter-mediated uptake of cholesterol and LCFAs.
Fatty Acid- and Cholesterol Transporter Protein Expression along the Human Intestinal Tract
BackgroundProtein distribution profiles along the human intestinal tract of transporters involved in the absorption of cholesterol and long-chain fatty acids (LCFA) have been scarcely...Full Text Available
Full Text Available.BackgroundThromboxane A2 (TxA2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can be partly attributed to TxA2-induced Ca2+ influx, which resulted in vascular contraction via Ca2+-calmodulin-MLCK pathway. This study aims to identify the channels that mediate TxA2-induced Ca2+ influx in vascular smooth muscle cells.Methodology/Principal FindingsApplication of U-46619, a thromboxane A2 mimic, resulted in a constriction in endothelium-denuded small mesenteric artery segments. The constriction relies on the presence of extracellular Ca2+, because removal of extracellular Ca2+ abolished the constriction. This constriction was partially inhibited by an L-type Ca2+ channel inhibitor nifedipine (0.5–1 µM). The remaining component was inhibited by L-cis-diltiazem, a selective inhibitor for CNG channels, in a dose-dependent manner. Another CNG channel blocker LY83583 [6-(phenylamino)-5,8-quinolinedione] had similar effect. In the primary cultured smooth muscle cells derived from rat aorta, application of U46619 (100 nM) induced a rise in cytosolic Ca2+ ([Ca2+]i), which was inhibited by L-cis-diltiazem. Immunoblot experiments confirmed the presence of CNGA2 protein in vascular smooth muscle cells.Conclusions/SignificanceThese data suggest a functional role of CNG channels in U-46619-induced Ca2+ influx and contraction of smooth muscle cells.
BackgroundThromboxane A2 (TxA2)-induced smooth muscle contraction has been implicated in cardiovascular, renal and respiratory diseases. This contraction can...Full Text Available
Cardiovascular Dementia - A Different Perspective
The number of dementia patients has been growing in recent years and dementia represents a significant threat to aging people all over the world. Recent research has shown that the number of people...Full Text Available
Cardiovascular Dementia - A Different Perspective
Full Text Available.The number of dementia patients has been growing in recent years and dementia represents a significant threat to aging people all over the world. Recent research has shown that the number of people affected by Alzheimer’s disease (AD) and dementia is growing at an epidemic pace. The rapidly increasing financial and personal costs will affect the world's economies, health care systems, and many families. Researchers are now exploring a possible connection among AD, vascular dementia (VD), diabetes mellitus (type 2, T2DM) and cardiovascular diseases (CD). This correlation may be due to a strong association of cardiovascular risk factors with AD and VD, suggesting that these diseases share some biologic pathways. Since heart failure is associated with an increased risk of AD and VD, keeping the heart healthy may prove to keep the brain healthy as well. The risk for dementia is especially high when diabetes mellitus is comorbid with severe systolic hypertension or heart disease. In addition, the degree of coronary artery disease (CAD) is independently associated with cardinal neuropathological lesions of AD. Thus, the contribution of T2DM and CD to AD and VD implies that cardiovascular therapies may prove useful in preventing AD and dementia.
Full Text Available.BackgroundThe major toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) appear to result from dysregulation of mRNA levels mediated by the aryl hydrocarbon receptor (AHR). Dioxin-like chemicals alter expression of numerous genes in liver, but it remains unknown which lie in pathways leading to major toxicities such as hepatotoxicity, wasting and lethality. To identify genes involved in these responses we exploited a rat genetic model. Rats expressing an AHR splice-variant lacking a portion of the transactivation domain are highly resistant to dioxin-induced toxicities. We examined changes in hepatic mRNA abundances 19 hours after TCDD treatment in two dioxin-resistant rat strains/lines and two dioxin-sensitive rat strains/lines.ResultsResistant rat strains/lines exhibited fewer transcriptional changes in response to TCDD than did rats with wildtype AHR. However, well-known AHR-regulated and dioxin-inducible genes such as CYP1A1, CYP1A2, and CYP1B1 remained fully responsive to TCDD in all strains/lines. Pathway analysis indicated that the genes which respond differently to TCDD between sensitive and resistant rats are mainly involved in lipid metabolism, cellular membrane function and energy metabolism. These pathways previously have been shown to respond differently to dioxin treatment in dioxin-sensitive versus dioxin-resistant rats at a biochemical level and in the differential phenotype of toxicologic responses.ConclusionThe transactivation-domain deletion in dioxin-resistant rats does not abolish global AHR transactivational activity but selectively interferes with expression of subsets of genes that are candidates to mediate or protect from major dioxin toxicities such as hepatotoxicity, wasting and death.
