5-LIPOXYGENASE PRODUCTS MODULATE THE ACTIVITY OF THE 85-KDA PHOSPHOLIPASE A(2) IN HUMAN NEUTROPHILS

Addition of submicromolar concentrations of arachidonic acid (AA) to human neutrophils induced a a-fold increase in the activity of a cytosolic phospholipase A(2) (PLA(2)) when measured using sonicated vesicles of 1-stearoyl-2-[C-14]arachidonoylphosphatidylcholine as substrate, A similar increase in cytosolic PLA(2) activity was induced by stimulation of neutrophils with leukotriene B-4 (LTB(4)), 5-oxoeicosatetraenoic acid, or 5-hydroxyeicosatetraenoic acid (5-HETE), LTB(4) was the most potent of the agonists, showing maximal effect at 1 nM. Inhibition of 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevented the AA-induced increase in PLA(2) activity but had no effect on the response induced by LTB(4), Furthermore, pretreatment of neutrophils with a LTB(4)-receptor antagonist, LY 255283, blocked the AA- and LTB(4)-induced activation of PLA(2) but did not influence the action of 5-HETE, Treatment of neutrophils with pancreatic PLA(2) also induced an increase in the activity of the cytosolic PLA(2); this response was inhibited by both eicosatetraynoic acid or LY 255283, The increases in PLA(2) activity in response to stimulation correlated with a shift in electrophoretic mobility of the 85-kDa PLA(2), as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA(2) likely underlies its increase in catalytic activity, Although stimulation of neutrophils with individual lipoxygenase metabolites did not induce significant mobilization of endogenous AA, they greatly enhanced the N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA as determined by mass spectrometry analysis. Our findings support a positive feedback model in which stimulus-induced release of AA or exocytosis of secretory PLA(2) modulate the activity of the cytosolic 85-kDa PLA(2) by initiating the formation of LTB(4). The nascent LTB, is then released to act on the LTB(4) receptor and thereby promote further activation of the 85-kDa PLA(2). Since 5-HETE and LTB, are known to prime the synthesis of platelet-activating factor, the findings suggest that 85-kDa PLA(2) plays a role in platelet-activating factor synthesis.