Essential requirement of cytosolic phospholipase A(2) for stimulation of NADPH oxidase-associated diaphorase activity in granulocyte-like cells.
We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient differentiated PLB-985 cells (PLB-D cells) and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for NADPH oxidase activation. In this study we used this model to investigate the physiological role of cPLA(2) in regulation of NADPH oxidase-associated diaphorase activity. A novel diaphorase activity assay, using 4-iodonitrotetrazolium violet as an electron acceptor, was used in permeabilized neutrophils and PLB-985 cells differentiated toward the granulocytic or monocytic phenotypes. Phorbol 12-myristate 13-acetate, guanosine 5'-3-O- (thio)triphosphate (GTP gamma S), or FMLP stimulated a similar diphenylene iodonium-sensitive diaphorase activity pattern in neutrophils and in differentiated parent PLB-985 cells. This diaphorase activity was not detected in undifferentiated cells, but developed during differentiation. Furthermore, diaphorase activity could not be stimulated in permeabilized neutrophils from X-linked CGD patients and in differentiated gp91(phox)-targeted PLB-985 cells that lacked normal expression of gp91(phox), but was restored to these cells following transduction with retrovirus encoding gp91(phox). The differentiated PLB-D cells showed no diaphorase activity when stimulated by either GTP gamma S or FMLP, and only partial activation when stimulated with phorbol 12-myristate 13-acetate. Diaphorase activity in response to either agonists was fully restored by the addition of 10 microm free AA. The permeabilized cell 4-iodonitrotetrazolium violet reduction assay offers a unique tool for the evaluation of NADPH oxidase-associated diaphorase activity in stimulated whole cells. These results establish an essential and specific physiological requirement of cPLA(2)-generated AA in activation of electron transfer through the FAD reduction center of NADPH oxidase.
Pessach
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Infectious Diseases Laboratory, Department of Clinical Biochemistry, Faculty of Health Sciences, Ben-Gurion University of the Negev and Soroka Medical Center, Beer Sheva 84105, Israel.
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Malech
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D002273:Carcinogens; D002454:Cell Differentiation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003574:Cytochrome c Group / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D008058:Dihydrolipoamide Dehydrogenase / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004579:Electron Transport; D004583:Electrons; D004789:Enzyme Activation; D006098:Granulocytes / Q000201:enzymology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D006801:Humans; D008562:Membrane Glycoproteins / Q000378:metabolism; D008954:Models, Biological; D009000:Monocytes / Q000201:enzymology; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology; D010641:Phenotype; D010741:Phospholipases A / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D013481:Superoxides / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013778:Tetrazolium Salts / Q000494:pharmacology*; D013997:Time Factors
D002273:Carcinogens; D003574:Cytochrome c Group; D008562:Membrane Glycoproteins; D013778:Tetrazolium Salts; D013481:Superoxides; C013574:iodonitrotetrazolium; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D008058:Dihydrolipoamide Dehydrogenase; D010741:Phospholipases A; D013755:Tetradecanoylphorbol Acetate