BackgroundThe major toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) appear to result from dysregulation of mRNA levels mediated by the aryl hydrocarbon...Full Text Available
An insight into the sialome of Glossina morsitans morsitans
Full Text Available.BackgroundBlood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules that disarm their host's anti-bleeding defenses (hemostasis), inflammatory and immune reactions. Recent sialotranscriptome analyses (from the Greek sialo = saliva) of blood feeding insects and ticks have revealed that the saliva contains hundreds of polypeptides, many unique to their genus or family. Adult tsetse flies feed exclusively on vertebrate blood and are important vectors of human and animal diseases. Thus far, only limited information exists regarding the Glossina sialome, or any other fly belonging to the Hippoboscidae.ResultsAs part of the effort to sequence the genome of Glossina morsitans morsitans, several organ specific, high quality normalized cDNA libraries have been constructed, from which over 20,000 ESTs from an adult salivary gland library were sequenced. These ESTs have been assembled using previously described ESTs from the fat body and midgut libraries of the same fly, thus totaling 62,251 ESTs, which have been assembled into 16,743 clusters (8,506 of which had one or more EST from the salivary gland library). Coding sequences were obtained for 2,509 novel proteins, 1,792 of which had at least one EST expressed in the salivary glands. Despite library normalization, 59 transcripts were overrepresented in the salivary library indicating high levels of expression. This work presents a detailed analysis of the salivary protein families identified. Protein expression was confirmed by 2D gel electrophoresis, enzymatic digestion and mass spectrometry. Concurrently, an initial attempt to determine the immunogenic properties of selected salivary proteins was undertaken.ConclusionsThe sialome of G. m. morsitans contains over 250 proteins that are possibly associated with blood feeding. This set includes alleles of previously described gene products, reveals new evidence that several salivary proteins are multigenic and identifies at least seven new polypeptide families unique to Glossina. Most of these proteins have no known function and thus, provide a discovery platform for the identification of novel pharmacologically active compounds, innovative vector-based vaccine targets, and immunological markers of vector exposure.
An insight into the sialome of Glossina morsitans morsitans
BackgroundBlood feeding evolved independently in worms, arthropods and mammals. Among the adaptations to this peculiar diet, these animals developed an armament of salivary molecules...Full Text Available
2010-01-01
The mossy fiber synapses onto hippocampal CA3 neurons show unique molecular features and a wide dynamic range of plasticity. Although acute stress has been well recognized to alter bidirectional long-term synaptic plasticity in the hippocampal CA1 region and dentate gyrus, it remains unclear whether the same effect may also occur at the mossy fiber-CA3 synapses. Here, we report that hippocampal slices prepared from adult mice that had experienced an acute unpredictable and inescapable restraint tail-shock stress showed a marked impairment of long-term potentiation (LTP) induced by high-frequency stimulation or adenylyl cyclase activator forskolin. This effect was prevented when animals were submitted to bilateral adrenalectomy or given the glucocorticoid receptor antagonist RU38486 before ...
