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To clarify which adenosine receptor subtype(s) are responsible for regulation of coronary flow through endogenous adenosine, coronary vascular responses were examined in isolated hearts from wild-type (WT) and A(2A) knockout (A(2A)KO) mice. Adenosine deaminase inhibitor, erythro-9-hydroxy-nonyl-adenine (EHNA), and adenosine kinase inhibitor, iodotubericidine (ITU), were used to examine the effects of endogenous adenosine. Combined infusion of EHNA and ITU in Balb/c hearts produced comparable increases in coronary flow as exerted by exogenous adenosine while they markedly decreased the heart rate, and these effects were reversed by adenosine receptor antagonist, 8-p-sulfophenyl-theophylline (8-SPT). Similarly, EHNA and ITU increased coronary flow in WT hearts to 422% of baseline, whereas this response was reduced to 144% of baseline in A(2A)KO hearts. Heart rate was equally reduced (approximately 50% of baseline) in both groups. Alloxazine (A(2B) receptor antagonist) abolished EHNA- and ITU-induced coronary flow in A(2A)KO hearts without altering the reduced heart rate. Selective A(1) receptor antagonist, 8-cyclopentyl-1-1,3-dipropylxanthine (DPCPX), reversed EHNA- and ITU-induced decreases in heart rate without altering the elevated coronary flow. These findings suggest that coronary vascular responses to endogenous adenosine mimic those produced by exogenous adenosine and are mediated at least by activation of both A(2A) and A(2B) receptors in isolated mouse hearts.

To determine whether adenosine A(3) receptors participate in adenosine-induced changes in coronary flow, isolated hearts from wild-type (WT) and A(3) receptor knockout (A(3)KO) mice were perfused under constant pressure and effects of nonselective and selective agonists were examined. Adenosine and the selective A(2A) agonist 2-[p-(2-carboxyethyl)]phenylethylamino-5'-N-ethylcarboxamidoadenosine (CGS-21680) produced augmented maximal coronary vasodilation in A(3)KO hearts compared with WT hearts. Selective activation of A(3) receptors with 2-chloro-N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA) at nanomolar concentrations did not effect coronary flow, but at higher concentrations it produced coronary vasodilation both in WT and A(3)KO hearts. Cl-IB-MECA-induced increases in coronary flow were susceptible to both pharmacological blockade and genetic deletion of A(2A) receptors. Because deletion or blockade of adenosine A(3) receptors augmented coronary flow induced by nonselective adenosine and the selective A(2A) receptor agonist CGS-21680, we speculate that this is due to removal of an inhibitory influence associated with the A(3) receptor subtype. These data indicate that the presence of adenosine A(3) receptors may either inhibit or negatively modulate coronary flow mediated by other adenosine receptor subtypes.

To clarify the relative roles of A(2) adenosine receptor subtypes in the regulation of coronary flow and myocardial contractility, coronary vascular and functional responses to adenosine and its analogs were examined in isolated wild-type (WT) and A(2A) receptor knockout (A(2A)KO) mouse hearts. Nonselective agonists adenosine and 5'-N-ethyl-carboxamido-adenosine (NECA) increased coronary flow in A(2A)KO hearts, albeit with a rightward shift of concentration-response curves and decreased maximal vasodilation compared with WT hearts. 2-p-(2-Carboxy-ethyl)phenethylamino-5'-N-ethyl-carboxamidoadenosine (CGS-21680, a selective A(2A) receptor agonist) increased coronary flow in WT hearts but did not affect A(2A)KO hearts. Adenosine and NECA each elicited equal maximal increases in developed pressure in WT and A(2A)KO hearts, whereas CGS-21680 did not affect developed pressure in A(2A)KO hearts. Alloxazine, a selective A(2B) receptor antagonist, attenuated NECA-induced coronary vasodilation (from 202 +/- 14% to 128 +/- 9% of baseline, P < 0.05) and NECA-induced increases in developed pressure (from 133 +/- 8% to 112 +/- 7% of baseline, P < 0.05) in A(2A)KO hearts. Together, these findings support the conclusion that A(2B) adenosine receptor activation increases coronary flow and developed pressure in isolated murine hearts.

The present study was designed to characterize and compare the vascular effects of adenosine and its analogs in the murine heart and aorta. Mouse hearts perfused under constant pressure in standard Langendorff fashion demonstrated concentration-dependent increases in coronary flow to adenosine, 2-chloradenosine (CAD), 5'-(N-ethyl-carboxamido)-adenosine (NECA), and 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxam-idoadenosine (CGS-21680). All agonists produced comparable increases in coronary flow with the following order of potency: CGS-21680 = NECA >> CAD > or = adenosine. In l-phenylephrine hydrochloride (phenylephrine) precontracted aortic rings, all nonselective agonists (NECA, CAD, and adenosine) produced marked concentration-dependent relaxation, whereas the adenosine A(2A) selective agonist CGS-21680 did not. Adenosine receptor agonists were >100 times more potent for coronary vasodilation than aortic vasorelaxation. The selective A(2A) receptor antagonist 5-amino-7-(beta-phenylethyl)-2-(8-furyl)pyrazolo-[4,3-e]-1,2,4-triazolo-[1,5-c]pyrimidine (SCH-58261) blocked both CGS-21680- and NECA-induced increases in coronary flow, whereas the A(2B) receptor antagonist benzo[g]pteridine-2,4(1H,3H)-dione (alloxazine) inhibited NECA-induced aortic relaxation. These data indicate a differential response to adenosine agonists in murine coronary vasculature and aorta where coronary vasodilation is mediated predominantly by activation of A(2A) adenosine receptors.