Adenosine receptor agonists: Binding and adenylate cyclase stimulation in rat liver plasma membranes

Summary N⁶-Cyclohexyl³H]adenosine(³H]CHA),³H]adenosine, and 5N-ethylcarboxamide³H]adenosine (³H]NECA), potent agonists in adenosine-responsive cellular systems, have been used to identify adenosine binding sites in rat liver plasma membranes. Endogenous ligands were removed by prior dialysis of the membranes. Specific binding of the ligands tested was characterized by rapid forward and reverse kinetics and heterogeneity as indicated by curvilinear Scatchard plots. The K _D in the high affinity range was 80 nM for ³H]adenosine, 84 nM for ³H]NECA, and 168 nM for ³H]CHA; the respective binding capacities of 1.19, 1.03, and 1.05 pmol/mg protein were of virtually the same magnitude, suggesting labeling of identical sites. However, all ligands also displayed binding to large numbers of low affinity sites. This high level of apparently non-receptor binding markedly influenced the adenosine structure-activity profile of ³H]CHA displacement, which differs with pharmacological findings. — NECA and CHA stimulated hepatic adenylate cyclase with an apparent ED₅₀ of 60 and 580 nM, respectively; adenosine was stimulatory at a concentration range from 0.1–2.0 M, but inhibitory at higher concentrations. Hence, estimation of the true ED₅₀ was not possible. Because the K _D of high affinity binding and the ED₅₀ of the biological effect of NECA and CHA are in the same range, it may be reasonable to assume that the high affinity sites represent adenosine receptors, recently classified as R_a-site receptors.Preliminary reports of this study have been presented at the 22nd Spring Meeting of the Deutsche Pharmakologische Gesellschaft (Naunyn-Schmiedeberg's Arch Pharmacol 316, R10, 1981) and at the 8th International Congress of Pharmacology Tokyo 1981 (P 1459)]