Reactive Nitrogen Species Is Required for the Activation of the AMP-activated Protein Kinase by Statin in Vivo

Reactive nitrogen species, such as peroxynitrite, can nitrate tyrosine in proteins to form nitrotyrosine. Nitrotyrosine is metabolized to 3-nitro-4-hydroxyphenylacetic acid (NHPA), which is excreted in the urine. This has led to the notion that measurement of urinary NHPA may provide a time-integrated index of nitrotyrosine formation in vivo. However, it is not known whether NHPA is derived exclusively from metabolism of nitrotyrosine, or whether it can be formed by nitration of circulating para-hydroxyphenylacetic acid (PHPA), a metabolite of tyrosine. In the present study, we have developed a gas chromatography MS assay for NHPA and PHPA to determine whether or not NHPA can be formed directly by nitration of PHPA. Following the injection of nitrotyrosine, 0.5±0.16% of injected dose was recovered unchanged as nitrotyrosine, and 4.3±0.2% as NHPA in the urine. To determine whether or not NHPA could be formed by the nitration of PHPA, deuterium-labelled PHPA ([2H6]PHPA) was injected, and the formation of deuterated NHPA ([2H5]NHPA) was measured. Of the infused [2H6]PHPA, 78±2% was recovered in the urine unchanged, and approx. 0.23% was recovered as [2H5]NHPA. Since the plasma concentration of PHPA is markedly higher than free nitrotyrosine (approx. 400-fold), the nitration of high-circulating endogenous PHPA to form NHPA becomes very significant and accounts for the majority of NHPA excreted in urine. This is the first study to demonstrate that NHPA can be formed by nitration of PHPA in vivo, and that this is the major route for its formation.

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Inhibition of Reactive Nitrogen Species Effects in Vitro and in Vivo by Isoflavones and Soy-Based Food Extracts

Journal of Agricultural and Food Chemistry ◽

10.1021/jf034876b ◽

2003 ◽

Vol 51 (27) ◽

pp. 7892-7900 ◽

Cited By ~ 33

Author(s):

Gow-Chin Yen ◽

Hsi-Huai Lai

Keyword(s):

Reactive Nitrogen Species ◽

Reactive Nitrogen ◽

Nitrogen Species ◽

Species Effects

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REACTIVE NITROGEN SPECIES IN BRAIN AFTER IN VIVO EXPOSURE TO ARSENIC

International Journal of Advanced Research ◽

10.21474/ijar01/11179 ◽

2020 ◽

Vol 8 (6) ◽

pp. 980-992

Author(s):

Julian G. Bonetto ◽

◽

Juan C. Perazzo ◽

Susana Puntarulo ◽

◽

...

Keyword(s):

Reactive Nitrogen Species ◽

Reactive Nitrogen ◽

Nitrogen Species

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The reactive nitrogen species peroxynitrite is a potent inhibitor of renal Na-K-ATPase activity

AJP Renal Physiology ◽

10.1152/ajprenal.90296.2008 ◽

2008 ◽

Vol 295 (4) ◽

pp. F1191-F1198 ◽

Cited By ~ 21

Author(s):

Matthew S. Reifenberger ◽

Krista L. Arnett ◽

Craig Gatto ◽

Mark A. Milanick

Keyword(s):

Atpase Activity ◽

Reactive Nitrogen Species ◽

Potent Inhibitor ◽

In Vivo Studies ◽

Reactive Nitrogen ◽

Tyrosine Nitration ◽

Nitrogen Species ◽

Single Bolus ◽

Atpase Inhibition

Peroxynitrite is a reactive nitrogen species produced when nitric oxide and superoxide react. In vivo studies suggest that reactive oxygen species and, perhaps, peroxynitrite can influence Na-K-ATPase function. However, the direct effects of peroxynitrite on Na-K-ATPase function remain unknown. We show that a single bolus addition of peroxynitrite inhibited purified renal Na-K-ATPase activity, with IC50 of 107 ± 9 μM. To mimic cellular/physiological production of peroxynitrite, a syringe pump was used to slowly release (∼0.85 μM/s) peroxynitrite. The inhibition of Na-K-ATPase activity induced by this treatment was similar to that induced by a single bolus addition of equal cumulative concentration. Peroxynitrite produced 3-nitrotyrosine residues on the α, β, and FXYD subunits of the Na pump. Interestingly, the flavonoid epicatechin, which prevented tyrosine nitration, was unable to blunt peroxynitrite-induced ATPase inhibition, suggesting that tyrosine nitration is not required for inhibition. Peroxynitrite led to a decrease in iodoacetamidofluorescein labeling, implying that cysteine modifications were induced. Glutathione was unable to reverse ATPase inhibition. The presence of Na+ and low MgATP during peroxynitrite treatment increased the IC50 to 145 ± 10 μM, while the presence of K+ and low MgATP increased the IC50 to 255 ± 13 μM. This result suggests that the EPNa conformation of the pump is slightly more sensitive to peroxynitrite than the E(K) conformation. Taken together, these results show that peroxynitrite is a potent inhibitor of Na-K-ATPase activity and that peroxynitrite can induce amino acid modifications to the pump.

