STIMULATION OF PHOSPHOLIPASE-D ACTIVITY BY PHORBOL ESTERS IN CULTURED ASTROCYTES

Abstract: The phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D‐catalyzed transphosphatidylation, was formed. The half‐maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol‐treated cells was accompanied by an inhibition of the TPA‐induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoylglycerol (100 μM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol‐treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades. Copyright © 1990, Wiley Blackwell. All rights reserved