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.

The effects of delta9-tetrahydrocannabinol and 2-arachidonoylglycerol on the intracellular free Ca2+ concentration ([Ca2+]i) in NG108-15 cells were examined in detail. We found that delta9-tetrahydrocannabinol induces a rapid, modest increase in [Ca2+]i. The response was detectable with 3 nM delta9-tetrahydrocannabinol. We also found that very low concentrations of 2-arachidonoylglycerol elicit a rapid, more prominent increase in [Ca2+]i. Such a response was observed not only in NG108-15 cells but also in N18TG2 cells. The response induced by 2-arachidonoylglycerol in either NG108-15 cells or N18TG2 cells was abolished by pretreatment of the cells with a cannabinoid CB1 receptor specific antagonist, SR141716A, suggesting that 2-arachidonoylglycerol interacts with the CB1 receptor to induce the response. The results of an experiment involving a phospholipase C inhibitor suggested that phospholipase C is involved in the rapid increase in [Ca2+]i induced by 2-arachidonoylglycerol. We also found that 1(3)-arachidonoylglycerol exhibits similar activity to that of 2-arachidonoylglycerol, although its activity at low concentrations was somewhat weak compared with that of 2-arachidonoylglycerol. We further confirmed that several structural analogues of 2-arachidonoylglycerol were less active compared with 2-arachidonoylglycerol. These results suggest that the structure of 2-arachidonoylglycerol is strictly recognized by the CB1 receptor, which raises the possibility that the CB1 receptor is originally a 2-arachidonoylglycerol receptor.

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CoA-dependent transacylation activity in microsomes catalyzes the transfer of fatty acid between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acid. We examined the mechanism of the transacylation system using partially purified acyl-CoA:lysophosphatidylinositol (LPI) acyltransferase (LPIAT) from rat liver microsomes to test our hypothesis that both the reverse and forward reactions of acyl-CoA:lysophospholipid acyltransferases are involved in the CoA-dependent transacylation process. The purified LPIAT fraction exhibited ATP-independent acyl-CoA synthetic activity and CoA-dependent LPI generation from PI, suggesting that LPIAT could operate in reverse to form acyl-CoA and LPI. CoA-dependent acylation of LPI by the purified LPIAT fraction required PI as the acyl donor. In addition, the combination of purified LPIAT and recombinant lysophosphatidic acid acyltransferase could reconstitute CoA-dependent transacylation between PI and phosphatidic acid. These results suggest that the CoA-dependent transacylation system consists of the following: 1) acyl-CoA synthesis from phospholipid through the reverse action of acylCoA:lysophospholipid acyltransferases; and 2) transfer of fatty acyl moiety from the newly formed acyl-CoA to lysophospholipid through the forward action of acylCoA:lysophospholipid acyltransferases.The fatty acyl moieties of phospholipids are not static but instead are dynamically turned over. Acyltransferases and transacylases catalyze the acylation of lysophospholipids and are involved in the biosynthesis and fatty acid remodeling of phospholipids (1-4). A CoA-dependent transacylation system (or CoA-dependent transacylase) catalyzes the transfer of fatty acids from phospholipids to lysophospholipids in the presence of CoA without generation of free fatty acids (5-11). In rabbit liver microsomes, arachidonic acid (20:4n-6), linoleic acid (18: 2n-6), and stearic acid (18:0), esterified in phospholipids, are the main species transferred to lysophospholipids in the presence of CoA, indicating that this system shows some fatty acid specificity (11). Phosphatidylcholine (PC), 1 phosphatidylethanolamine (PE), and phosphatidylinositol (PI) have been shown to serve as donor phospholipids, PI being the preferred acyl donor (10, 11). Despite the importance of CoA-dependent transacylation system in fatty acid remodeling of phospholipids, the mechanism underlying the transacylation system is not fully understood yet. In fact, the identities of the enzymes responsible for CoA-dependent transacylation reactions have not been established. Although we and other investigators (5,6,11,(12)(13)(14) considered previously that acyl-CoA:lysophospholipid acyltransferases may be involved in CoA-dependent transacylation reactions, direct and conclusive evidence has not been obtained because of the lack of success in purification and cloning of membrane-bound lysophospholipid acyltransferases and transacylases, including the CoA-dependent transacylation system.Previously, we de...