Hydrogen peroxide-induced phospholipase D activation in rat pheochromocytoma PC12 cells: possible involvement of Ca2+-dependent protein tyrosine kinase.

The mechanism for hydrogen peroxide (H2O2)-induced phospholipase D (PLD) activation was investigated in [3H]palmitic acid-labeled PC12 cells. In the presence of butanol, H2O2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H2O2 of cell lysates exerted no effect on PLD activity. Treatment with H2O2 had only a marginal effect on phospholipase C (PLC) activation. A protein kinase C (PKC) inhibitor, Ro 31-8220, did not inhibit but rather slightly enhanced H2O2-induced PLD activity. Thus, H2O2-induced PLD activation is considered to be independent of the PLC-PKC pathway in PC12 cells. In contrast, pretreatment with tyrosine kinase inhibitor herbimycin A, genistein, or ST638 resulted in a concentration-dependent inhibition of H2O2-induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H2O2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca2+ abolished H2O2-induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca2+ potentiated H2O2-induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca2+-dependent protein tyrosine kinase(s) somehow participates in H2O2-induced PLD activation in PC12 cells.