CHARACTERIZATION AND LOCALIZATION OF ADENOSINE A(2) RECEPTORS IN BOVINE ROD OUTER SEGMENTS

Previous work from this laboratory has shown that retinal adenosine A(2) binding sites are localized over outer and inner segments of photoreceptors in rabbit and mouse retinal sections. In the present study, adenosine receptor binding has been characterized and localized in membranes from bovine rod outer segments (ROS). Saturation studies with varying concentrations (10-150 nM) of 5'-(N-[2,8-H-3]ethylcarboxamido)adenosine ([H-3]NECA) and 100 mu g of ROS membrane protein show a single site with a K-D of 103 nM and a B-max of 1.3 pM/mg of protein. Cold Scatchards, which used nonradiolabeled NECA (concentrations ranging from 10 nM to 250 mu M) in competition with a fixed amount of [H-3]NECA (30 nM), demonstrated the presence of a low-affinity site (K-D, 50 mu M) in addition to the high-affinity site. To confirm the presence of A(2a) binding sites, saturation analyses with 2-p-(2-[H-3]carboxyethyl)phenylamino-5'-N-ethylcarboxamido adenosine (0-80 nM) also revealed a single population of high-affinity A(2a) receptors (K-D, 9.4 nM). The binding sites labeled by [H-3]NECA appear to be A(2) receptor sites because binding was displaced by increasing concentrations of 5'-(N-methylcarboxamido)adenosine and 2-chloroadenosine. ROS were fractionated into plasma and disk membranes for localization studies. Receptor binding assays, used to determine specific binding, showed that the greatest concentration of A(2) receptors was on the plasma membranes. Therefore, adenosine A(2) receptors are in a position to respond to changes in the concentration of extracellular adenosine, which may exhibit a circadian rhythm.