Adenosine A1 receptor agonists as clinically viable agents for treatment of ischemic brain disorders.
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Adenosine↗
Biomedical subjects
K A Jacobson
Publications and source records attributed to K A Jacobson.
Regulation of A1 adenosine receptors by amiodarone and electrical stimulation in rat myocardial cells in vitro.
The effects of conditions that either increase or decrease heart rate on the pharmacological properties of adenosine receptors in cultured rat myocytes were examined. Levels of A1 adenosine receptors, following prolonged treatment with electrical stimulation (ES) or the antiarrhythmic drug amiodarone, were determined using radioligand binding with the specific A1 receptor antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine (CPX). The effects of lowering temperature were also explored. Exposure to amiodarone for 4 days reduced the density of A1 receptors by 19% (from 24.7 +/- 0.4 to 20.09 +/- 0.3 fmol/dish) and inhibited the rate of contraction by 60% (from 188 +/- 16 to 76 +/- 30 beats/min), without changing the receptor affinity, protein content, creatine kinase (CK) activity or cell number. Electrical stimulation at 25 degrees C elevated the density of A1 adenosine receptors by 185% (from 4.1 +/- 0.4 to 11.69 +/- 2.1 fmol/dish). Four days of reduced temperature (from 37 degrees C to either 30 or 25 degrees C) lowered the density of A1 adenosine receptors by 69 or 86%, respectively (from 24.1 +/- 1.2 to 7.4 +/- 0.4 or 3.4 +/- 0.3 fmol/dish), with no significant change in the receptor affinity, activity of CK, or lactate dehydrogenase (LDH), protein content or cell number. The observed up- and down-regulation of A1 adenosine receptors in primary myocyte cultures in response to conditions that exogenously alter the rate of contraction, is indicative of the role of adenosine receptors in adaptation of heart cells to stress.
Amiodarone↗
Mutagenesis reveals structure-activity parallels between human A2A adenosine receptors and biogenic amine G protein-coupled receptors.
Structure-affinity relationships for ligand binding at the human A2A adenosine receptor have been probed using site-directed mutagenesis in the transmembrane helical domains (TMs). The mutant receptors were expressed in COS-7 cells and characterized by binding of the radioligands [3H]CGS21680, [3H]NECA, and [3H]XAC. Three residues, at positions essential for ligand binding in other G protein-coupled receptors, were individually mutated. The residue V(3.32) in the A2A receptor that is homologous to the essential aspartate residue of TM3 in the biogenic amine receptors, i.e., V84(3.32), may be substituted with L (present in the A3 receptor) but not with D (in biogenic amine receptors) or A. H250(6.52), homologous to the critical N507 of rat m3 muscarinic acetylcholine receptors, may be substituted with other aromatic residues or with N but not with A (Kim et al. J. Biol. Chem. 1995, 270, 13987-13997). H278(7.43), homologous to the covalent ligand anchor site in rhodopsin, may not be substituted with either A, K, or N. Both V84L(3.32) and H250N(6.52) mutant receptors were highly variable in their effect on ligand competition depending on the structural class of the ligand. Adenosine-5'-uronamide derivatives were more potent at the H250N(6.52) mutant receptor than at wild type receptors. Xanthines tended to be close in potency (H250N(6.52)) or less potent (V84L(3.32)) than at wild type receptors. The affinity of CGS21680 increased as the pH was lowered to 5.5 in both the wild type and H250N(6.52) mutant receptors. Thus, protonation of H250(6.52) is not involved in this pH dependence. These data are consistent with a molecular model predicting the proximity of bound agonist ligands to TM3, TM5, TM6, and TM7.
Adenosine↗
Structure-activity relationships of 4-(phenylethynyl)-6-phenyl-1,4-dihydropyridines as highly selective A3 adenosine receptor antagonists.
