CB1 cannabinoid receptor-mediated increases in cyclic AMP accumulation are correlated with reduced Gio function.
CB1 cannabinoid receptors (CB1Rs) stimulate Gio-dependent signaling pathways. CB1R-mediated cAMP increases were proposed to result from Gs activation, but CB1R-stimulated GTPγS binding to Gs has not heretofore been investigated. Three models of CB1R-stimulated cAMP production were tested pertussis toxin disruption of Gio in N18TG2 cells L341AA342L-CB1R expressed in Chinese hamster ovary (CHO) cells and CB1 and D2 dopamine receptors endogenously co-expressed in MN9D cells. cAMP was assayed by 3HcAMP binding competition. G protein activation was assayed by the antibody-targeted scintillation proximity assay. In L341AA342L-CB1-CHO cells, cannabinoid agonists significantly stimulated cAMP accumulation over vehicle (-)-3-2-hydroxyl-4-(1,1-dimethylheptyl)phenyl-4-3-hydroxyl propyl cyclohexan-1-ol (CP55940)-stimulated 35SGTPγS binding to Gi123 was reversed, whereas binding to Gs was not different from CB1R. In MN9D cells, CB1 agonist HU210 or D2 agonist quinpirole alone inhibited forskolin-activated cAMP accumulation, whereas HU210 plus quinpirole increased cAMP accumulation above basal. HU210 alone stimulated 35SGTPγS binding to Gi123, whereas co-stimulation with quinpirole reversed HU210-stimulated 35SGTPγS binding to Gi123. CB1R couples to Gs but with low efficacy compared to Gio. The L341AA342L mutation in CB1R reversed CP55940 activation of Gi to an inhibition, but had no effect on Gs. Combined CB1 plus D2 agonists in MN9D cells converted the CB1 agonist-mediated activation of Gi to inhibition of Gi. In these models, the CB1 agonist response was converted to an inverse agonist response at Gi activation. Cannabinoid agonist-stimulated cAMP accumulation can be best explained as reduced activation of Gi, thereby attenuating the tonic inhibitory influence of Gi on the major isoforms of adenylyl cyclase.