SOLUBILIZED RAT-BRAIN ADENOSINE RECEPTORS HAVE 2 HIGH-AFFINITY BINDING-SITES FOR 1,3-DIPROPYL-8-CYCLOPENTYLXANTHINE

The specific binding of L-N6-[H-3]phenylisopropyladenosine (L-[H-3]PIA) to solubilized receptors from rat brain membranes was studied. The interaction of these receptors with relatively low concentrations of L-[H-3]PIA (0.5-12.0 nM) in the presence of Mg2+ showed the existence of two binding sites for this agonist, with respective dissociation constant (K(D)) values of 0.24 and 3.56 nM and respective receptor number (B(max)) values of 0.28 +/- 0.03 and 0.66 +/- 0.05 pmol/mg of protein. In the presence of GTP, the binding of L-[H-3]PIA also showed two sites with K(D) values of 24.7 and 811.5 nM and B(max) values of 0.27 +/- 0.09 and 0.93 +/- 0.28 pmol/mg of protein for the first and the second binding site, respectively. Inhibition of specific L-[H-3]PIA binding by 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) (0.1-300 nM) performed with the same preparations revealed two DPCPX binding sites with K(i) values of 0.29 and 13.5 nM, respectively. [H-3]DPCPX saturation binding experiments also showed two binding sites with respective K(D) values of 0.81 and 10.7 nM and respective B(max) values of 0.19 +/- 0.02 and 0.74 +/- 0.06 pmol/mg of protein. The results suggest that solubilized membranes from rat brain possess two adenosine receptor subtypes: one of high affinity with characteristics of the A1 subtype and another with lower affinity with characteristics of the A3 subtype of adenosine receptor.