4-(Phenylethynyl)-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine) in the submicromolar range. In this study, structure-activity relationships at various positions of the dihydropyridine ring (the 3- and 5-acyl substituents, the 4-aryl substituent, and 1-methyl group) were probed synthetically. Using the combined protection of the 1-ethoxymethyl and the 5-[2-(trimethylsilyl)ethyl] ester groups, a free carboxylic acid was formed at the 5-position allowing various substitutions. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure-activity analysis at adenosine receptors indicated that pyridyl, furyl, benzofuryl, and thienyl groups at the 4-position resulted in, at most, only moderate selectivity for A3 adenosine receptors. Ring substitution (e.g., 4-nitro) of the 4-phenylethylnyl group did not provide enhanced selectivity, as it did for the 4-styryl-substituted dihydropyridines. At the 3-position of the dihydropyridine ring, esters were much more selective for A3 receptors than closely related thioester, amide, and ketone derivatives. A cyclic 3-keto derivative was 5-fold more potent at A3 receptors than a related open-ring analogue. At the 5-position, a homologous series of phenylalkyl esters and a series of substituted benzyl esters were prepared and tested. (Trifluoromethyl)-, nitro-, and other benzyl esters substituted with electron-withdrawing groups were specific for A3 receptors with nanomolar Ki values and selectivity as high as 37000-fold. A functionalized congener bearing an [(aminoethyl)amino]carbonyl group was also prepared as an intermediate in the synthesis of biologically active conjugates.
Adenosine↗
8-cyclopentyl-1,3-dipropylxanthine and other xanthines differentially bind to the wild-type and delta F508 first nucleotide binding fold (NBF-1) domains of the cystic fibrosis transmembrane conductance regulator.
Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR). Certain alkyl xanthines such as CPX (8-cyclopentyl-1,3-dipropylxanthine) stimulate Cl- efflux from cells bearing the delta F508 genotype common to most cases of cystic fibrosis. We have hypothesized that the CFTR molecule itself might be the site for CPX action, perhaps in the region of the first nucleotide binding fold (NBF-1) domain. Therefore, to test this hypothesis directly we have used a rapid membrane filtration assay to measure the kinetics of association and dissociation of [3H]CPX to both recombinant NBF-1 and recombinant NBF-1 bearing the delta F508 mutation. We report that [3H]CPX binds with higher affinity to the delta F508-NBF-1 of CFTR (Kd = 1.0 nM) than to the wild-type NBF-1 of CFTR (Kd = 17.0 nM). These Kd values were calculated from direct measurements of the association and dissociation rate constants. The rate constants for the dissociation reaction of the wild-type NBF-1 and delta F508-NBF-1 of CFTR were not different from each other. However, the corresponding rate constants for the association reaction were k(+1) (NBF-1) = 4.7 +/- 0.9 x 10(4) M(-1) s(-1) and k(+1) (delta F508-NBF-1) = 1.6 +/- 0.3 x 10(5) M(-1) s(-1), respectively. These Kd values were corroborated by equilibrium-binding experiments, which gave very similar values. We have also measured the relative displacement of various xanthines from both wild-type NBF-1 and delta F508-NBF-1, in anticipation that the order of potencies for binding might parallel the action of the different xanthines on CF cells. For wild-type NBF-1, the rank order was DA-CPX > DAX > CPX > caffeine > adenosine >> IBMX > 2-thioCPX. For delta F508-NBF-1, the rank order was DAX > CPX > caffeine > DA-CPX > adenosine >> IBMX > 2-thioCPX. These relative potencies show close parallels with previously observed relative potencies of these drugs on CF cells, and thus lend strong support to the hypothesis that the mechanism of action on CF cells may involve direct interaction with the CFTR molecule itself.
Adenosine↗
Adenosine A3 receptor agonists protect HL-60 and U-937 cells from apoptosis induced by A3 antagonists.
The effects of novel, selective adenosine (ADO) A3 receptor antagonists of diverse structure on cells of the human HL-60 leukemia and U-937 lymphoma cell lines were examined. Both 3-ethyl 5-benzyl 2-methyl-6-phenyl-4-phenylethynyl-1,4-(+/-)-dihydropyridine-3, 5-dicarboxylate (MRS 1191, 0.5 microM) and 6-carboxy-methyl-5, 9-dihydro-9-methyl-2-phenyl-[1,2,4]-triazolo [5,1-a][2,7]naphthyridine (L-249313, 0.5 microM) induced apoptotic cell death and expression of bak protein. Low concentrations of the A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (Cl-IB-MECA, 10 nM or 1 microM) protected against antagonist-induced cell death. At concentrations > or = 10 microM, the agonist alone produced apoptosis and bak expression in various cell lines. It is suggested that there exists a tonic low level of A3 receptor activation, possibly induced by release of endogenous adenosine, that results in cell protection.
Adenosine↗
Activation of hippocampal adenosine A3 receptors produces a desensitization of A1 receptor-mediated responses in rat hippocampus.