A functional human Poly(A) site requires only a potent DSE and an A-rich upstream sequence
2010-05-05
We have analysed the sequences required for cleavage and polyadenylation in the intronless melanocortin 4 receptor (MC4R) pre-mRNA. Unlike other intronless genes, 3′end processing of the MC4R...Full Text Available
A functional human Poly(A) site requires only a potent DSE and an A-rich upstream sequence
2010-05-05
Full Text Available.We have analysed the sequences required for cleavage and polyadenylation in the intronless melanocortin 4 receptor (MC4R) pre-mRNA. Unlike other intronless genes, 3′end processing of the MC4R primary transcript is independent of any auxiliary sequence elements and only requires the core poly(A) sequences. Mutation of the AUUAAA hexamer had little effect on MC4R 3′end processing but small changes in the short DSE severely reduced cleavage efficiency. The MC4R poly(A) site requires only the DSE and an A-rich upstream sequence to direct efficient cleavage and polyadenylation. Our observation may be highly relevant for the understanding of how human noncanonical poly(A) sites are recognised. This is supported by a genome-wide analysis of over 10 000 poly(A) sites where we show that many human noncanonical poly(A) signals contain A-rich upstream sequences and tend to have a higher frequency of U and GU nucleotides in their DSE compared with canonical poly(A) signals. The importance of A-rich elements for noncanonical poly(A) site recognition was confirmed by mutational analysis of the human JUNB gene, which contains an A-rich noncanonical poly(A) signal.
Adenosine: Tipping the balance towards hepatic steatosis and fibrosis
2010-01-01
COMMENTARY ON: Adenosine signaling contributes to ethanol induced fatty liver in mice. Zhongsheng Peng et al. J Clin Invest 9 2009; 119:582-94. doi: 10.1172/7CI37409. Copyright 2009 by AMERICAN SOCIETY FOR CLINICAL INVESTIGATION. Reproduced with permission of AMERICAN SOCIETY FOR CLINICAL INVESTIGATION in the format Journal via Copyright Clearance Center. Abstract: Fatty liver is commonly associated with alcohol ingestion and abuse. While the molecular pathogenesis of these fatty changes is well understood, the histochemical and pharmacological mechanisms by which ethanol stimulates these molecular changes remain unknown. During ethanol metabolism, adenosine is generated by the enzyme ecto-5prime-nucleotidase, and adenosine production and adenosine receptor activation are known to play cri...
2007-07-01
Sleep is an essential physiological process. However, the functions of sleep and the endogenous mechanisms involved in sleep regulation are only partially understood. Convergent lines of evidence support the hypothesis that the build-up of sleep propensity during wakefulness and its decline during sleep are associated with alterations in brain adenosine levels and adenosine receptor concentrations. The non-selective A{sub 1} and A{sub 2A} adenosine receptor antagonist caffeine stimulates alertness and is known to attenuate changes in the waking and sleep electroencephalogram (EEG) typically observed after prolonged waking. Several findings point to an important function of the adenosine A{sub 1} receptor (A{sub 1}AR) in the modulation of vigilance states. The A{sub 1}AR is densely expressed in brain regions involved in sleep regulation, and pharmacological manipulations affecting the A{sub 1}AR were shown to influence sleep propensity and sleep depth. However, an involvement of the A{sub 2A} adenosine receptor (A{sub 2A}AR) is also assumed. The distinct functions of the A{sub 1} and A{sub 2A} receptor subtypes in sleep-wake regulation and in mediating the effects of caffeine have not been identified so far. The selective adenosine A{sub 1} receptor antagonist, 8-cyclopentyl-3-(3-{sup 18}Ffluoropropyl)- 1-propylxanthine ({sup 18}F-CPFPX), offers the opportunity to get further insights into adenosinergic mechanisms by in vivo imaging of the A{sub 1}AR subtype with positron emission tomography (PET). The aim of this thesis was to elucidate the role of adenosine A{sub 1} receptors in human sleep regulation, combining {sup 18}F-CPFPX PET brain imaging and EEG recordings, the gold standard in sleep research. It was hypothesized that sleep deprivation would induce adenosine accumulation and/or changes in A{sub 1}AR density. Thus, the question was addressed whether these effects of prolonged wakefulness can be visualized by altered {sup 18}F-CPFPX binding. Moreover, it was investigated whether radioligand uptake might be influenced by caffeine, since caffeine is known to bind to both the A{sub 1} and A{sub 2A} adenosine receptors. A further objective was to examine a possible relationship between A{sub 1}AR binding