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Important roles of endothelial caveolin-1 in endothelium-dependent hyperpolarization and ischemic angiogenesis in mice

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00589.2018 ◽

2019 ◽

Vol 316 (4) ◽

pp. H900-H910 ◽

Cited By ~ 3

Author(s):

Akiyo Ito ◽

Takashi Shiroto ◽

Shigeo Godo ◽

Hiroki Saito ◽

Shuhei Tanaka ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cardiovascular Diseases ◽

Reactive Nitrogen Species ◽

Reactive Nitrogen ◽

Oxygen Species ◽

Nitrogen Species ◽

Hindlimb Ischemia ◽

Protein Modifications ◽

Caveolin 1

Although increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of cardiovascular diseases, the importance of physiological ROS has also been emerging. We have previously demonstrated that endothelium-derived H2O2 is an endothelium-dependent hyperpolarization (EDH) factor and that loss of endothelial caveolin-1 reduces EDH/H2O2 in the microcirculation. Caveolin-1 (Cav-1) is a scaffolding/regulatory protein that interacts with diverse signaling pathways, including angiogenesis. However, it remains unclear whether endothelial Cav-1 plays a role in ischemic angiogenesis by modulating EDH/H2O2. In the present study, we thus addressed this issue in a mouse model of hindlimb ischemia using male endothelium-specific Cav-1 (eCav-1) knockout (KO) mice. In isometric tension experiments with femoral arteries from eCav-1-KO mice, reduced EDH-mediated relaxations to acetylcholine and desensitization of sodium nitroprusside-mediated endothelium-independent relaxations were noted ( n = 4~6). An ex vivo aortic ring assay also showed that the extent of microvessel sprouting was significantly reduced in eCav-1-KO mice compared with wild-type (WT) littermates ( n = 12 each). Blood flow recovery at 4 wk assessed with a laser speckle flowmeter after femoral artery ligation was significantly impaired in eCav-1-KO mice compared with WT littermates ( n = 10 each) and was associated with reduced capillary density and muscle fibrosis in the legs ( n = 6 each). Importantly, posttranslational protein modifications by reactive nitrogen species and ROS, as evaluated by thiol glutathione adducts and nitrotyrosine, respectively, were both increased in eCav-1-KO mice ( n = 6~7 each). These results indicate that endothelial Cav-1 plays an important role in EDH-mediated vasodilatation and ischemic angiogenesis through posttranslational protein modifications by nitrooxidative stress in mice in vivo. NEW & NOTEWORTHY Although increased levels of reactive oxygen species (ROS) are involved in the pathogenesis of cardiovascular diseases, the importance of physiological ROS has also been emerging. The present study provides a line of novel evidence that endothelial caveolin-1 plays important roles in endothelium-dependent hyperpolarization and ischemic angiogenesis in hindlimb ischemia in mice through posttranslational protein modifications by reactive nitrogen species and ROS in mice in vivo.

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The Effect Of NO Donor on Calcium Uptake and Reactive Nitrogen Species Production in Mitochondria

Cellular Physiology and Biochemistry ◽

10.1159/000445616 ◽

2016 ◽

Vol 39 (1) ◽

pp. 193-204 ◽

Cited By ~ 8

Author(s):

Olga Akopova ◽

Anatoly Kotsiuruba ◽

Yulia Korkach ◽

Liudmila Kolchinskaya ◽

Valentina Nosar ◽

...

Keyword(s):

Reactive Nitrogen Species ◽

Calcium Uptake ◽

Mitochondrial Permeability Transition ◽

Permeability Transition ◽

Living Organism ◽

Reactive Nitrogen ◽

Nitrogen Species ◽

No Donor ◽

Dose Dependent

Background/Aims: NO and reactive nitrogen species (RNS) are thought to be physiologically important effectors of mitochondrial calcium transport, but this issue was not studied in a living organism. According to literature, the modulation of Ca2+ uptake could influence RNS production via the action on mitochondrial NO synthase (mtNOS). The aim of this work was to study the effect of in vivo administration of NO donor nitroglycerine (NG) on matrix Ca2+ accumulation, RNS production and mtNOS activity. Methods: Ca2+ uptake was studied spectrophotometrically with arsenazo-III. The amounts of stable RNS (nitrite, nitrate and nitrosothiols) and L-citrulline, the product of enzymatic NOS activity, were determined analytically. Results: NG administration resulted in dose-dependent short-term increase in Ca2+-uptake accompanied by essential rise in L-citrulline and RNS content in mitochondria. In parallel, dose-dependent elevation of hydroperoxide production was detected. Ca2+-uniporter activity was not affected, but mitochondrial permeability transition pore (MPTP) was effectively blocked by NO. Conclusion: Our results indicate that MPTP blockage by NO was the primary cause for the increase in calcium uptake which eventually resulted in the activation of mtNOS and RNS production. Improved Ca2+ accumulation in mitochondria, together with MPTP blockage, may contribute to well-known cardioprotective effects of pharmacological donors of nitric oxide.

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