The adenosine A3 receptor is expressed in brain, but the consequences of activation of this receptor on electrophysiological activity are unknown. We have characterized the actions of a selective adenosine A3 receptor agonist, 2-chloro-N6-(3-lodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), and a selective A3 receptor antagonist, 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191), in brain slices from rat hippocampus. In the CA1 region, activation of A3 receptors had no direct effects on synaptically evoked excitatory responses, long-term potentiation, or synaptic facilitation. However, activation of A3 receptors with Cl-IB-MECA antagonized the adenosine A1 receptor-mediated inhibition of excitatory neurotransmission. The effects of Cl-IB-MECA were blocked by pretreatment with MRS 1191, which by itself had no effect on A1 receptor-mediated responses. The presynaptic inhibitory effects of baclofen and carbachol, mediated via GABA(B) and muscarinic receptors, respectively, were unaffected by Cl-IB-MECA. The maximal response to adenosine was unchanged, suggesting that the primary effect of Cl-IB-MECA was to reduce the affinity of adenosine for the receptor rather than to uncouple it. Similar effects could be demonstrated after brief superfusion with high concentrations of adenosine itself. Under normal conditions, endogenous adenosine in brain is unlikely to affect the sensitivity of A1 receptors via this mechanism. However, when brain concentrations of adenosine are elevated (e.g., during hypoxia, ischemia, or seizures), activation of A3 receptors and subsequent heterologous desensitization of A1 receptors could occur, which might limit the cerebroprotective effects of adenosine under these conditions.
Adenosine↗
Differential effects of flavonoids on testosterone-metabolizing cytochrome P450s.
Flavonoids are widely distributed phytochemicals, whose modulation of cytochrome P450 mediated carcinogen metabolism is well established. Less well studied is their effect on P450 dependent metabolism of endogenous substrates. To address this question we evaluated a series of twelve flavonoids and hematoxylin for their effect on P450-mediated steroid hydroxylation by rat liver microsomes. Site-specific 7alpha-, 6beta- and 2alpha-hydroxylation of testosterone by P450s 2A1, 3A2 and 2C11, respectively, was measured. Highly selective patterns of inhibition or activation of these P450s were observed. 3,6-dichloro-2'-isopropyloxy-4'-methylflavone was the most potent inhibitor of P450 2C11 while cyanidin chloride most potently inhibited P450s 2A1 and 3A2. The flavonoid analogue hematoxylin was unique in that it activated 2C11 (by 2.5 fold) yet inhibited both 2A1 and 3A2 (by 60%). These results indicate that consumption of dietary flavonoids may likewise alter the metabolite profile of steroids and other physiological P450 substrates.
Animals↗
Pharmacological characterization of novel A3 adenosine receptor-selective antagonists.
The effects of putative A3 adenosine receptor antagonists of three diverse chemical classes (the flavonoid MRS 1067, the 6-phenyl-1,4-dihydropyridines MRS 1097 and MRS 1191, and the triazoloquinazoline MRS 1220) were characterized in receptor binding and functional assays. MRS1067, MRS 1191 and MRS 1220 were found to be competitive in saturation binding studies using the agonist radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide) at cloned human brain A3 receptors expressed in HEK-293 cells. Antagonism was demonstrated in functional assays consisting of agonist-induced inhibition of adenylate cyclase and the stimulation of binding of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to the associated G-proteins. MRS 1220 and MRS 1191, with KB values of 1.7 and 92 nM, respectively, proved to be highly selective for human A3 receptor vs human A1 receptor-mediated effects on adenylate cyclase. In addition, MRS 1220 reversed the effect of A3 agonist-elicited inhibition of tumor necrosis factor-alpha formation in the human macrophage U-937 cell line, with an IC50 value of 0.3 microM.
Adenosine↗
Towards a revised nomenclature for P1 and P2 receptors.
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Animals↗
Persistent activation by and receptor reserve for an irreversible A1-adenosine receptor agonist in DDT1 MF-2 cells and in guinea pig heart.
