Evidence That 2-Arachidonoylglycerol but Not N-Palmitoylethanolamine or Anandamide Is the Physiological Ligand for the Cannabinoid CB2 Receptor

Abstract

We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intra- cellular free Ca2 concentrations in HL-60 cells that ex- press the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient in- crease in intracellular free Ca2 concentrations in HL-60 cells. The response was affected by neither cy- clooxygenase inhibitors nor lipoxygenase inhibitors, suggesting that arachidonic acid metabolites are not involved. Consistent with this notion, free arachidonic acid was devoid of any agonistic activity. Importantly, the Ca2 transient induced by 2-arachidonoylglycerol was blocked by pretreatment of the cells with SR144528, a CB2 receptor-specific antagonist, but not with SR141716A, a CB1 receptor-specific antagonist, indicat- ing the involvement of the CB2 receptor but not the CB1 receptor in this cellular response. Gi or Go is also as- sumed to be involved, because pertussis toxin treatment of the cells abolished the response. We further examined the structure-activity relationship. We found that 2-arachidonoylglycerol is the most potent compound among a number of naturally occurring cannabimimetic molecules. Interestingly, anandamide and N-palmi- toylethanolamine, other putative endogenous ligands, were found to be a weak partial agonist and an inactive ligand, respectively. These results strongly suggest that the CB2 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic natural ligand for the CB2 receptor that is abundant in the immune system.