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

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 PKCα by prolonged treatment with 4β-phorbol 12-myristate 13-acetate inhibited the H₂O₂-stimulated PLD₂ activity, which points to the involvement of PKCα. 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 PKCα activation.