Regulation of neutrophil NADPH oxidase activation in a cell-free system by guanine nucleotides and fluoride. Evidence for participation of a pertussis and cholera toxin-insensitive G protein.
Guanine nucleotide-binding regulatory proteins (G proteins) transduce a remarkably diverse group of extracellular signals to a relatively limited number of intracellular target enzymes. In the neutrophil, transduction of the signal following fMet-Leu-Phe receptor-ligand interaction is mediated by a pertussis toxin substrate (Gi) that activates inositol-specific phospholipase C. We have utilized a plasma membrane-containing fraction from unstimulated human neutrophils as the target enzyme to explore the role of G proteins in arachidonate and cytosolic cofactor-dependent activation of the NADPH-dependent O-2-generating oxidase. When certain guanine nucleotides or their nonhydrolyzable analogues were present during arachidonate and cytosolic cofactor-dependent activation, they exerted substantial dose-dependent effects. The GTP analogue, GTP gamma S, caused a 2-fold increase in NADPH oxidase activation (half-maximal stimulation, 1.1 microM). Either GDP or its nonhydrolyzable analogue, GDP beta S, inhibited up to 80% of the basal NADPH oxidase activation (Ki GDP = 0.12 mM, GDP beta S = 0.23 mM). GTP caused only slight and variable stimulation, whereas F-, an agent known to promote the active conformation of G proteins, caused a 1.6-fold stimulation of NADPH oxidase activation. NADPH oxidase activation in the cell-free system was absolutely and specifically dependent on Mg2+. Although O2- production in response to fMet-Leu-Phe was inhibited greater than 90% in neutrophils pretreated with pertussis toxin, cytosolic cofactor and target oxidase membranes from neutrophils treated with pertussis toxin showed no change in basal- or GTP gamma S-stimulated NADPH oxidase activation. Cholera toxin treatment of neutrophils also had no effect on the cell-free activation system. Our results suggest a role for a G protein that is distinct from Gs or Gi in the arachidonate and cytosolic cofactor-dependent NADPH oxidase cell-free activation system.
D002474:Cell-Free System; D002772:Cholera Toxin / Q000494:pharmacology*; D002850:Chromatography, Gel; D004789:Enzyme Activation; D005459:Fluorides / Q000494:pharmacology*; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006150:Guanine Nucleotides / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000201:enzymology*; D037342:Pertussis Toxin; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D006150:Guanine Nucleotides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D013481:Superoxides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009240:N-Formylmethionine Leucyl-Phenylalanine; D006160:Guanosine Triphosphate; D002772:Cholera Toxin; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D008274:Magnesium; D005459:Fluorides