Hydrogen peroxide-induced phospholipase D-2 activation in lymphocytic leukemic L1210 cells

Abstract

Extracellular hydrogen peroxide (H2O2) has been implicated in the activation of phospholipase D (PLD), However, it was still unclear how this activation occurs and what the molecular identity of the H2O2-stimulated PLD isozyme is. This study shows that H2O2: potently increases the PLD activity in mouse lymphocytic leukemic L1210 cells, which contain exclusively PLD2, In addition, H2O2 increased PLD activity only in PLD2-transfected COS-7 cells and not in PLD1-transfected cells, This suggests that PLD2 is selectively activated by H2O2 Depletion of extracellular Ca2+ with EGTA completely blocked the H2O2-induced PLD activation, indicating that Ca2+ influx is required. Moreover, pretreatment of the cells with the protein kinase C (PKC) inhibitors GF-109203X and RO-31-8220 and down-regulation of PKC alpha by prolonged treatment with 4 beta-phorbol 12-myristate 13-acetate inhibited the H2O2-stimulated PLD2 activity, which points to the involvement of PKC alpha. Based on these new findings we suggest that PLD2 activity is specifically up-regulated by H2O2 and that the H2O2-induced PLD2 activation is mediated by Ca2+ influx and PKC alpha activation.