Characterization of adenosine A1-receptor binding sites in bovine retinal membranes.
Using retinas prepared from freshly dissected bovine eyes, we have characterized the binding of the A1-selective agonist, [3H]PIA (N6-R-[3H](2-phenylisopropyl)adenosine). Specific binding was linear over a range of membrane protein concentrations from 0.10 to 1.0 mg, and accounted for an average of 80-90% of the total binding. At room temperature (24 degrees C), binding reached equilibrium at 60 min, and was reversible upon addition of an excess of cold ligand. Saturation analysis and Scatchard transformation revealed two apparent populations of receptor binding sites. The higher affinity site exhibited a Kd of 0.134 +/- 0.007 nM and Bmax of 26.18 +/- 3.06 fmol-1 mg protein. The lower affinity site exhibited a Kd of 21.83 +/- 4.39 nM and Bmax of 53.94 +/- 15.80 fmol mg-1 protein. Kinetic analysis of association and dissociation rates, performed at a low concentration of [3H]PIA, yielded a calculated affinity constant for the high affinity site of 0.2 nM, in agreement with saturation studies. Competition experiments with a number of purine nucleoside agonists and antagonists were performed, using radioligand concentrations of 1 nM or less to examine binding at the high affinity site, and revealed a rank order of potency consistent with the reported pharmacology of A1 receptors. We have also assayed for adenylate cyclase activity in this same preparation and determined that PIA inhibited forskolin-activated adenylate cyclase in a dose-dependent manner. Maximum inhibition (40%) was observed with 1 nM PIA, while 10 microM 8-cyclopentyl-1,3-dipropylxanthine, an A1 selective antagonist, completely inhibited this modulation by PIA.
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St Louis, MO 63110.
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D000241:Adenosine / Q000378:metabolism*; D000067956:Adenylyl Cyclase Inhibitors; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D066298:In Vitro Techniques; D010660:Phenylisopropyladenosine / Q000378:metabolism; D011983:Receptors, Purinergic / Q000032:analysis*; D012160:Retina / Q000201:enzymology / Q000378:metabolism*; D013997:Time Factors
D000067956:Adenylyl Cyclase Inhibitors; D011983:Receptors, Purinergic; D010660:Phenylisopropyladenosine; D000241:Adenosine