On the high affinity binding site for [3H]‐1,3‐dipropyl‐8‐cyclopentylxanthine in frog brain membranes

Abstract

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 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 μm) 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 K_d value of [³H]-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 [³H]-R-PIA (15 nm) was decreased to 37 ± 6% by GTP (100 μm) 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 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 [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 A₁ or A₂ adenosine receptors.