Cannabinoid receptor antagonists and G protein subtypes

Abstract

The therapeutic importance of inverse agonism for cannabinoid receptors (CB1R) has been proposed. The underlying mechanisms of inverse cannabimimetic effects by the CB1R antagonist/inverse agonist, SR141716 were investigated in both N18TG2 mouse neuroblastoma and human embryonic kidney (HEK)‐293 cells stably expressing CB1Rs (CB1‐HEK). We utilized cell membranes in ligand‐modulated [35S]GTPγS binding and scintillation proximity assay (SPA) (Eldeeb et al., 2016 and 2017). The CB1R agonist, CP55940 stimulated [35S]GTPγS binding (from 1nM: 2.6±5.5% to 1μM: 54±7.3% over basal). Co‐incubation with 1μM of SR141716 inhibited CP55940‐mediated [35S]GTPγS binding to −27±3.2% and −5.6±7%, respectively. In N18TG2 cells treated with the diacylglycerol lipase inhibitor, tetrahydrolipstatin (THL, orlistat), 1μM for 2h, SR141716 antagonizing effects were unchanged. In membranes from cells pretreated with pertussis toxin (PT) (100 ng/ml) for 20 h, SR14171 inhibition of [35S]GTPγS binding was attenuated in PT‐pretreated membranes, indicating that SR141716 effects were Gi/o protein‐dependent. Using SPA, SR14176 alone inhibited [35S]GTPγS binding by Gi/o protein subtypes, primarily Gαi1 and Gαi3 (−46.5±6.6% and −46.7±6.2% with respect to basal). Both previously reported CB1R neutral antagonists, AM4113 and PIMSR, at 1μM were able to reduce maximal CP55940‐stimulated [35S]GTPγS binding for total and individual Gi/o protein subtypes, primarily Gαi1 and Gαi3. Neither AM4113 nor PIMSR (1μM) were able to competitively reverse the SR141716‐mediated inhibition of [35S]GTPγS binding in N18TG2 neuronal cells membranes. In CB1‐HEK cell membranes neither AM4113 nor PIMSR(1μM) behaved as neutral antagonist to the CP55940‐mediated stimulation of [35S]GTPγS binding. The CB1R constitutive activity is mediated by Gi/o protein stimulation, predominantly Gαi1 and Gαi3, and in our experiments, was not associated with endogenously released endocannabinoids. Further research into the molecular mechanism of the competitive antagonist inverse agonist and/or negative allosteric modulator effects of SR141716 and its analogs will be necessary to consider in developing “neutral antagonists” for the CB1 receptor.Support or Funding InformationNIH grants R01‐DA03690, R01‐DA042157, P50‐DA006634, K12‐GM102773This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.