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 [H-3]arachidonic acid ([H-3]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 [H-3]AA showed that ionomycin stimulation leads to the sequential formation of [H-3]phosphatidic acid ([H-3]PA), [H-3]DAG, and [H-3]2-AG. [H-3]2-AG biosynthesis in N18TG2 cells prelabeled with [H-3]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 [H-3]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 [H-3]phosphatidylethanol and did not counteract the formation of [H-3]PA, thus arguing against the activation of PLD by the Ca2+ ionophore, Selective inhibitors of secretory phospholipase A, and the acyl-CoA acylase inhibitor thimerosal significantly reduced [H-3]2-AG biosynthesis, The implications of these latter findings, and of the PA-dependent pathways of 2-AG formation described here, are discussed.