Nitrite Generates an Oxidant Stress and Increases Nitric Oxide in EA.hy926 Endothelial Cells

Nitrite is a breakdown product of nitric oxide that in turn is oxidized to nitrate in cells. In this work, we investigated whether reactive oxidant species might be generated during nitrite metabolism in cultured EA.hy926 endothelial cells. Nitrite was taken up by the cells in a time- and concentration-dependent manner and oxidized to nitrate, which accumulated in cells to concentrations almost 10-fold those of nitrite. Conversion of low millimolar concentrations of nitrite to nitrate was associated with increased oxidant stress in the cells. This manifested as increased oxidation of dihydrofluorescein in tandem with depletion of both GSH and ascorbate. Further, loading cells with ascorbate or treatment with desferrioxamine prevented nitrite-induced dihydrofluorescein oxidation. Nitrite within cells also increased the fluorescence of 4-amino-5-methylamino-2′,7′-difluorofluorescein and inhibited the activity of cellular glyceraldehyde 3-phosphate dehydrogenase, which are markers of intracellular nitrosation reactions. Intracellular ascorbate partially prevented both of these effects of nitrite. Although ascorbate can reduce nitrite to nitric oxide at low pH, in endothelial cells loaded with ascorbate, its predominant effect at high nitrite concentrations is to prevent potentially damaging nitrosation reactions.     

Nitric oxide-induced oxidant stress in endothelial cells: amelioration by ascorbic acid

Nitric oxide has multiple beneficial effects in the blood vessel wall. However, high concentrations of nitric oxide in the presence of hydroperoxides have been shown to damage cultured cells. In this work, the effect of relatively high concentrations of nitric oxide alone on the function and antioxidant status of a human endothelial cell line (EA.hy926) was tested. Nitric oxide generated from 0.1 to 0.5mM spermine NONOate generated reactive species in the cells detected by triazole formation from diaminofluorescein and by oxidation of dihydrofluorescein. Intracellular ascorbic acid decreased this oxidant stress. Spermine NONOate also decreased intracellular ascorbate concentrations, although reduced glutathione was not affected unless cells had also been caused to reduce dehydroascorbic acid to ascorbate. Nitric oxide predictably inhibited both endothelial nitric oxide synthase and glyceraldehyde 3-phosphate dehydrogenase, and ascorbate partially prevented inhibition of the latter enzyme. These results suggest that relatively high concentrations of nitric oxide can cause oxidant stress in endothelial cells that is ameliorated by ascorbic acid.     

Ascorbic acid blunts oxidant stress due to menadione in endothelial cells

Endothelial cells are exposed to potentially damaging reactive oxygen species generated both within the cells and in the bloodstream and underlying vessel wall. In this work, we studied the ability of ascorbic acid to protect cultured human-derived endothelial cells (EA.hy926) from oxidant stress generated by the redox cycling agent menadione. Menadione caused intracellular oxidation of dihydrofluorescein, which required the presence of D-glucose in the incubation medium, and was inhibited by intracellular ascorbate and desferrioxamine. At concentrations of 100 microM and higher, menadione depleted the cells of both GSH and ascorbate, and ascorbate loading partially prevented the decrease in GSH due to menadione. Menadione increased L-arginine uptake by the cells, but inhibited endothelial nitric oxide synthase, an effect that was prevented by acute loading with ascorbate. Ascorbate blunts menadione-induced oxidant stress in EA.hy926 cells, which may help to preserve nitric oxide synthase activity under conditions of excessive oxidant stress.     

Oxidative stress and inhibition of oxidative phosphorylation induced by peroxynitrite and nitrite in rat brain subcellular fractions

Nitrite and nitrate, two endogenous oxides of nitrogen, are toxic in vivo. Furthermore, the reaction of superoxide (produced by all aerobic cells) with nitric oxide (NO) generates peroxynitrite, a potent oxidizing agent, that can cause biological oxidative stress. Using subcellular fractions from rat brain hemispheres we studied oxidative stress induced by these nitrogen compounds with special emphasis on nitrite. The consumption of Vitamin C (ascorbate) and Vitamin E (alpha tocopherol), two of the important nutritional antioxidants, was followed in synaptosomes (nerve-ending particles) and mitochondria along with changes in parameters of mitochondrial oxidative phosphorylation. Nitrite, but not nitrate, oxidized ascorbate without oxidizing alpha tocopherol in both synaptosomes and mitochondria whereas peroxynitrite oxidized both ascorbate and alpha tocopherol. Functionally, both nitrite and peroxynitrite inhibited mitochondrial oxidative phosphorylation. Nitrite was less potent than peroxynitrite when the effects of equal concentrations of the two were compared. However, since nitrite is much more stable than peroxynitrite the impact of nitrite as an oxidant in vivo could be as much or even more significant than peroxynitrite. Nitrate would not have similar action unless it is reduced to nitrite. It is possible that nitrite may impair oxidative phosphorylation through modulating levels of nitric oxide, changing the activity of heme proteins or a mild uncoupling of mitochondria.     

