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 were accompanied by an ∼ 4-fold increase in in vitro phospholipase (PL) A2 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 PLA2 inhibitor), each suppressed fMLP-stimulated arachidonate release. Treatment with RHC-80267 (a diacylglycerol lipase inhibitor), however, had no such effect. The cytosolic PLA2 (cPLA2) inhibitor, arachidonoyl trifluoromethyl ketone, suppressed PLA2 activity in PMN homogenates and arachidonate release by fMLP-treated PMN. These results suggest that fMLP-elicited arachidonate release is mediated by cPLA2 but not diacylglycerol lipase, and that the activation of cPLA2 is downstream of the PLD-dependent signaling pathway.
