Phospholipase D is involved in cytosolic phospholipase A(2)-dependent selective release of arachidonic acid by fMLP-stimulated rat neutrophils

When rat polymorphonuclear neutrophils (PMN) were treated with N-formyl-Met-Leu-Phe (fMLP), the release of arachidonic acid in preference to other fatty acids was observed. Levels of arachidonic acid reached a plateau within 5 min, and mere accompanied by an similar to 4-fold increase in in vitro phospholipase (PL) A(2) and PLD activities in PMN lysates. Treatment of PMN with ethanol (an inhibitor of PLD-mediated phosphatidic acid formation), propranolol (a phosphatidic acid phosphatase inhibitor), or 4-bromophenacylbromide (a PLA(2) inhibitor), each suppressed fMLP-stimulated arachidonate release, Treatment with RHC-80267 (a diacylglycerol lipase inhibitor), however, had no such effect. The cytosolic PLA(2) (cPLA(2)) inhibitor, arachidonoyl trifluoromethyl ketone, suppressed PLA(2) activity in PMN homogenates and arachidonate release by fMLP-treated PMN. These results suggest that fMLP-elicited arachidonate release is mediated by cPLA(2) but not diacylglycerol lipase, and that the activation of cPLA(2) is downstream of the PLD-dependent signaling pathway.