Activation and priming of neutrophil nicotinamide adenine dinucleotide phosphate oxidase and phospholipase A(2) are dissociated by inhibitors of the kinases p42(ERK2) and p38(SAPK) and by methyl arachidonyl fluorophosphonate, the dual inhibitor of cytosolic and calcium-independent phospholipase A(2).
Arachidonic acid (AA) generated by phospholipase A(2) (PLA(2)) is thought to be an essential cofactor for phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity. Both enzymes are simultaneously primed by cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-alpha). The possibility that either unprimed or cytokine-primed responses of PLA(2) or NADPH oxidase to the chemotactic agents formyl-methionyl-leucyl-phenylalanine (FMLP) and complement factor 5a (C5a) could be differentially inhibited by inhibitors of the mitogen-activated protein (MAP) kinase family members p42(ERK2) (PD98059) and p38(SAPK) (SB203580) was investigated. PD98059 inhibited the activation of p42(ERK2) by GM-CSF, TNF-alpha, and FMLP, but it did not inhibit FMLP-stimulated superoxide production in either unprimed or primed neutrophils. There was no significant arachidonate release from unprimed neutrophils stimulated by FMLP, and arachidonate release stimulated by calcium ionophore A23187 was not inhibited by PD98059. In contrast, PD98059 inhibited both TNF-alpha- and GM-CSF-primed PLA(2) responses stimulated by FMLP. On the other hand, SB203580 inhibited FMLP-superoxide responses in unprimed as well as TNF-alpha- and GM-CSF-primed neutrophils, but failed to inhibit TNF-alpha- and GM-CSF-primed PLA(2) responses stimulated by FMLP, and additionally enhanced A23187-stimulated arachidonate release, showing that priming and activation of PLA(2) and NADPH oxidase are differentially dependent on both the p38(SAPK) and p42(ERK2) pathways. Studies using C5a as an agonist gave similar results and confirmed the findings with FMLP. In addition, methyl arachidonyl fluorophosphonate (MAFP), the dual inhibitor of c and iPLA(2) enzymes, failed to inhibit superoxide production in primed cells at concentrations that inhibited arachidonate release. These data demonstrate that NADPH oxidase activity can be dissociated from AA generation and indicate a more complex role for arachidonate in neutrophil superoxide production.
Mollapour
E
E
Department of Haematology, The Royal Free and University College, London Medical Schools, London, England.
Linch
D C
DC
Roberts
P J
PJ
D016718:Arachidonic Acid / Q000378:metabolism / Q000502:physiology*; D001095:Arachidonic Acids / Q000494:pharmacology*; D000001:Calcimycin / Q000494:pharmacology; D015936:Complement C5a / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005419:Flavonoids / Q000494:pharmacology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D054522:Group VI Phospholipases A2; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007476:Ionophores / Q000494:pharmacology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000378:metabolism*; D048728:MAP Kinase Kinase Kinase 1; D020935:MAP Kinase Signaling System / Q000187:drug effects; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors* / Q000502:physiology; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors* / Q000502:physiology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000037:antagonists & inhibitors / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology* / Q000378:metabolism; D063065:Organophosphonates / Q000494:pharmacology*; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011725:Pyridines / Q000494:pharmacology; D016897:Respiratory Burst / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases
D001095:Arachidonic Acids; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D007476:Ionophores; D007527:Isoenzymes; D063065:Organophosphonates; D011725:Pyridines; D014409:Tumor Necrosis Factor-alpha; C104810:methyl arachidonylfluorophosphonate; D013481:Superoxides; D016718:Arachidonic Acid; D000001:Calcimycin; D009240:N-Formylmethionine Leucyl-Phenylalanine; D015936:Complement C5a; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D019255:NADPH Oxidases; D017346:Protein Serine-Threonine Kinases; D019950:Mitogen-Activated Protein Kinase 1; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; C482576:MAP3K1 protein, human; D010741:Phospholipases A; D054522:Group VI Phospholipases A2; C516475:PLA2G6 protein, human; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one