Phosphatidylethanol Biosynthesis in Ethanol - exposed NG 108 - 15 Neuroblastoma X Glioma Hybrid Cells

12-0-Tetradecanoylphorbol-13-acetate (TPA) stimulates the release of free choline from intact NG10815 cells into the medium, without affecting the release of phosphocholine (Liscovitch, M., Blusztajn, J. K., Freese, A., and Wurtman, R. J. (1987) Biochem. J. 241, 81-86). To test the hypothesis that this response reflects activation of cellular phospholipase D, via protein kinase C (Ca’+/phospholipid-dependent enzyme), I examined in NG108-15 cells the biosynthesis of the abnormal phospholipid phosphatidylethanol, produced by phospholipase D in the presence of ethanol by transphosphatidylation. Phosphatidylethanol production was quantitated by measuring the incorporation of phosphatidyl moieties (prelabeled metabolically with [3H]oleic acid) into phosphatidylethanol. The production of phosphatidylethanol in NGlO8-15 cells was virtually dependent on stimulation by TPA, in a timeand concentration-dependent manner (ECao = 18 nM). The rate of 3H-phosphatidylethanol formation reached a peak after 10 min of incubation with TPA and declined gradually thereafter. The levels of ‘H-phosphatidylethanol in TPA-treated cells were directly related to ethanol concentration in the physiologically attainable range (20-80 mM). Phosphatidylethanol production was activated only by phorbol derivatives that are activators of protein kinase C (zk TPA, 48-phorbol12,13-dibutyrate, and 4j3-phorbol-12,13-didecanoate) and could be mimicked by a cell-permeant diacylglycerol, 1,2-dioctanoyl-an-glycerol, in a nonadditive manner. The effect of TPA was inhibited by the protein kinase C inhibitor l-(5-isoquinolinesulfonyl)-2-methylpiperazine (0.1 mM) by 70% but not by N-(Z-guanidinoethyl)-5-isoquinolinesulfonamide. Phosphatidylethanol formation was completely abolished in cells in which protein kinase C was down-regulated by pretreatment of the cells with TPA. These results indicate that phosphatidylethanol biosynthesis in NGlO8-15 cells depends largely on activation of protein kinase C. In contrast to its effects on the release of free choline and on the accumulation of phosphatidylethanol, TPA did not affect the levels of phosphatidic acid in NG10815 cells. It is therefore proposed that protein kinase C selectively activates the phosphatidyl transferase activity of phospholipase D, reflecting a signal termination mechanism which may be operative in phospholipase D-mediated signal transduction cascades.