Ascorbic acid decreases oxidant stress in endothelial cells caused by the nitroxide tempol

Stable nitroxide radicals have been considered as therapeutic antioxidants because they can scavenge more toxic radicals in biologic systems. However, as radicals they also have the potential to increase oxidant stress in cells and tissues. We studied the extent to which this occurs in cultured EA.hy926 endothelial cells exposed to the nitroxide Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl). Tempol was rapidly reduced by the cells, as manifest by an increase in the ability of the cells to reduce extracellular ferricyanide and by disappearance of the Tempol EPR signal. Cells loaded with ascorbic acid, which directly reacts with Tempol, showed increased rates of Tempol-dependent ferricyanide reduction, and a more rapid loss of the Tempol EPR signal than cells not containing ascorbate. In this process, intracellular ascorbate was oxidized, and was depleted at lower Tempol concentrations than was GSH, another important intracellular low molecular weight antioxidant. Further evidence that Tempol concentrations of 100-1000 μM induced an oxidant stress was that it caused an increase in the oxidation of dihydrofluorescein in cells and inhibited ascorbate transport at concentrations as low as 50-100 μM. The presence of intracellular ascorbate both prevented dihydrofluorescein oxidation and spared GSH from oxidation by Tempol. Such sparing was not observed when GSH was depleted by other mechanisms, indicating that it was likely due to protection against oxidant stress. These results show that whereas Tempol may scavenge other more toxic radicals, care must be taken to ensure that it does not itself induce an oxidant stress, especially with regard to depletion of ascorbic acid.     

Oxidized lipoprotein induces the macrophage ascorbate transporter (SVCT2): Protection by intracellular ascorbate against oxidant stress and apoptosis

To assess whether ascorbic acid decreases the cytotoxicity of oxidized human low density lipoprotein (oxLDL) in cells involved in atherosclerosis, its interaction with oxLDL was studied in murine RAW264.7 macrophages. Macrophages took up ascorbate to millimolar intracellular concentrations and retained it with little loss over 18 h in culture. Culture of the macrophages with oxLDL enhanced ascorbate uptake. This was associated with increased expression of the ascorbate transporter (SVCT2), which was prevented by ascorbate and by inhibiting the NF-κB pathway. Culture of RAW264.7 macrophages with oxLDL increased intracellular dihydrofluorescein oxidation and lipid peroxidation, both of which were decreased by intracellular ascorbate. Ascorbate also protected the cells against oxLDL-induced cytotoxicity and apoptosis, but it did not affect macrophage accumulation of lipid from oxLDL or oxLDL-induced increases in macrophage cytokine secretion. These results suggest that ascorbate protects macrophages against oxLDL-induced oxidant stress and subsequent apoptotic death without impairing their function.     

Nitrite uptake and metabolism and oxidant stress in human erythrocytes

Nitric oxide, when released into the bloodstream, is quicklyscavenged by Hb in erythrocytes or oxidized to nitrite. Nitrite canalso enter erythrocytes and oxidize Hb. The goals of this work were todetermine the mechanism of erythrocyte nitrite uptake and whether thisuptake causes oxidant stress in these cells. Erythrocytes took up 0.8 mM nitrite with a half-time of 11 min. Nitrite uptake was sensitive totemperature and to the pH and ionic composition of the medium but wasnot inhibited by the specific anion-exchange inhibitor DIDS. About 25%of nitrite uptake occurred on the sodium-dependent phosphatetransporter and the rest as diffusion of nitrous acid or other speciesacross the plasma membrane. Methemoglobin formation increased inproportion to the intracellular nitrite concentration. Nitritereacted with erythrocyte ascorbate, but ascorbate loading of cellsdecreased nitrite-induced methemoglobin formation only at high nitriteconcentrations. In conclusion, nitrite rapidly enters erythrocytes andreacts with oxyhemoglobin but does not exert a strong oxidant stress onthese cells.

     

Dietary nitrite restores NO homeostasis and is cardioprotective in endothelial nitric oxide synthase-deficient mice

Endothelial production of nitric oxide (NO) is critical for vascular homeostasis. Nitrite and nitrate are formed endogenously by the stepwise oxidation of NO and have, for years, been regarded as inactive degradation products. As a result, both anions are routinely used as surrogate markers of NO production, with nitrite as a more sensitive marker. However, both nitrite and nitrate are derived from dietary sources. We sought to determine how exogenous nitrite affects steady-state concentrations of NO metabolites thought to originate from nitric oxide synthase (NOS)-derived NO as well as blood pressure and myocardial ischemia-reperfusion (I/R) injury. Mice deficient in endothelial nitric oxide synthase (eNOS-/-) demonstrated decreased blood and tissue nitrite, nitrate, and nitroso proteins, which were further reduced by low-nitrite (NOx) diet for 1 week. Nitrite supplementation (50 mg/L) in the drinking water for 1 week restored NO homeostasis in eNOS-/- mice and protected against I/R injury. Nitrite failed to alter heart rate or mean arterial blood pressure at the protective dose. These data demonstrate the significant influence of dietary nitrite intake on the maintenance of steady-state NO levels. Dietary nitrite and nitrate may serve as essential nutrients for optimal cardiovascular health and may provide a novel prevention/treatment modality for disease associated with NO insufficiency.     

