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 h uman neutrophils induced a a-fold increase in the activity of a cytoso lic 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 stimulat ion of neutrophils with leukotriene B-4 (LTB(4)), 5-oxoeicosatetraenoi c acid, or 5-hydroxyeicosatetraenoic acid (5-HETE), LTB(4) was the mos t potent of the agonists, showing maximal effect at 1 nM. Inhibition o f 5-lipoxygenase with either eicosatetraynoic acid or zileuton prevent ed 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 in hibited by both eicosatetraynoic acid or LY 255283, The increases in P LA(2) activity in response to stimulation correlated with a shift in e lectrophoretic mobility of the 85-kDa PLA(2), as determined by Western blot analysis, suggesting that phosphorylation of the 85-kDa PLA(2) l ikely underlies its increase in catalytic activity, Although stimulati on of neutrophils with individual lipoxygenase metabolites did not ind uce significant mobilization of endogenous AA, they greatly enhanced t he N-formylmethionyl-leucyl-phenylalanine-induced mobilization of AA a s determined by mass spectrometry analysis. Our findings support a pos itive feedback model in which stimulus-induced release of AA or exocyt osis 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 the n released to act on the LTB(4) receptor and thereby promote further a ctivation of the 85-kDa PLA(2). Since 5-HETE and LTB, are known to pri me the synthesis of platelet-activating factor, the findings suggest t hat 85-kDa PLA(2) plays a role in platelet-activating factor synthesis .