Central nervous system responses to cannabis are primarily mediated by CB1 receptors, which couple preferentially to Gi/o G proteins. Here, we used calcium photometry to monitor the effect of CB1 activation on intracellular calcium concentration. Perfusion with 5 μM CB1 aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB1 and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB1 antagonist, SR141716A, and was absent in nontransfected human embryonic kidney 293 cells. The calcium rise was WIN-specific, being essentially absent in cells treated with other classes of cannabinoid agonists, including Δ9-tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of Gi/o, because it was present in pertussis toxin-treated cells. Indeed, pertussis toxin pretreatment enhanced the potency and efficacy of WIN to increase intracellular calcium. The calcium increases appeared to be mediated by Gq G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative Gq or treated with the phospholipase C inhibitors U73122and ET-18-OCH3 and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca2+ pump inhibitor thapsigargin, the inositol trisphosphate receptor inhibitor xestospongin D, and the ryanodine receptor inhibitors dantrolene and 1,1′-diheptyl-4,4′-bipyridinium dibromide, but not by removal of extracellular calcium, showing that WIN releases calcium from intracellular stores. In summary, these results suggest that WIN stabilizes CB1 receptors in a conformation that enables Gq signaling, thus shifting the G protein specificity of the receptor.
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