and changes in waking and sleep EEG during prolonged wakefulness and after caffeine intake. The radiosynthesis of {sup 18}F-CPFPX, performed in analogy to a reported two-step reaction sequence, was validated according to the requirements of good manufacturing practice (GMP). {sup 18}F-CPFPX was obtained in a radiochemical yield of about 6 %, and the specific activity exceeded 80 GBq/{mu}mol. The final product fulfilled all the specifications concerning sterility, apyrogenicity, isotonicity and radiochemical and chemical purity, and thus could be safely applied in humans. A study was performed in ten young healthy male volunteers. The protocol consisted of two baseline nights, followed by a 40-h sleep deprivation and a recovery night. During the nights, sleep was recorded polysomnographically. Across the sleep deprivation period, waking EEG recordings were conducted at 3-h intervals. After 16 h of wakefulness, 300 mg slow-release caffeine or placebo were administered to the participants in a randomized, double-blind design. PET brain imaging with application of {approx} 80 MBq {sup 18}F-CPFPX was performed {approx} 8 h before the second baseline night and the recovery night. Generally, high {sup 18}F-CPFPX accumulation was observed in brain areas with known high A{sub 1}AR density, such as cerebral cortex, thalamus and basal ganglia. In contrast, low radioligand binding occurred in the cerebellum and brainstem, regions with a low A{sub 1}AR concentration. The distribution volumes (DV) of the radioligand, calculated with Logan's graphical analysis (GA), did not differ significantly between the PET scans before and after sleep deprivation in both the placebo and caffeine groups. EEG spectra in the placebo group revealed the typical sleep deprivation induced increase in the markers of sleep homeostasis, namely theta (5-8 Hz) power in waking and delta (1-4 Hz) power in the namuron sleep. These effects were not observed in the subjects who received caffeine. Correlation analysis revealed no relationship between {sup 18}F-CPFPX binding and theta and delta EEG power in waking and nonREM sleep. However, significant negative associations were found between the {sup 18}F-CPFPX uptake and alpha (8-12 Hz) power in both wakefulness and nonREM sleep under baseline conditions (before sleep deprivation). Moreover, in the placebo group a positive correlation was observed between the change in radioligand binding and the change in waking EEG alpha power during sleep deprivation. Such correlations were not found in the caffeine group. Our findings confirm high A{sub 1}AR concentration in brain regions involved in sleep. The results suggest that sleep deprivation and caffeine have no significant effects on A{sub 1}AR occupancy, and challenge the hypothesis of a prominent involvement of the A{sub 1}AR in the homeostatic regulation of sleep. However, they indicate an association between the EEG alpha power density and cerebral A{sub 1}AR binding. EEG alpha activity is known to exhibit high interindividual variability, and power within the alpha frequency range in waking is modulated by the level of alertness. The correlations found for {sup 18}F-CPFPX binding and alpha activity support a role of the A{sub 1}AR in modulating vigilance in humans, but the A{sub 1}AR does not appear to be responsible for the effects of caffeine on waking and sleep EEG. A test and retest study in a larger group of volunteers would be necessary in order to demonstrate reproducibility and also to consolidate the statistical significance of the data. Furthermore, a PET study with the selective A{sub 2A}AR antagonist {sup 11}C-TMSX could be an important step forward in specifying the functions of the A{sub 2A}AR in the regulation of the sleep-wake cycle. (author)
Adenosine receptor subtypes in airways responses of sensitized guinea-pigs to inhaled ovalbumin
2010-01-01
Endogenous adenosine is released in asthmatic patients’ lungs by inhaled allergen, however, its exact role in asthmatic responses or the receptors mediating these responses has not been determined. Our hypothesis was that adenosine released during allergen challenge contributes to the airways responses to inhaled allergen. The effects of selective antagonists of the four adenosine receptor subtypes were investigated on the airways responses of sensitized guinea-pigs to inhaled ovalbumin to ascertain the role of adenosine in these allergen responses, and compared with a corticosteroid, dexamethasone. Early (EAR) and late asthmatic responses (LAR) to inhaled ovalbumin (10 mg/ml) of sensitized, conscious guinea-pigs were recorded by whole body plethysmography following administration o...
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