Characterization of Adenosine A 2 Receptors in Bovine Retinal Membranes

Two classes of extracellular receptors for adenosine. A 1 and A 2, have been demonstrated in the mammalian retina. Our laboratory has previously reported the pharmacological characteristics of the mammalian retinal A 1 receptors. We now report our characterization of retinal A 2 receptors based on data obtained from both adenylate cyclase assays and radioligand binding studies. [ 3H]-5′-N-ethylcarboxamidoadenosine (NECA) in the presence of 10 nM cyclopentyladenosine (CPA, which selectively binds to A 1 receptors) or [ 3H]-CGS 21680 were used to label the A 2 binding sites. Using [ 3H]-NECA (plus CPA), two populations of binding sites, having K ds of 106 nM and 9·4 μM, were determined. [ 3H]-CGS 21680, a derivative of NECA which has been demonstrated to be highly selective for A 2 receptors in brain synaptic membrane preparations was more potent than NECA at the higher affinity population of A 2 sites, and saturation analysis revealed the presence of both a high affinity site, K d of 18 nM, and a lower affinity site having a K d of 4·3 μM. The high affinity site labeled by [ 3H]-CGS 21680 corresponds to the A 2a receptor. Using either radioligand, guanosine triphosphate-dependent shifts to a single population of binding sites were observed. Despite the differences in affinities revealed by the two radioligands for the high affinity A 2 site, both [ 3H]-CGS 21680 and [3H]-NECA were competitively displaced by increasing concentrations of a variety of adenosine receptor agonists and antagonists, and exhibited an identical rank order of potency that is consistent with that reported for high affinity A 2a receptors. Receptor-mediated modulation o[ adenylate cyclase activities in retinal synaptic membranes was also assessed, and while NECA or N 6-methyladenosine elicited decreases in forskolin-activated cyclase activity at concentrations between 0·1-50 nM, this inhibition was reversed, and enzyme stimulated by higher agonist concentrations. CGS 21680 elicited only a stimulation of either basal and forskolin-activated adenylate cyclase activities at concentrations above 50 nM. The stimulatory modulation of adenylate cyclase at these concentrations is consistent with mediation by the A 2a and/or A 2b receptors.