title: 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.
abstract: In inflammatory cells, agonist-stimulated arachidonic acid (AA) release is thought to be induced by activation of group IV Ca(2+)-dependent cytosolic phospholipase A(2) (cPLA(2)) through mitogen-activated protein kinase (MAP kinase)- and/or protein kinase C (PKC)-mediated phosphorylation and Ca(2+)-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 microM)-induced AA release from neutrophil-like db-cAMP-differentiated HL-60 cells. U 73122 (1 microM), an inhibitor of phosphatidyl-inositol-4,5-biphosphate-specific phospholipase C, or the membrane-permeant Ca(2+)-chelator 1, 2-bis¿2-aminophenoxyĕthane-N,N,N',N'-tetraacetic acid (10 microM) abolished fMLP-mediated Ca(2+) signaling, but had no effect on fMLP-induced AA release. The protein kinase C-inhibitor Ro 318220 (5 microM) or the inhibitor of cPLA(2) arachidonyl trifluoromethyl ketone (AACOCF(3); 10-30 microM) did not inhibit fMLP-induced AA release. In contrast, AA release was stimulated by the Ca(2+) ionophore A23187 (10 microM) plus the PKC activator phorbol myristate acetate (PMA) (0.2 microM). 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. fMLP-mediated AA release therefore appeared to be independent of Ca(2+) signaling and PKC and MAP kinase activation. However, fMLP-mediated AA release was reduced by approximately 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 microM), decreased fMLP-mediated AA release by approximately 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 cPLA(2) pathway, but may originate from activation of PC-PLC and PLD.
authors.affiliation: Physiologisches Institut, Universität des Saarlandes, Homburg/Saar, D-66421, Germany.
authors.forename: L
authors.initials: L
authors.lastname: Sternfeld
authors.forename: F
authors.initials: F
authors.lastname: Thévenod
authors.forename: I
authors.initials: I
authors.lastname: Schulz
mesh_terms: D020001:1-Butanol / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism*; D001095:Arachidonic Acids / Q000494:pharmacology; D001426:Bacterial Proteins; D001427:Bacterial Toxins / Q000494:pharmacology; D001952:Bridged-Ring Compounds / Q000494:pharmacology; D003994:Bucladesine / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000201:enzymology; D003600:Cytosol / Q000201:enzymology*; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004963:Estrenes / Q000494:pharmacology; D018922:HL-60 Cells; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D008954:Models, Biological; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000378:metabolism*; D009636:Norbornanes; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D054801:Phosphoinositide Phospholipase C; D010739:Phospholipase D / Q000378:metabolism; D010741:Phospholipases A / Q000378:metabolism*; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011760:Pyrrolidinones / Q000494:pharmacology; D013859:Thiocarbamates; D013871:Thiones / Q000494:pharmacology; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism* / Q000494:pharmacology
chemical_list: D001095:Arachidonic Acids; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001952:Bridged-Ring Compounds; D004791:Enzyme Inhibitors; D004963:Estrenes; D007211:Indoles; D007476:Ionophores; D009636:Norbornanes; D019269:Phosphatidylinositol 4,5-Diphosphate; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D013859:Thiocarbamates; D013871:Thiones; C031815:toxB protein, Clostridium difficile; C081565:arachidonyltrifluoromethane; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D016718:Arachidonic Acid; D000001:Calcimycin; D004533:Egtazic Acid; D009240:N-Formylmethionine Leucyl-Phenylalanine; C046498:tricyclodecane-9-yl-xanthogenate; D003994:Bucladesine; D020001:1-Butanol; D010741:Phospholipases A; D010727:Phosphoric Diester Hydrolases; D010738:Type C Phospholipases; D054801:Phosphoinositide Phospholipase C; C105598:phosphatidylcholine-specific phospholipase C; D010739:Phospholipase D; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium; C064758:Ro 31-8220