Phorbol-12-myristate-13-acetate activation of phospholipase D in human neutrophils leads to the production of phosphatides and diglycerides

The contribution of phospholipase D (PLD) to the production of phosphatides (PA) and diglycerides (DG) in phorbol-12-myristate-13-acetate (PMA)-stimulated human neutrophils was studied. Neutrophils were double labeled with 1-O-[ 3H]alkyl-phosphatidylcholine ([ 3H]alkyl-PC and alkyl-[ 32P]PC. Upon stimulation with PMA, these cells produced 1-O-alkyl-PA (alkyl-PA) and, in the presence of ethanol, 1-O-alkyl-phosphatidylethanol (alkyl-PEt) both containing 3H and 32P. Lagging behind alkyl-PA and alkyl-PEt formation was the production of 1-O-[ 3H]alkyl-diglyceride ([ 3H]alkyl-DG) and [ 32P]orthophosphate ([ 32P]PO 4), suggesting dephosphorylation of alkyl-PA by PA phosphohydrolase (PPH). Furthermore, the PPH inhibitor, propranolol, inhibited the formation of both [ 3H]alkyl-DG and [ 32P]PO 4, while increasing alkyl-PA levels (containing both 3H and 32P). PMA-induced DG mass accumulation was also inhibited by propranolol. The results of this study demonstrate that PMA activates PLD in neutrophils leading to the generation of PA and that the bulk of the DG mass accumulation is derived from the sequential actions of PLD and PPH on PC.