The p- and m-isothiocyanate adenosine derivatives N6-[4-[[[4-[[[[2-[[[(p-(m)-isothiocyanatophenyl)amino]thiocarbonyl ]am ino]ethyl]amino]carbonyl]methyl]anilino]carbonyl]methyl]phenyl] adenosine (p- and m-DITC-ADAC) were examined for irreversible agonist effects at the A1-adenosine receptor (A1-AdoR) in DDT1 MF-2 (DDT) cells and a functional A1-AdoR response in the guinea pig isolated heart. The p- and m-DITC-ADAC inhibited (-)-isoproterenol stimulated cAMP accumulation in DDT cells in the low nanomolar range, and the maximal responses elicited by both compounds were similar to that for N6-cyclopentyladenosine. Once established, the p-DITC-ADAC-mediated inhibition of cAMP accumulation in DDT cells was not affected by the addition of the AdoR antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX). Pretreatment of DDT cells with p-DITC-ADAC (1 microM), followed by washing, reduced [3H]CPX binding to the A1-AdoR by 44% without altering the Kd value for the radioligand to the remaining receptors. The relationship between irreversible A1-AdoR occupancy by p-DITC-ADAC and inhibition of cAMP accumulation revealed a relatively large receptor reserve (64%) for the maximal response. In guinea pig isolated hearts, m-DITC-ADAC (5 microM) prolonged the stimulus to His bundle (SH) interval by 2.1-fold; this response could be prevented by the antagonist 8-cyclopentyltheophylline (5 microM). However, after the SH interval prolongation was established, extensive washout or the addition of 8-cyclopentyltheophylline had little reversal effect on the m-DITC-ADAC response. Binding of [3H]CPX to the guinea pig ventricular membranes after m-DITC-ADAC treatment and washing was reduced by 35%. The A1-AdoR occupancy response relationship for m-DITC-ADAC to prolong the SH interval indicated a small (10-20%) receptor reserve. Both p -and m-DITC-ADAC seem to be irreversible full agonists at the A1-AdoR and may prove to be useful probes to further investigate A1-AdoR structure-function relationships.
Adenosine↗
A mutational analysis of residues essential for ligand recognition at the human P2Y1 receptor.
We conducted a mutational analysis of residues potentially involved in the adenine nucleotide binding pocket of the human P2Y1 receptor. Mutated receptors were expressed in COS-7 cells with an epitope tag that permitted confirmation of expression in the plasma membrane, and agonist-promoted inositol phosphate accumulation was assessed as a measure of receptor activity. Residues in transmembrane helical domains (TMs) 3, 5, 6, and 7 predicted by molecular modeling to be involved in ligand recognition were replaced with alanine and, in some cases, by other amino acids. The potent P2Y1 receptor agonist 2-methylthio-ATP (2-MeSATP) had no activity in cells expressing the R128A, R310A, and S314A mutant receptors, and a markedly reduced potency of 2-MeSATP was observed with the K280A and Q307A mutants. These results suggest that residues on the exofacial side of TM3 and TM7 are critical determinants of the ATP binding pocket. In contrast, there was no change in the potency or maximal effect of 2-MeSATP with the S317A mutant receptor. Alanine replacement of F131, H132, Y136, F226, or H277 resulted in mutant receptors that exhibited a 7-18-fold reduction in potency compared with that observed with the wild-type receptor. These residues thus seem to subserve a less important modulatory role in ligand binding to the P2Y1 receptor. Because changes in the potency of 2-methylthio-ADP and 2-(hexylthio)-AMP paralleled the changes in potency of 2-MeSATP at these mutant receptors, the beta- and gamma-phosphates of the adenine nucleotides seem to be less important than the alpha-phosphate in ligand/P2Y1 receptor interactions. However, T221A and T222A mutant receptors exhibited much larger reductions in triphosphate (89- and 33-fold versus wild-type receptors, respectively) than in diphosphate or monophosphate potency. This result may be indicative of a greater role of these TM5 residues in gamma-phosphate recognition. Taken together, the results suggest that the adenosine and alpha-phosphate moieties of ATP bind to critical residues in TM3 and TM7 on the exofacial side of the human P2Y1 receptor.
Adenosine Monophosphate↗
A novel cardioprotective function of adenosine A1 and A3 receptors during prolonged simulated ischemia.
The possible cardioprotective roles of adenosine A1 and A3 receptors were investigated in a cardiac myocyte model of injury. The adenosine A3 receptor is a novel cardiac receptor capable of mediating potentially important cardioprotective functions. Prolonged hypoxia with glucose deprivation was used to simulate ischemia and to induce injury in cardiac ventricular myocytes cultured from chick embryos 14 days in ovo. When present during the prolonged hypoxia, the adenosine A3 agonists N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (IB-MECA) and 2-chloro-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide (CI-IB-MECA) caused a dose-dependent reduction in the extent of hypoxia-induced injury as manifested by a decrease in the amount of creatine kinase released and the percentage of myocytes killed. The adenosine A1 agonists 2-chloro-N6-cyclopentyladenosine (CCPA), N6-cyclohexyladenosine, and adenosine amine congener were also able to cause a decrease in the extent of myocyte injury. The A1 receptor-selective antagonist 8-cyclopentyl-1,3-dipropylxanthine blocked the cardioprotective effect of the A1 but not of the A3 agonists. Conversely, the selective A3 antagonists MRS-1191 and MRS-1097 blocked the protection induced by CI-IB-MECA but had minimal effect on that caused by CCPA. Thus the cardioprotective effects of A1 and A3 agonists were mediated by their respective receptors. This study defines a novel cardioprotective function of the cardiac A3 receptor and provides conclusive evidence that activation of both A1 and A3 receptors during hypoxia can attenuate myocyte injury.
