fMLP-induced arachidonic acid release in db-cAMP-differentiated HL-60 cells is independent of phosphatidylinositol-4,5-bisphosphate-specific phospholipase C activation and cytosolic phospholipase A(2) activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca2+-dependent cytosolic ph ospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP ki nase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca2+-depe ndent translocation of the enzyme to the membrane. Here we investigated the role of phospholipases in N-formylmethionyl-L-leucyl-L-phenylalanine (fMLP ; 1 nM-10 mu M)-induced AA release from neutrophil-like db-cAMP-differentia ted HL-60 cells. U 73122 (1 mu M), an inhibitor of phosphatidyl-inosito1-4, 5-biphosphate-specific phospholipase C, or the membrane-permeant Ca2+-chela tor 1,2-bis[2-aminophenoxy]ethane-N,N,N',N'-tetraacetic acid (10 mu M) abol ished fMLP-mediated Ca2+ signaling, but had no effect on fMLP-induced AA re lease. The protein kinase C-inhibitor Ro 318220 (5 mu M) or the inhibitor o f cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 mu M) did no t inhibit fMLP-induced AA release. In contrast, AA release was stimulated b y the Ca2+ ionophore A23187 (10 mu M) plus the PKC activator phorbol myrist ate acetate (PMA) (0.2 mu M). This effect was inhibited by either Ro 318220 or AACOCF(3). Accordingly, a translocation of cPLA(2) from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fM LP-mediated AA release therefore appeared to be independent of Ca2+ signali ng and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximate to 45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor o f phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 mu M), d ecreased fMLP-mediated AA release by approximate to 35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude sim ilar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid pho sphohydrolase, This suggests that the bulk of AA generated by fMLP stimulat ion of db-cAMP-differentiated HL-60 cells is independent of the cPLA(2) pat hway, but may originate from activation of PC-PLC and PLD, (C) 2000 Academi c Press.