Phosphatidic Acid as the Biosynthetic Precursor of the Endocannabinoid 2‐Arachidonoylglycerol in Intact Mouse Neuroblastoma Cells Stimulated with Ionomycin

Abstract

Abstract: In mouse neuroblastoma N18TG2 cells prelabeled with [3 H]arachidonic acid ([3 H]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 [3 H]AA showed that ionomycin stimulation leads to the sequential formation of [3 H]phosphatidic acid ([3 H]PA), [3 H]DAG, and [3 H]2‐AG. [3 H]2‐AG biosynthesis in N18TG2 cells prelabeled with [3 H]AA was counteracted by propranolol and N‐ethylmaleimide, two inhibitors of the Mg 2+/Ca 2+‐dependent brain PA phosphohydrolase. Pretreatment of cells with exogenous phospholipase D (PLD) led to a strong potentiation of ionomycin‐induced [3 H]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 [3 H]phosphatidylethanol and did not counteract the formation of [3 H]PA, thus arguing against the activation of PLD by the Ca 2+ ionophore. Selective inhibitors of secretory phospholipase A 2 and the acyl‐CoA acylase inhibitor thimerosal significantly reduced [3 H]2‐AG biosynthesis. The implications of these latter findings, and of the PA‐dependent pathways of 2‐AG formation described here, are discussed.