Hydrogen peroxide regulates phospholipase D-mediated hydrolysis of phosphatidylethanolamine and phosphatidylcholine by different mechanisms in NIH 3T3 fibroblasts.

Abstract A major goal of this work was to determine in NIH 3T3 fibroblasts whether the recently described effects of H 2 O 2 on phospholipase D-mediated hydrolysis of phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho) are mediated by similar or different mechanisms. While exposure of NIH 3T3 fibroblasts to H 2 O 2 stimulated the hydrolysis of both PtdEtn and PtdCho, the following important differences were noted: (i) prolonged (24 h) treatment of fibroblasts with 400 nM phorbol 12-myristate 13-acetate (PMA) blocked the stimulatory effect of H 2 O 2 on PtdEtn, but not on PtdCho, hydrolysis; (ii) PMA-induced hydrolysis of PtdEtn, but not PtdCho, was inhibited by H 2 O 2 ; (iii) the stimulatory effect of H 2 O 2 was additive with that of sphingosine or staurosporine, inhibitors of protein kinase C, on the hydrolysis of PtdCho, but not PtdEtn; (iv) with membranes isolated from H 2 O 2 -treated fibroblasts, the hydrolysis of PtdCho, but not PtdEtn, was increased compared to values obtained with control membranes. These results imply that H 2 O 2 regulates PtdEtn and PtdCho hydrolysis by different mechanisms. Stimulation of PtdEtn hydrolysis by H 2 O 2 , sphingosine, and staurosporine may commonly involve, at least in part, neutralization of an inhibitory protein kinase C isozyme.