ON THE HIGH-AFFINITY BINDING-SITE FOR [H-3] 1,3-DIPROPYL-8-CYCLOPENTYLXANTHINE IN FROG BRAIN MEMBRANES

1 Radioligand binding properties of the adenosine receptor ligands, [H-3]-1,3-dipropyl-8-cyclopentylxanthine ([H-3]-DPCPX), and [H-3]-R-phenylisopropyladenosine ([H-3]-R-PIA) were investigated in frog brain membranes. 2 The specific binding of the adenosine antagonist, [H-3]-DPCPX to frog brain membranes showed one binding site with K(d) and B(max) values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 muM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [H-3]-DPCPX (40 nM) binding to frog brain, membranes. 3 [H-3]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the K(d) value of [H-3]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. B(max) values were not significantly (P > 0.05) modified (0.261 +/-0.018 pmol mg-1 protein, with Mg2+, and 0.266 +/- 0.026 pmol mg-1 protein, in presence of GTP) by the presence of Mg2+ or GTP. 4 The specific binding of [H-3]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 muM) and increased to 123 +/- 4% by Mg2+ (8 mM). [H-3]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with K(d) and B(max) values of 0.9 nM and 0.229 +/- 0.008 pmol mg-1 of protein, respectively. 5 The concentration-inhibition curves of adenosine agonists and antagonists versus [H-3]-DPCPX binding showed the following order of potencies: CPA > R-PIA congruent-to NECA > S-PIA >> CGS 21680, for the agonists, and XAC congruent-to DPCPX >> XCC > PACPX, for the antagonists. 6 The present results suggest that the adenosine binding site in the frog brain membranes is G-protein coupled, but that the antagonist affinities and the pharmacological profile is different from the A1 or A2 adenosine receptors.