MASTOPARAN-INDUCED PHOSPHATIDYLCHOLINE HYDROLYSIS BY PHOSPHOLIPASE-D ACTIVATION IN HUMAN ASTROCYTOMA-CELLS

1 The effect of mastoparan on phosphatidylcholine hydrolysis was examined in 1321N1 human astrocytoma cells. Mastoparan (3-30 mu M) caused an accumulation of diacylglycerol (DG) and phosphatidic acid (PA) accompanied by choline release in a concentration- and time-dependent manner. 2 In the presence of 2% n-butanol, mastoparan (3-100 mu M) induced phosphatidylbutanol (PBut) accumulation in a concentration- and time-dependent manner, suggesting that mastoparan activates phospholipase D (PLD). Propranolol (30-300 mu M), a phosphatidate phosphohydrolase inhibitor, inhibited DG accumulation induced by mastoparan, supporting this idea. 3 Depletion of extracellular free calcium ion did not alter the effect of mastoparan on PLD activity. 4 A protein kinase C (PKC) inhibitor, calphostin C (1 mu M), did not inhibit mastoparan-induced PLD. activation but the ability of mastoparan to stimulate phospholipase D activity was decreased in the PKC down regulated cells. 5 PLD activity stimulated by mastoparan was not prevented by pretreatment of the cells with pertussis toxin (PT) or C3 ADP-ribosyltransferase. Furthermore, guanine nucleotides did not affect PLD activity stimulation by mastoparan in membrane preparations. 6 Mastoparan stimulated PLD in several cell lines such as RBL-2H3, RBL-1, HL-60, P388, endothelial cells, as well as 1321N1 human astrocytoma cells. 7 These results suggest that mastoparan induces phosphatidylcholine (PC) hydrolysis by activation of PLD, not by activation of phosphatidylcholine-specific phospholipase C (PC-PLC); mastoparan-induced PLD activation is not mediated by G proteins.