Hydrogen Peroxide-Induced Phospholipase D Activation in Rat Pheochromocytoma PC12 Cells: Possible Involvement of Ca2+-Dependent Protein Tyrosine Kinase

Abstract: 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.