Cannabinoid receptor stimulation of guanosine-5′-O-(3-[ 35S]thio)triphosphate binding in rat brain membranes

Abstract

Cannabinoid receptors belong to the class of G-protein-coupled receptors which inhibit adenylyl cyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5′-O-(3-[ 35S]thio)triphosphate ([ 35S]GTPγS) binding in the presence of excess GDP. The present study examined the effect of cannabinoid agonists on [ 35S]GTPγS binding in rat brain membranes. Assays were conducted with 0.05 nM [ 35S]GTPγS, incubated with rat cerebellar membranes, 1–30 μM GDP and the cannabinoid agonist WIN 55212-2. Results showed that the ability of WIN 55212-2 to stimulate [ 35S]GTPγS binding increased with increasing concentrations of GDP, with 10–30 μM GDP providing approximately 150–200% stimulation by the cannabinoid agonist. The pharmacology of cannabinoid agonist stimulation of [ 35S]GTPγS binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [ 35S]GTPγS binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [ 35S]GTPγS binding by WIN 55212-2 in a number of brain areas, consistent with in vitro [ 35S]GTPγS autoradiography. These results demonstrate that [ 35S]GTPγS binding in the presence of excess GDP is an effective measure of cannabinoid receptor coupling to G-proteins in brain membranes.