Abstract
BackgroundInorganic 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. MethodsMouse 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. ResultsNOX 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. ConclusionsOur 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.
Highlights
Immune cells, like macrophages, are major sources of reactive oxygen species (ROS) in the body
We demonstrate here that nitrite-mediated attenuation of NOXderived superoxide production in LPS-activated macrophages is associated with xanthine oxidoreductase (XOR)-dependent nitric oxide (NO) production
We found that nitrite-mediated reduction of superoxide production does not involve S-nitrosation of the membrane-bound NOX subunits Nox2 and p22phox, but can partially be attributed to changes in protein expression of the cytosolic subunits p47phox and p67phox
Summary
Like macrophages, are major sources of reactive oxygen species (ROS) in the body. Oxidative stress represents a major risk factor and cause of both metabolic and cardiovascular diseases [1] It can be ameliorated either by a decrease in ROS production or an increase in NO bioavailability. In addition to NO production via the classic nitric oxide synthase (NOS) pathway, fueling the alternative nitrate-nitrite-NO pathway with inorganic nitrate and nitrite has shown beneficial effects in many disorders and experimental disease models via restoring NO bioavailability [2,3,4]. It is still not clear how this effect is mediated and signaling events may occur in a cell-, tissue- and environment-. 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
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
