Characterization of Adenosine A 2 Receptors in Bovine Retinal Pigment Epithelial Membranes

Abstract

The pharmacological characteristics of adenosine A 2 receptors are described for membranes prepared from bovine retinal pigmented epithelial (RPE) cells. RPE cells were isolated after removal of retina, lysed by freeze-thawing, and membranes separated from cytoplasmic components. A single population of adenosine binding sites is present in RPE membranes, as determined from saturation analysis and competition binding assays. From Scatchard plots, this single class of binding sites exhibited low affinity for adenosine receptor agonists. These low affinity sites were labeled by [ 3H]-N-ethylcarboxamidoadenosine (NECA) or [ 3H]-CGS 21680 and K ds of 423 and 5·3 μM were determined for each radioligand, respectively. NECA-mediated stimulation of adenylate cyclase demonstrated that these binding sites represent adenosine receptors. No high affinity A 2a binding sites were detected in RPE membranes by either saturation studies, or by competition with adenosine A 1-selective agonists which only displaced radioligand binding at high micromolecular concentrations. The low affinity A 2 receptor on RPE differs from the high affinity A 2a receptor characterized in bovine retinal membranes, but may be similar or identical to the lower affinity A 2b receptor detected in retinal membranes as well as other tissues.