Guanine Nucleotide Effects on 8‐Cyclopentyl‐1,3‐[ 3 H]Dipropylxanthine Binding to Membrane‐Bound and Solubilized A 1 Adenosine Receptors of Rat Brain

Abstract: The effects of guanine nucleotides on binding of 8‐cyclopentyl‐1,3‐[ 3 H]dipropylxanthine ([ 3 H]DPCPX), a highly selective A 1 adenosine receptor antagonist, have been investigated in rat brain membranes and solubilized A 1 receptors. GTP, which induces uncoupling of receptors from guanine nucleotide binding proteins, increased binding of [ 3 H]DPCPX in a concentration‐dependent manner. The rank order of potency for different guanine nucleotides for increasing [ 3 H]DPCPX binding was the same as for guanine nucleotide‐induced inhibition of agonist binding. Therefore, a role for a guanine nucleotide binding protein, e.g., G i , in the regulation of antagonist binding is suggested. This was confirmed by inactivation of G i by N ‐ethylmaleimide (NEM) treatment of membranes, which resulted in an increase in [ 3 H]DPCPX binding similar to that seen with addition of GTP. Kinetic and equilibrium binding studies showed that the GTP‐ or NEM‐induced increase in antagonist binding was not caused by an affinity change of A 1 receptors for [ 3 H]DPCPX but by an increased B max value. Guanine nucleotides had similar effects on membrane‐bound and solubilized receptors, with the effects in the solubilized system being more pronounced. In the absence of GTP, when most receptors are in a high‐affinity state for agonists, only a few receptors are labeled by [ 3 H]DPCPX. It is suggested that [ 3 H]DPCPX binding is inhibited when receptors are coupled to G i . Therefore, uncoupling of A 1 receptors from G i by guanine nucleotides or by inactivation of G i with NEM results in an increased antagonist binding.