Phosphatidic acid as the biosynthetic precursor of the endocannabinoid 2-arachidonoylglycerol in intact mouse neuroblastoma cells stimulated with ionomycin.
In mouse neuroblastoma N18TG2 cells prelabeled with [3H]arachidonic acid ([3H]AA) the biosynthesis of 2-arachidonoylglycerol (2-AG) is induced by ionomycin in a fashion sensitive to an inhibitor of diacylglycerol (DAG) lipase, RHC 80267, but not to four different phospholipase C (PLC) blockers. Pulse experiments with [3H]AA showed that ionomycin stimulation leads to the sequential formation of [3H]phosphatidic acid ([3H]PA), [3H]DAG, and [3H]2-AG. [3H]2-AG biosynthesis in N18TG2 cells prelabeled with [3H]AA was counteracted by propranolol and N-ethylmaleimide, two inhibitors of the Mg2+/Ca2(+)-dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin-induced [3H]2-AG formation. These data indicate that DAG precursors for 2-AG in intact N18TG2 cells are obtained from the hydrolysis of PA and not through the activation of PLC. The presence of 2% ethanol during ionomycin stimulation failed to elicit the synthesis of [3H]phosphatidylethanol and did not counteract the formation of [3H]PA, thus arguing against the activation of PLD by the Ca2+ ionophore. Selective inhibitors of secretory phospholipase A2 and the acyl-CoA acylase inhibitor thimerosal significantly reduced [3H]2-AG biosynthesis. The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.
Bisogno
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Istituto per la Chimica di Molecole di Interesse Biologico, Consiglio Nazionale delle Ricerche, Arco Felice, Napoli, Italy.
Melck
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De Petrocellis
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Di Marzo
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D000818:Animals; D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D003512:Cyclohexanones / Q000494:pharmacology; D004075:Diglycerides / Q000037:antagonists & inhibitors / Q000378:metabolism; D063388:Endocannabinoids; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005989:Glycerides / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D006868:Hydrolysis; D015759:Ionomycin / Q000494:pharmacology*; D007476:Ionophores / Q000494:pharmacology*; D008071:Lipoprotein Lipase / Q000037:antagonists & inhibitors; D051379:Mice; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D010712:Phosphatidic Acids / Q000378:metabolism*; D010739:Phospholipase D / Q000494:pharmacology; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors; D011355:Prodrugs / Q000378:metabolism*; D011480:Protease Inhibitors / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D010738:Type C Phospholipases / Q000037:antagonists & inhibitors
D001095:Arachidonic Acids; D063385:Cannabinoid Receptor Modulators; D003512:Cyclohexanones; D004075:Diglycerides; D063388:Endocannabinoids; D004791:Enzyme Inhibitors; D005989:Glycerides; D007476:Ionophores; D010712:Phosphatidic Acids; D011355:Prodrugs; D011480:Protease Inhibitors; D015759:Ionomycin; C036535:1,6-bis(cyclohexyloximinocarbonyl)hexane; C094503:glyceryl 2-arachidonate; D008071:Lipoprotein Lipase; D010744:Phosphoric Monoester Hydrolases; D010738:Type C Phospholipases; D010739:Phospholipase D