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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...

The levels of N-arachidonoylethanolamine (anandamide), an endogenous cannabinoid-receptor ligand, and a relevant molecule, N-arachidonoylphosphatidylethanolamine (N-arachidonoylPtdEtn), in rat brain were investigated using a newly developed sensitive analytical method. We found that rat brain contains small but significant amounts of these two types of N-arachidonoyl lipids (4.3 pmol/g tissue and 50.2 pmol/g tissue, respectively). Then, we investigated how N-arachidonoylethanolamine (anandamide) is produced in the brain. We found that anandamide can be formed enzymatically via two separate synthetic pathways in the brain : enzymatic condensation of free arachidonic acid and ethanolamine; and formation of N-arachidonoylPtdEtn from PtdEtn and arachidonic acid esterified at the I-position of phosphatidyl-choline (PtdCho), and subsequent release of anandamide from N-arachidonoylPtdEtn through the action of a phosphodiesterase. We confirmed that rat brain contains both the enzyme activities and lipid substrates involved in these reactions. Several lines of evidence strongly suggest that the second pathway, rather than the first one, meets the requirements and conditions for the synthesis of various species of N-acylethanolamine including anandamide in the brain.

G2A is a G protein-coupled receptor that is predominantly expressed in lymphoid tissues and macrophages. G2A can be induced by diverse stimuli to cause cell cycle arrest in the G 2 /M phase in pro-B and T cells. G2A is also expressed in macrophages within atherosclerotic lesions, suggesting G2A involvement in atherosclerosis. Recently, G2A was discovered to possess proton-sensing ability. In this paper, we report another function of G2A, that is, as a receptor for 9-hydroxyoctadecadienoic acid (9-HODE) and other oxidized free fatty acids. G2A, expressed in CHO-K1 or HEK293 cells, showed 9-HODE-induced intracellular calcium mobilization, inositol phosphate accumulation, inhibition of cAMP accumulation, [35 S]guanosine 5-3-O-(thio)triphosphate binding, and MAP kinase activation. Furthermore, G2A was activated by various oxidized derivatives of linoleic and arachidonic acids, but it was weakly activated by cholesteryl-9-HODE. Oxidized phosphatidylcholine (1-palmitoyl-2-linoleoyl) when hydrolyzed with phospholipase A 2 also evoked intracellular calcium mobilization in G2A-expressing cells. These results indicate that G2A is activated by oxidized free fatty acids produced by oxidation and subsequent hydrolysis of phosphatidylcholine or cholesteryl linoleate. Thus, G2A might have a biological role in diverse pathological conditions including atherosclerosis.

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An endogenous cannabimimetic molecule, 2-arachidonoylglycerol, induces a rapid, transient increase in intracellular free Ca 2؉ concentrations in NG108 -15 cells through a cannabinoid CB1 receptor-dependent mechanism. We examined the activities of 24 relevant compounds (2-arachidonoylglycerol, its structural analogues, and several synthetic cannabinoids). We found that 2-arachidonoylglycerol is the most potent compound examined so far: its activity was detectable from as low as 0.3 nM, and the maximal response induced by 2-arachidonoylglycerol exceeded the responses induced by others. Activities of HU-210 and CP55940, potent cannabinoid receptor agonists, were also detectable from as low as 0.3 nM, whereas the maximal responses induced by these compounds were low compared with 2-arachidonoylglycerol. Anandamide was also found to act as a partial agonist in this assay system. We confirmed that free arachidonic acid failed to elicit a response. Furthermore, we found that a metabolically stable ether-linked analogue of 2-arachidonoylglycerol possesses appreciable agonistic activity, although its activity was apparently lower than that of 2-arachidonoylglycerol. We also confirmed that pretreating cells with various cannabinoid receptor agonists nullified the response induced by 2-arachidonoylglycerol, whereas pretreating cells with other neurotransmitters or neuromodulators did not affect the response. These results strongly suggested that the cannabinoid CB1 receptor is originally a 2-arachidonoylglycerol receptor, and 2-arachidonoylglycerol is the intrinsic physiological ligand for the cannabinoid CB1 receptor.It is well known that ⌬ 9 -tetrahydrocannabinol (⌬ 9 -THC), 1 a psychoactive ingredient of marijuana, possesses a variety of pharmacological activities in vitro and in vivo (1), although, until recently, the mechanism of the action of ⌬ 9 -THC had long been unelucidated. In 1988, Devane et al. (2) provided evidence that a specific binding site(s) for cannabinoids is present in the brain. Soon after, Matsuda et al. (3) cloned a cDNA encoding a cannabinoid receptor (CB1) from a rat brain cDNA library. These findings raised the possibility that at least part of the action of ⌬ 9 -THC is mediated through such a specific receptor and prompted the search for endogenous cannabinoid receptor ligands in mammalian tissues.In 1992, Devane et al. (4) isolated N-arachidonoylethanolamine (anandamide) from porcine brain as the first endogenous cannabinoid receptor ligand. They demonstrated that anandamide exhibits several cannabimimetic activities in vitro and in vivo (4, 5). So far, a number of studies have been carried out on anandamide, and it has been assumed that anandamide is one of the important lipid mediators in the nervous system as well as in other systems (5). However, we (6, 7) and others (8 -11) have found that the levels of anandamide are very low in several mammalian tissues. In addition, the biosynthetic pathways for anandamide, either the N-acylphosphatidylethanolamine pathway (6,7,(11)(12)(13)(14) or t...

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The molecular species compositions of monoacylglycerols obtained from various rat tissues were examined by reverse-phase high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analyses. We confirmed that 2-arachidonoylglycerol, an endogenous cannabinoid receptor agonist, is one of the most abundant molecular species of monoacylglycerols in the brain. Substantial amounts of 2-arachidonoylglycerol were also found in the liver, spleen, lung and kidney, but the levels were considerably lower than that in the brain. We found that a small amount of 2-arachidonoylglycerol was generated in a brain homogenate during incubation in the absence of Ca P+ . Importantly, the generation of 2-arachidonoylglycerol was markedly augmented in the presence of Ca P+ , suggesting that Ca P+ plays a key role in regulation of the generation of 2-arachidonoylglycerol in this tissue.z 1998 Federation of European Biochemical Societies.