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

Extracellular hydrogen peroxide (H 2 O 2 ) 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 H 2 O 2 ‐stimulated PLD isozyme is. This study shows that H 2 O 2 potently increases the PLD activity in mouse lymphocytic leukemic L1210 cells, which contain exclusively PLD 2 . In addition, H 2 O 2 increased PLD activity only in PLD 2 ‐transfected COS‐7 cells and not in PLD 1 ‐transfected cells. This suggests that PLD 2 is selectively activated by H 2 O 2 . Depletion of extracellular Ca 2+ with EGTA completely blocked the H 2 O 2 ‐induced PLD activation, indicating that Ca 2+ 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 PKCct by prolonged treatment with 4β‐phorbol 12‐myristate 13‐acetate inhibited the H 2 O 2 ‐stimulated PLD 2 activity, which points to the involvement of PKCα. Based on these new findings we suggest that PLD 2 activity is specifically up‐regulated by H 2 O 2 and that the H 2 O 2 ‐induced PLD 2 activation is mediated by Ca 2+ influx and PKCα activation. J. Leukoc. Biol. 67: 630–636; 2000.