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 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...

Nonpsychotropic Cannabinoid Receptors Regulate Microglial Cell Migration

“…In 1992, when Devane et al identified AEA as a ligand for CB1 receptors, it seemed reasonable to expect that this lipid was only the first representative of a larger family of endocannabinoids (Devane et al, 1992). Indeed, ensuing studies identified a second endocannabinoid, 2-AG, that acts as a full agonist on CB1 and CB2 receptors (Mechoulam et al, 1995;Sugiura et al, 1995Sugiura et al, , 2000Stella et al, 1997). In this study, we show that neurons and microglial cells produce four endocannabinoids: the two well known endocannabinoids, AEA and 2-AG, as well as the two new endocannabinoids, HEA and DEA.…”

Hematopoietic cells expressing the peripheral cannabinoid receptor migrate in response to the endocannabinoid 2-arachidonoylglycerol

“…19,20,22,56 In the present study we compared the effects of 2-AG with that of the other endocanabinoids (AEA, PEA, POEA), the natural cannabinoids (␦ 8 -THC, ␦ 9 -THC, cannabinol, cannabidiol), and the synthetic molecules (WIN55212-2, CP55940).Among the endocannabinoids, 2-AG was the most potent inducer of migration, supporting the idea that 2-AG is the true endogenous ligand for the peripheral cannabinoid receptor. [56][57][58][59] AEA, which was the first endogenous cannabinoid ligand isolated, 22 shows similar binding affinity to the Cb2 receptor as 2-AG does. 59 However, AEA only weakly stimulated migration of Cb2-expressing cells.…”

Anandamide-induced cell death in primary neuronal cultures: role of calpain and caspase pathways

“…[5][6][7] Anandamide was first isolated from brain and initially characterized as an endogenous ligand for cannabinoid receptors. Although agonist activity at both CB1 and CB2 receptors has been demonstrated, 8,9 anandamide also acts as an agonist at a vanilloid (VR1) or capsaicin receptor. [10][11][12][13] In addition, direct modulation of NMDA receptor activity by AEA has also been described.…”