Adenosine↗
Polarization of adenosine effects on intracellular pH in A6 renal epithelial cells.
The effect of adenosine on Na+/H+ exchange activity was examined in cultured A6 renal epithelial cells. Adenosine and its analogue N6-cyclopentyladenosine (CPA) had different effects on Na+/H+ exchange activity depending on the side of addition. Basolateral CPA induced a stimulation of Na+/H+ exchange activity that was completely prevented by preincubation with an A2A-selective antagonist, 8-(3-chlorostyryl)caffeine, whereas apical CPA induced a slight but significant inhibition of Na+/H+ exchange activity that was significantly reduced by the A1-receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine. Protein kinase C activation may be involved in mediating the apical CPA inhibition of Na+/H+ exchange activity; this inhibition was prevented by the protein kinase C inhibitor calphostin C. Treatment with either forskolin or 8-bromo-cAMP significantly stimulated Na+/H+ exchange activity; only basolateral CPA addition induced an increase in cAMP level. These observations together with the finding that the CPA-dependent stimulation of exchange activity was prevented by the protein kinase A inhibitor H-89 support the hypothesis that basolateral CPA stimulates Na+/H+ exchange via adenylate cyclase/protein kinase A activation. Basolateral CPA also increased transepithelial Na+ transport, and this stimulation was prevented by the Na+/H+ exchange inhibitor HOE-694, suggesting that changes in pHi during hormone action can act as an intermediate in the second-messenger cascade.
Adenosine↗
Postischemic administration of adenosine amine congener (ADAC): analysis of recovery in gerbils.
Although adenosine receptor-based treatment of cerebral ischemia and other neurodegenerative disorders has been frequently advocated, cardiovascular side effects and an uncertain therapeutic time window of such treatment have constituted major obstacles to clinical implementation. Therefore, we have investigated the neuroprotective effects of the adenosine A1 receptor agonist adenosine amine congener (ADAC) injected after either 5 or 10 min ischemia at 100 micrograms/kg. When the drug was administered at either 6 or 12 h following 5 min forebrain ischemia, all animals were still alive on the 14th day after the occlusion. In both ADAC treated groups neuronal survival was approximately 85% vs. 50% in controls. Administration of a single dose of ADAC at times 15 min to 12 h after 10 min ischemia resulted in a significant improvement of survival in animals injected either at 15 or 30 min, or at 1, 2, or 3 h after the insult. In all 10 min ischemia groups, administration of ADAC resulted in a significant protection of neuronal morphology and preservation of microtubule associated protein 2 (MAP-2). However, postischemic Morris' water maze tests revealed full preservation of spatial memory and learning ability in animals injected at 6 h. On the other hand, the performance of gerbils treated at 12 h postischemia was indistinguishable from that of the controls. Administration of ADAC at 100 micrograms/kg in non-ischemic animals did not result in bradycardia, hypotension, or hypothermia. The data indicate that when ADAC is used postischemically, the most optimal level of protection is obtained when drugs are given at 30 min to 6 h after the insult. Although the mechanisms involved in neuroprotective effects of adenosine A1 receptor agonists require further studies, the present results demonstrate the feasibility of their clinical applications.
Adenosine↗
6-phenyl-1,4-dihydropyridine derivatives as potent and selective A3 adenosine receptor antagonists.
