Characterization and localization of adenosine A2 receptors in bovine rod outer segments.
Previous work from this laboratory has shown that retinal adenosine A2 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-[2,8-3H]ethylcarboxamido)adenosine ([3H]NECA) and 100 micrograms of ROS membrane protein show a single site with a KD of 103 nM and a Bmax of 1.3 pM/mg of protein. Cold Scatchards, which used nonradiolabeled NECA (concentrations ranging from 10 nM to 250 microM) in competition with a fixed amount of [3H]NECA (30 nM), demonstrated the presence of a low-affinity site (KD, 50 microM) in addition to the high-affinity site. To confirm the presence of A2a binding sites, saturation analyses with 2-p-(2-[3H]-carboxyethyl)phenylamino-5'-N-ethylcarboxamido adenosine (0-80 nM) also revealed a single population of high-affinity A2a receptors (KD, 9.4 nM). The binding sites labeled by [3H]NECA appear to be A2 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 A2 receptors was on the plasma membranes. Therefore, adenosine A2 receptors are in a position to respond to changes in the concentration of extracellular adenosine, which may exhibit a circadian rhythm.
McIntosh
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Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Blazynski
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D015762:2-Chloroadenosine / Q000378:metabolism; D000241:Adenosine / Q000031:analogs & derivatives / Q000378:metabolism; D019830:Adenosine-5'-(N-ethylcarboxamide); D000818:Animals; D001667:Binding, Competitive; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D010627:Phenethylamines / Q000378:metabolism; D018047:Receptors, Purinergic P1 / Q000378:metabolism*; D012374:Rod Cell Outer Segment / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
D010627:Phenethylamines; D018047:Receptors, Purinergic P1; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D015762:2-Chloroadenosine; C046756:5'-N-methylcarboxamideadenosine; D019830:Adenosine-5'-(N-ethylcarboxamide); D000241:Adenosine