Abstract

Cannabinoid receptors are members of the superfamily of G protein-coupled receptors. Their activation has previously been shown to stimulate guanosine 5′-O-(3-[³⁵S]thio)-triphosphate ([³⁵S]GTPγS) binding in a range of brain regions using both membrane preparations and autoradiography. This study evaluates the activities of structurally diverse cannabinoid receptor ligands in the GTPγS binding assay, comparing the relationship between receptor binding and activation and also examining efficacy differences between compounds. Using rat cerebellar membrane preparations, the effects of GDP concentration on GTPγS binding and the activities of a range of cannabinoid receptor ligands, including the CB₁ selective antagonist SR141716A, were investigated. GDP concentration was found to have differing effects on cannabinoid-stimulated [³⁵S]GTPγS binding depending on the nature of the agonist used. The stimulation produced by high efficacy compounds, such as CP 55,940 and WIN 55212–2, was increased by raising the GDP concentration, but that of a low efficacy agonist, (−)-Δ-tetrahydrocannabinol, was decreased. Of the cannabinoid compounds tested, a wide range of potencies (EC₅₀) and levels of maximal stimulation (E_max) were observed. These ranged from CP 55,244 (E_max of 165, 148–183%, and an EC₅₀ of 0.47, 0.22–0.96, nM) through (−)-Δ-tetrahydrocannabinol, cannabinol and anandamide, which produced no concentration-dependent stimulation of [³⁵S]GTPγS binding under the same conditions. SR141716A competitively antagonized all the agonists against which it was tested, providing equilibrium dissociation constants (K_d values) in the sub-nanomolar range (0.06–0.40 nM), implicating a CB₁ receptor mediated response. These results provide a more detailed characterization of the cannabinoid-stimulated [³⁵S]GTPγS binding assay than has previously been reported.