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We examined the effect of 2-arachidonoylglycerol, an endogenous cannabinoid receptor ligand, on the intracellular free Ca 2؉ concentrations in HL-60 cells that express the cannabinoid CB2 receptor. We found that 2-arachidonoylglycerol induces a rapid transient increase in intracellular free Ca 2؉ concentrations in HL-60 cells. The response was affected by neither cyclooxygenase 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 Ca 2؉ 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, 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 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-palmitoylethanolamine, 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. ⌬ 9 -Tetrahydrocannabinol (⌬ 9 -THC), 1 a major psychoactive constituent of marijuana, is known to exert a variety of pharmacological effects on laboratory animals and humans. When administered orally or intravenously, ⌬ 9 -THC induces reduced spontaneous motor activity, analgesia, heightened sensory awareness, euphoria, and impairment of short-term memory (1). ⌬ 9 -THC is also known to exert profound effects on several biological systems other than the central nervous system; for example, ⌬ 9 -THC exhibits potent immunosuppressive activities (2-12). The mechanism underlying such diverse actions of ⌬ 9 -THC remained obscure until the late 1980s. -THC itself bind to a specific receptor site(s) thereby eliciting a variety of responses. To date, two types of cannabinoid receptors have been identified as follows: the CB1 receptor expressed primarily in the nervous system (14), and the CB2 receptor expressed mainly in the immune system (15). Both are seven transmembrane, G protein-coupled receptors, and they share 44% overall identity (68% identity for transmembrane domains) (15).The discovery of specific receptors for cannabinoids prompted the search for endogenous ligands. To date, two types of arachidonic acid-containing molecules, anandamide (16) and 2-arachidonoylglycerol (2-AG) (17, 18), have been reported as putative endogenous cannabinoid receptor ligands. Anandamide was isolated from porcine brain (16), and 2-AG was isolated from rat brain (17) and canine gut...

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2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2) and has beenshown to exhibit a variety of cannabimimetic activities in vitro and in vivo. Recently, we proposed that 2-arachidonoylglycerol is the true endogenous ligand for the cannabinoid receptors, and both receptors (CB1 and CB2) are primarily 2-arachidonoylglycerol receptors. The CB1 receptor is assumed to be involved in the attenuation of neurotransmission. On the other hand, the physiological roles of the CB2 receptor, which is abundantly expressed in several types of leukocytes such as macrophages, still remain unknown. In this study, we examined the effects of 2-arachidonoylglycerol on the motility of HL-60 cells differentiated into macrophage-like cells. We found that 2-arachidonoylglycerol induces the migration of differentiated HL-60 cells. The migration induced by 2-arachidonoylglycerol was blocked by treatment of the cells with either SR144528, a CB2 receptor antagonist, or pertussis toxin, suggesting that the CB2 receptor and G i /G o are involved in the 2-arachidonoylglycerol-induced migration. Several intracellular signaling molecules such as Rho kinase and mitogen-activated protein kinases were also suggested to be involved. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, failed to induce the migration. The 2-arachidonoylglycerol-induced migration was also observed for two other types of macrophage-like cells, the U937 cells and THP-1 cells, as well as human peripheral blood monocytes. These results strongly suggest that 2-arachidonoylglycerol induces the migration of several types of leukocytes such as macrophages/monocytes through a CB2 receptor-dependent mechanism thereby stimulating inflammatory reactions and immune responses.