The cannabinoid agonist WIN55,212-2 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins

Central nervous system responses to cannabis are primarily mediated by CB 1 receptors, which couple preferentially to G i/o G proteins. Here, we used calcium photometry to monitor the effect of CB 1 activation on intracellular calcium concentration. Perfusion with 5 μM CB 1 aminoalkylindole agonist, WIN55,212-2 (WIN), increased intracellular calcium by several hundred nanomolar in human embryonic kidney 293 cells stably expressing CB 1 and in cultured hippocampal neurons. The increase was blocked by coincubation with the CB 1 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 Δ 9 -tetrahydrocannabinol, HU-210, CP55,940, 2-arachidonoylglycerol, methanandamide, and cannabidiol. The increase in calcium elicited by WIN was independent of G i/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 G q G proteins and phospholipase C, because they were markedly attenuated in cells expressing dominant-negative G q or treated with the phospholipase C inhibitors U73122 and ET-18-OCH 3 and were accompanied by an increase in inositol phosphates. The calcium increase was blocked by the sarco/endoplasmic reticulum Ca 2+ 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 CB 1 receptors in a conformation that enables G q signaling, thus shifting the G protein specificity of the receptor. aminoalkylindole inositol phosphate neurons phospholipase C