Evidence that 2-arachidonoylglycerol but not N-palmitoylethanolamine or anandamide is the physiological ligand for the cannabinoid CB2 receptor - Comparison of the agonistic activities of various cannabinoid receptor ligandsin HL-60 cells

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca2+ concentrations in HL-60 ce lls that ex press the cannabinoid CB2 receptor. We found that 2-arachidonoy lglycerol induces a rapid transient increase in intracellular free Ca2+ con centrations in HL-60 cells. The response was affected by neither cyclooxyge nase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic ac id metabolites are not involved. Consistent with this notion, free arachido nic acid was devoid of any agonistic activity. Importantly, the Ca2+ transi ent induced by 2-arachidonoylglycerol was blocked by pretreatment of the ce lls with SR144528, CB2 receptor-specific antagonist, but not with SR141716A , a CB1 receptor-specific antagonist, indicating the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. G(i) or G(o) is also assumed to be involved, because pertussis toxin treatment of the ce lls abolished the response. We further examined the structure-activity rela tionship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interesting ly, anandamide and N-palmitoylethanolamine, other putative endogenous ligan ds, were found to be a weak partial agonist and an inactive ligand, respect ively, These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrins ic natural ligand for the CB2 receptor that is abundant in the immune syste m.