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

The mechanism for hydrogen peroxide (H 2 O 2 )-induced phospholipase D (PLD) activation was investigated in [ 3 H]palmitic acid-labeled PC12 cells. In the presence of butanol, H 2 O 2 caused a great accumulation of [3H]phosphatidylbutanol in a concentration- or time-dependent manner. However, treatment with H 2 O 2 of cell lysates exerted no effect on PLD activity. Treatment with H 2 O 2 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 H 2 O 2 -induced PLD activity. Thus, H 2 O 2 -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 H 2 O 2 -induced PLD activation. Western blot analysis revealed several apparent tyrosine-phosphorylated protein bands after the H 2 O 2 treatment and tyrosine phosphorylation of these proteins was inhibited by these tyrosine kinase inhibitors. Moreover, depletion of extracellular Ca 2+ abolished H 2 O 2 -induced PLD activation and protein tyrosine phosphorylation. Extracellular Ca 2+ potentiated H 2 O 2 -induced PLD activation in a concentration-dependent manner. Taken together, these results suggest that a certain Ca 2+ -dependent protein tyrosine kinase(s) somehow participates in H 2 O 2 -induced PLD activation in PC12 cells.