Adenosine receptors in an enriched fraction of plasma membranes from canine ventricular myocardium

[3H]Phenylisopropyladenosine ([3H]PIA) was used to characterize adenosine receptor sites in a sarcolemma-enriched membrane fraction from canine ventricle. Specific [3H]PIA binding to the cardiac membrane preparation was rapid, readily reversible, and saturable with increasing free ligand concentrations. Scatchard analysis indicated a single class of binding sites having a Bmax of 601 fmol/mg protein. The Kd of [3H]PIA for its binding site was 52-85 nM as determined independently from kinetic and equilibrium studies, respectively. Binding was stereospecific in that (-)PIA was ninefold more potent than (+)PIA in competing for [3H]PIA binding sites. Adenosine receptor agonists such as N6-cyclohexyladenosine, (-)PIA, 2-chloroadenosine, N6-methyladenosine, and adenosine-5'-ethylcarboxamide were the most potent agents found to compete for [3H]PIA binding sites and displayed IC50 values of 14-224 nM, while 2',5'-dideoxyadenosine, a potent P-site agonist, inhibited binding only weakly. Alkylxanthines also inhibited [3H]PIA binding with relative potency relationships that paralleled their known pharmacological activity as adenosine receptor antagonists. (-)PIA inhibited activation of membrane adenylate cyclase by isoproterenol in a concentration-dependent manner with a maximum of 22% inhibition occurring at 1 microM PIA. It is concluded that the specific binding of [3H]PIA to the sarcolemma-enriched fraction of canine ventricle represents an Ri adenosine receptor on the surface of the myocardial cell. Such a receptor has been postulated to mediate the adenosine-induced attenuation of the effects of catecholamines on intact ventricular myocardium.