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 A2 Activation

In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca2+-dependent cytosolic phospholipase A2 (cPLA2) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca2+-dependent 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 μM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 μM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca2+-chelator 1,2-bis[2-aminophenoxy]ethane-N,N,N′,N′-tetraacetic acid (10 μM) abolished fMLP-mediated Ca2+ signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 μM) or the inhibitor of cPLA2 arachidonyl trifluoromethyl ketone (AACOCF3; 10–30 μM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca2+ ionophore A23187 (10 μM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 μM). This effect was inhibited by either Ro 318220 or AACOCF3. Accordingly, a translocation of cPLA2 from the cytosol to the membrane fraction was observed with A23187 + PMA, but not with fMLP. fMLP-mediated AA release therefore appeared to be independent of Ca2+ signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by ≈45% by Clostridium difficile toxin B (10 ng/ml) or by 1-butanol; both block phospholipase D (PLD) activity. The inhibitor of phosphatidylcholine-specific phospholipase C (PC-PLC), D609 (100 μM), decreased fMLP-mediated AA release by ≈35%. The effect of D609 + 1-butanol on fMLP-induced AA release was additive and of a magnitude similar to that of propranolol (0.2 mM), an inhibitor of phosphatidic acid phosphohydrolase. This suggests that the bulk of AA generated by fMLP stimulation of db-cAMP-differentiated HL-60 cells is independent of the cPLA2 pathway, but may originate from activation of PC-PLC and PLD.