MRS-1097

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.

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.