title: Nitrite-mediated reduction of macrophage NADPH oxidase activity is dependent on xanthine oxidoreductase-derived nitric oxide but independent of S-nitrosation.
abstract: Inorganic nitrite has shown beneficial effects in cardiovascular and metabolic diseases partly via attenuation of NADPH-oxidase (NOX)-mediated oxidative stress. However, the exact mechanisms are still unclear. Here we investigated the role of S-nitrosation or altered expression of NOX subunits, and the role of xanthine oxidoreductase (XOR) in nitrite-derived nitric oxide (NO) production.
  
  
  
  Mouse macrophages were activated with LPS in the presence or absence of nitrite. NOX activity was measured by lucigenin-dependent chemiluminescence. Gene and protein expression of NOX2 subunits and XOR were investigated using qPCR and Western Blot. S-nitrosation of Nox2 and p22phox was studied with a Biotin Switch assay. Uric acid levels in cell culture medium were analyzed as a measure of XOR activity, and NO production was assessed by DAF-FM fluorescence.
  
  
  
  NOX activity in activated macrophages was significantly reduced by nitrite. Reduced NOX activity was not attributed to decreased NOX gene expression. However, protein levels of p47phox and p67phox subunits were reduced by nitrite in activated macrophages. Protein expression of Nox2 and p22phox was not influenced by this treatment and neither was their S-nitrosation status. Increased uric acid levels after nitrite and diminished NO production during XOR-inhibition with febuxostat suggest that XOR is more active during nitrite-treatment of activated macrophages and plays an important role in the bioactivation of nitrite.
  
  
  
  Our findings contribute to the mechanistic understanding about the therapeutic effects associated with nitrite supplementation in many diseases. We show that nitrite-mediated inhibition of NOX activity cannot be explained by S-nitrosation of the NOX enzyme, but that changes in NOX2 expression and XOR function may contribute.
authors.affiliation: Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden; Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
authors.forename: Christa
authors.initials: C
authors.lastname: Zollbrecht
authors.affiliation: Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
authors.forename: A Erik G
authors.initials: AE
authors.lastname: Persson
authors.affiliation: Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Jon O
authors.initials: JO
authors.lastname: Lundberg
authors.affiliation: Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Eddie
authors.initials: E
authors.lastname: Weitzberg
authors.affiliation: Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden. Electronic address: Mattias.Carlstrom@ki.se.
authors.forename: Mattias
authors.initials: M
authors.lastname: Carlström
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation; D017737:Macrophages, Peritoneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D009573:Nitrites / Q000494:pharmacology*; D015538:Nitrosation; D018384:Oxidative Stress; D014527:Uric Acid / Q000378:metabolism; D014968:Xanthine Dehydrogenase / Q000235:genetics / Q000378:metabolism*
chemical_list: D008070:Lipopolysaccharides; D009573:Nitrites; D014527:Uric Acid; D009569:Nitric Oxide; D014968:Xanthine Dehydrogenase; D019255:NADPH Oxidases
keywords: NADPH oxidase; Nitric oxide; Nitrite; Oxidative stress; S-nitrosation; Xanthine oxidoreductase