The requirement for phospholipase A2 for activation of the assembled NADPH oxidase in human neutrophils.
Phospholipase A2 (PLA2) inhibitors suppressed simultaneously, in a dose-dependent manner, the activation of NADPH oxidase and the release of 3H-labelled arachidonic acid ([3H]AA) stimulated by either phorbol 12-myristate 13-acetate (PMA) or opsonized zymosan (OZ) in human neutrophils. In spite of total inhibition of superoxide production in the presence of the PLA2 inhibitors, 10 microM bromophenacyl bromide (BPB) or 20 microM quinacrine, a maximal phosphorylation of p47 and translocation of p47 and p67 to the neutrophil membranes induced by PMA or OZ was observed. Addition of 10 microM free AA, which by itself did not stimulate superoxide generation, restored oxidase activity in neutrophils treated with PLA2 inhibitors. These findings indicate that phosphorylation and translocation of the cytosolic factors to the membranes are not sufficient for generating superoxide; a functional PLA2 is also needed to stimulate the oxidase activity. The inhibition of PLA2 activity did not prevent the phosphorylation of p47, suggesting that the location of PLA2 is downstream of and does not activate protein kinase C.
Dana
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Laboratory of Infectious Diseases and Clinical Biochemistry Unit, Faculty of Health Sciences, Soroka Medical Center of Kupat Holim, Ben-Gurion University of the Negev, Beer-Sheva, Israel.
Malech
H L
HL
Levy
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D000098:Acetophenones / Q000494:pharmacology; D000470:Alkaloids / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D004789:Enzyme Activation; D006801:Humans; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D009895:Opsonin Proteins; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000378:metabolism*; D054467:Phospholipases A2; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism; D011796:Quinacrine / Q000494:pharmacology; D019311:Staurosporine; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014316:Tritium; D015054:Zymosan / Q000494:pharmacology
D000098:Acetophenones; D000470:Alkaloids; D009895:Opsonin Proteins; D014316:Tritium; D013481:Superoxides; D016718:Arachidonic Acid; D015054:Zymosan; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D011493:Protein Kinase C; D010741:Phospholipases A; D054467:Phospholipases A2; D011796:Quinacrine; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; C006821:4-bromophenacyl bromide