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      Nitrite-mediated reduction of macrophage NADPH oxidase activity is dependent on xanthine oxidoreductase-derived nitric oxide but independent of S-nitrosation

      a , b , b , a , a , a , *

      Redox Biology


      DAF-FM DA, 4-amino-5-methylamino-2′,7′-difluorescein diacetate, DETA-NONOate, diethylenetriamine/nitric oxide adduct, Febuxostat, 2-(3-Cyano-4-isobutoxy-phenyl)-4-methyl-thiazole-5-carboxylic acid, CysNO, S-nitrosocysteine, L-NAME, Nω-nitro-l-arginine methylester hydrochloride, LPS, Lipopolysaccharides, NADH, β-Nicotinamide-adenine dinucleotide, NADPH, β-Nicotinamide-adenine dinucleotide 2′-phosphate, NO2−, nitrite, NO, nitric oxide, NOS, nitric oxide synthase, NOX, NADPH oxidase, ROS, reactive oxygen species, XOR, xanthine oxidoreductase, NADPH oxidase, Nitrite, Nitric oxide, Oxidative stress, S-nitrosation, Xanthine oxidoreductase

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          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.

          Graphical abstract


          • Inorganic nitrite reduces NADPH oxidase activity in LPS-activated macrophages.

          • Xanthine oxidoreductase plays an important role in nitrite reduction to NO.

          • The effects of inorganic nitrite are not mediated by S-nitrosation of Nox2 and p22phox.

          • Nitrite reduced protein expression of p47phox and p67phox.

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          Most cited references 41

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          Effects of dietary nitrate on blood pressure in healthy volunteers.

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            Dietary nitrate provides sustained blood pressure lowering in hypertensive patients: a randomized, phase 2, double-blind, placebo-controlled study.

            Single dose administration of dietary inorganic nitrate acutely reduces blood pressure (BP) in normotensive healthy volunteers, via bioconversion to the vasodilator nitric oxide. We assessed whether dietary nitrate might provide sustained BP lowering in patients with hypertension. We randomly assigned 68 patients with hypertension in a double-blind, placebo-controlled clinical trial to receive daily dietary supplementation for 4 weeks with either dietary nitrate (250 mL daily, as beetroot juice) or a placebo (250 mL daily, as nitrate-free beetroot juice) after a 2-week run-in period and followed by a 2-week washout. We performed stratified randomization of drug-naive (n=34) and treated (n=34) patients with hypertension aged 18 to 85 years. The primary end point was change in clinic, ambulatory, and home BP compared with placebo. Daily supplementation with dietary nitrate was associated with reduction in BP measured by 3 different methods. Mean (95% confidence interval) reduction in clinic BP was 7.7/2.4 mm Hg (3.6-11.8/0.0-4.9, P<0.001 and P=0.050). Twenty-four-hour ambulatory BP was reduced by 7.7/5.2 mm Hg (4.1-11.2/2.7-7.7, P<0.001 for both). Home BP was reduced by 8.1/3.8 mm Hg (3.8-12.4/0.7-6.9, P<0.001 and P<0.01) with no evidence of tachyphylaxis over the 4-week intervention period. Endothelial function improved by ≈20% (P<0.001), and arterial stiffness was reduced by 0.59 m/s (0.24-0.93; P<0.01) after dietary nitrate consumption with no change after placebo. The intervention was well tolerated. This is the first evidence of durable BP reduction with dietary nitrate supplementation in a relevant patient group. These findings suggest a role for dietary nitrate as an affordable, readily-available, adjunctive treatment in the management of patients with hypertension (funded by The British Heart Foundation).
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              Strategies to increase nitric oxide signalling in cardiovascular disease.

              Nitric oxide (NO) is a key signalling molecule in the cardiovascular, immune and central nervous systems, and crucial steps in the regulation of NO bioavailability in health and disease are well characterized. Although early approaches to therapeutically modulate NO bioavailability failed in clinical trials, an enhanced understanding of fundamental subcellular signalling has enabled a range of novel therapeutic approaches to be identified. These include the identification of: new pathways for enhancing NO synthase activity; ways to amplify the nitrate-nitrite-NO pathway; novel classes of NO-donating drugs; drugs that limit NO metabolism through effects on reactive oxygen species; and ways to modulate downstream phosphodiesterases and soluble guanylyl cyclases. In this Review, we discuss these latest developments, with a focus on cardiovascular disease.

                Author and article information

                Redox Biol
                Redox Biol
                Redox Biology
                28 September 2016
                December 2016
                28 September 2016
                : 10
                : 119-127
                [a ]Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
                [b ]Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden
                Author notes
                [* ]Corresponding author. Mattias.Carlstrom@ 123456ki.se
                © 2016 The Authors

                This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

                Research Paper


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