An approach to designing dihydropyridines that bind to adenosine receptors without binding to L-type calcium channels has been described. 1,4-Dihydropyridine derivatives substituted with beta-styryl or phenylethynyl groups at the 4-position and aryl groups at the 6-position were synthesized and found to be selective for human A3 receptors. Combinations of methyl, ethyl, and benzyl esters were included at the 3- and 5-positions. Affinity was determined in radioligand binding assays at rat brain A1 and A2A receptors using [3H]-(R)-PIA [[3H]-(R)-N6-(phenylisopropyl)adenosine] and [3H]CGS 21680 [[3H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5'- (N-ethylcarbamoyl)adenosine], respectively. Affinity was determined at cloned human and rat A3 receptors using [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-5'-(N-ethylcarbamoyl)-adenosine]. Structure-activity analysis indicated that substitution of the phenyl ring of the beta-styryl group but not of the 6-phenyl substituent was tolerated in A3 receptor selective agents. Replacement of the 6-phenyl ring with a 3-thienyl or 3-furyl group reduced the affinity at A3 receptors by 4- and 9-fold, respectively. A 5-benzyl ester 4-trans-beta-styryl derivative, 26, with a Ki value of 58.3 nM at A3 receptors, was > 1700-fold selective vs either A1 receptors or A2A receptors. Shifting the benzyl ester to the 3-position lowered the affinity at A3 receptors 3-fold. A 5-benzyl, 3-ethyl ester 4-phenylethynyl derivative, 28, displayed a Ki value of 31.4 nM at A3 receptors and 1300-fold selectivity vs A1 receptors. The isomeric 3-benzyl, 5-ethyl diester was > 600-fold selective for A3 receptors. Oxidation of 28 to the corresponding pyridine derivative reduced affinity at A3 receptors by 88-fold and slightly increased affinity at A1 receptors.
Animals↗
Activation of A3 adenosine receptors on human eosinophils elevates intracellular calcium.
Adenosine (ADO) is a potent bronchoconstrictor in allergic patients and has been shown to increase the release of histamine from human lung tissues. Antagonists of ADO A1 and A2A receptors are not effective in attenuating these effects. Therefore, involvement of ADO A3 receptors in the bronchoconstrictor and/or inflammatory effects have to be considered. Eosinophils also play a pivotal role in allergic diseases such as asthma, thus it is natural to consider a link between the A3 receptor and eosinophils. Human peripheral blood eosinophils express the ADO A3 receptor as indicated by detection of the transcript for A3 receptors in polymerase chain reaction-amplified cDNA derived from the cells. A3 receptors on eosinophil membranes were characterized using the A3 receptor agonist radioligand 125I-labeled AB-MECA, which yielded Bmax and Kd values of 1.31 pmol/mg protein and 3.19 nmol/L, respectively. Treatment of eosinophils with the highly potent and selective A3 receptor agonist CI-IB-MECA clearly induced Ca2+ release from intracellular Ca2+ pools followed by Ca2+ influx, suggesting the presence of phospholipase C-coupled A3 receptors. In contrast, the ADO receptor agonists CPA and CGS 21680, selective for A1 and A2A receptors, respectively, at concentrations of < or = 30 mumol/ L did not elevate the intracellular Ca2+ level. These results attest to the existence of ADO A3 receptors on eosinophils and suggest that ADO stimulates these cells to release Ca2+ from intracellular stores via the activation of A3 receptors.
Adenosine↗
Direct preconditioning of cultured chick ventricular myocytes. Novel functions of cardiac adenosine A2a and A3 receptors.
Preconditioning with brief ischemia before a sustained period of ischemia reduces infarct size in the perfused heart. A cultured chick ventricular myocyte model was developed to investigate the role of adenosine receptor subtypes in cardiac preconditioning. Brief hypoxic exposure, termed preconditioning hypoxia, prior to prolonged hypoxia, protected myocytes against injury induced by the prolonged hypoxia. Activation of the adenosine A1 receptor with CCPA or the A3 receptor with C1-IB-MECA can replace preconditioning hypoxia and simulate preconditioning, with a maximal effect at 100 nM. While activation of the A2a receptor by 1 microM CGS21680 could not mimic preconditioning, its stimulation during preconditioning hypoxia, however, attenuated the protection against hypoxia-induced injury. Blockade of A2a receptors with the selective antagonist CSC (1 microM) during preconditioning hypoxia enhanced the protective effect of preconditioning. Nifedipine, which blocked the A2a receptor-mediated calcium entry, abolished the A2a agonist-induced attenuation of preconditioning. Isoproterenol, forskolin, and BayK 8644, which stimulated calcium entry, also attenuated preconditioning. Nifedipine blocked the increase in calcium uptake by these agents as well as their attenuating effect on preconditioning. The present study provides the first evidence that the adenosine A3 receptor is present on ventricular myocytes and can mediate simulation of preconditioning. The data demonstrate, for the first time, that activation of the A2a receptor antagonizes the preconditioning effect of adenosine, with increased calcium entry during the preconditioning stimuli as a novel mechanism.
Animals↗