On the high affinity binding site for [3H]-1,3-dipropyl-8-cyclopentylxanthine in frog brain membranes.
1. Radioligand binding properties of the adenosine receptor ligands, [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), and [3H]-R-phenylisopropyladenosine ([3H]-R-PIA) were investigated in frog brain membranes. 2. The specific binding of the adenosine antagonist, [3H]-DPCPX to frog brain membranes showed one binding site with Kd and Bmax values of 43.8 nM and 0.238 +/- 0.016 pmol mg-1 protein, respectively. Guanosine 5'-triphosphate (GTP, 100 microM) decreased to 72 +/- 7% and Mg2+ (8 mM) increased to 121 +/- 3% [3H]-DPCPX (40 nM) binding to frog brain membranes. 3. [3H]-DPCPX saturation binding experiments performed in the presence of Mg2+ (8 mM), or in the presence of GTP showed that Mg2+ ions decreased the Kd value of [3H]-DPCPX to 14 nM, and GTP increased this value to 65.6 nM. Bmax 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 [3H]-R-PIA (15 nM) was decreased to 37 +/- 6% by GTP (100 microM) and increased to 123 +/- 4% by Mg2+ (8 mM). [3H]-R-PIA saturation binding experiments performed in the presence of Mg2+ (8 mM) showed one binding site with Kd and Bmax 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 [3H]-DPCPX binding showed the following order of potencies: CPA> R-PIA~ NECA> S-PIA> > CGS 21680, for the agonists, and XAC ~-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 Al or A2 adenosine receptors.
Oliveira
J C
JC
Laboratory of Pharmacology, Gulbenkian Institute of Science, Oeiras, Portugal.
Sebastião
A M
AM
Ribeiro
J A
JA
D000241:Adenosine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D010660:Phenylisopropyladenosine / Q000494:pharmacology; D011895:Rana ridibunda; D011983:Receptors, Purinergic / Q000187:drug effects / Q000378:metabolism*; D014970:Xanthines / Q000378:metabolism*
D011983:Receptors, Purinergic; D014970:Xanthines; D010660:Phenylisopropyladenosine; D006160:Guanosine Triphosphate; C051360:1,3-dipropyl-8-cyclopentylxanthine; D019204:GTP-Binding Proteins; D008274:Magnesium; D000241:Adenosine