CANNABINOID RECEPTOR STIMULATION OF GUANOSINE-5'-O-(3-[S-35]THIO)TRIPHOSPHATE BINDING IN RAT-BRAIN MEMBRANES

Cannabinoid receptors belong to the class of G-protein-coupled recepto rs which inhibit adenylylcyclase. Coupling of receptors to G-proteins can be assessed by the ability of agonists to stimulate guanosine-5'-O -(3-[S-35]thio)triphosphate ([S-35]GTP gamma S) binding in the presenc e of excess GDP. The present study examined the effect of cannabinoid agonists on [S-35]GTP gamma S binding in rat brain membranes. Assays w ere conducted with 0.05 nM [S-35]GTP gamma S, incubated with rat cereb ellar membranes, 1-30 mu M GDP and the cannabinoid agonist WIN 55212-2 . Results showed that the ability of WIN 55212-2 to stimulate [S-35]GT P gamma S binding increased with increasing concentrations of GDP, wit h 10-30 mu M GDP providing approximately 150-200% stimulation by the c annabinoid agonist. The pharmacology of cannabinoid agonist stimulatio n of [S-35]GTP gamma S binding paralleled that of previously reported receptor binding and adenylyl cyclase assays, and agonist stimulation of [S-35]GTP gamma S binding was blocked by the cannabinoid antagonist SR141716A. Brain regional studies revealed widespread stimulation of [S-35]GTP gamma S binding by WIN 55212-2 in a number of brain areas, c onsistent with in vitro [S-35]GTP gamma S autoradiography. These resul ts demonstrate that [S-35]GTP gamma S binding in the presence of exces s GDP is an effective measure of cannabinoid receptor coupling to G-pr oteins in brain membranes.