Hydrogen peroxide-induced phospholipase D2 activation in lymphocytic leukemic L1210 cells

Abstract: Extracellular hydrogen peroxide (H2O2) has been implicated in the activation of phospholi-pase 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 activ-ity 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 Ca21 with EGTA com-pletely blocked the H2O2-induced PLD activation, indicating that Ca21 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 PKCa by prolonged treat-ment with 4b-phorbol 12-myristate 13-acetate in-hibited the H2O2-stimulated PLD2 activity, which points to the involvement of PKCa. Based on these new findings we suggest that PLD2 activity is specifi-cally up-regulated by H2O2 and that the H2O2-induced PLD2 activation is mediated by Ca21 influ