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Phosphatidic Acid as the Biosynthetic Precursor of the Endocannabinoid 2‐Arachidonoylglycerol in Intact Mouse Neuroblastoma Cells Stimulated with Ionomycin
Author(s) -
Bisogno Tiziana,
Melck Dominique,
Petrocellis Luciano,
Marzo Vincenzo
Publication year - 1999
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.1999.0722113.x
Subject(s) - ionomycin , 2 arachidonoylglycerol , diacylglycerol lipase , phosphatidic acid , phospholipase d , arachidonic acid , phospholipase c , biochemistry , diacylglycerol kinase , phospholipase , phosphatidylethanol , endocannabinoid system , chemistry , biology , microbiology and biotechnology , signal transduction , enzyme , protein kinase c , cannabinoid receptor , phospholipid , receptor , agonist , membrane , intracellular
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.