Antioxidant effects of dihydrocaffeic acid in human EA.hy926 endothelial cells

Dihydrocaffeic acid (DHCA) is a metabolite of caffeic acid with potent antioxidant properties. Since DHCA has been detected in human plasma following coffee ingestion, we tested the hypothesis that DHCA protects the endothelium from oxidative stress in a model in human-derived EA.hy926 endothelial cells. During culture for 16-24 hours, the cells accumulated DHCA against a concentration gradient to low millimolar concentrations. In alpha-tocopherol-loaded cells, DHCA spared alpha-tocopherol during overnight culture in a dose-dependent manner. In response to oxidant stress induced by a water-soluble free radical initiator, both alpha-tocopherol and DHCA diminished oxidation of cis-parinaric acid that had been incorporated into the cells, although their antioxidant activities were not additive. DHCA also decreased intracellular oxidation of dihydrofluorescein due to redox cycling by menadione. This suggests that the protective effects of DHCA were caused by scavenging of intracellular reactive oxygen species. DHCA also increased nitric oxide synthase activity in a dose-dependent manner in cultured cells, which was associated with a comparable increase in endothelial nitric oxide synthase protein. Although the DHCA concentrations required for these effects are higher than those likely to be present in plasma or the interstitium, these results indicate that DHCA can function as an intracellular antioxidant.     

Dihydrofluorescein diacetate is superior for detecting intracellular oxidants: comparison with 2',7'-dichlorodihydrofluorescein diacetate, 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate, and dihydrorhodamine 123.

To detect intracellular oxidant formation during reoxygenation of anoxic endothelium, the oxidant-sensing fluorescent probes, 2',7'-dichlorodihydrofluorescein diacetate, dihydrorhodamine 123, or 5(and 6)-carboxy-2',7'-dichlorodihydrofluorescein diacetate were added to human umbilical vein endothelial cells during reoxygenation. None of these fluorescent probes were able to differentiate the controls from the reoxygenated cells in the confocal microscope. However, dihydrofluorescein diacetate demonstrated fluorescence of linear structures, consistent with mitochondria, in reoxygenated endothelium. This work tests the hypothesis that dihydrofluorescein diacetate is a better fluorescent probe for detecting intracellular oxidants because it is more reactive toward specific oxidizing species. To investigate this, dihydrofluorescein diacetate was exposed to various oxidizing species (hydrogen peroxide, superoxide [KO2], peroxynitrite, nitric oxide, horseradish peroxidase, ferric iron, xanthine oxidase, cytochrome c, and lipoxygenase) and compared with the three other popular probes. Though oxidized dihydrofluorescein has higher molar fluorescence, comparison of the reactions of dihydrofluorescein with these other three probes in a cell-free system indicates that dihydrofluorescein is sometimes less fluorescent than the other probes. In addition, we find that the reactivity of all of the probes is very complex. Based on the results reported here, it is no longer appropriate to think of these probes as detecting a specific oxidizing species in cells, such as H2O2, but rather as detectors of a broad range of oxidizing reactions that may be increased during intracellular oxidant stress. Cell-loading studies indicate that dihydrofluorescein achieves higher intracellular concentrations than the second brightest intracellular probe, 2',7'-dichlorodihydrofluorescein. This fact and its higher molar fluorescence may account for the superior brightness of dihydrofluorescein diacetate. Dihydrofluorescein diacetate may be a superior fluorescent probe for many cell-based studies.     

Nitrite disrupts multiple physiological functions in aquatic animals

Nitrite is a potential problem in aquatic environments. Freshwater fish actively take up nitrite across the gills, leading to high internal concentrations. Seawater fish are less susceptible but do take up nitrite across intestine and gills. Nitrite has multiple physiological effects. Its uptake is at the expense of chloride, leading to chloride depletion. Nitrite also activates efflux of potassium from skeletal muscle and erythrocytes, disturbing intracellular and extracellular K(+) levels. Nitrite transfer across the erythrocytic membrane leads to oxidation of haemoglobin to methaemoglobin (metHb), compromising blood O(2) transport. Other haem proteins are also oxidised. Hyperventilation is observed, and eventually tissue O(2) shortage becomes reflected in elevated lactate concentrations. Heart rate increases rapidly, before any significant elevations in metHb or extracellular potassium occur. This suggests nitrite-induced vasodilation (possibly via nitric oxide generated from nitrite) that is countered by increased cardiac pumping to re-establish blood pressure. Nitrite can form and/or mimic nitric oxide and thereby interfere with processes regulated by this local hormone. Steroid hormone synthesis may be inhibited, while changes in ammonia and urea levels and excretion rates reflect an influence of nitrite on nitrogen metabolism. Detoxification of nitrite occurs via endogenous oxidation to nitrate, and elimination of nitrite takes place both via gills and urine. The susceptibility to nitrite varies between species and in some cases also within species. Rainbow trout fall into two groups with regard to susceptibility and physiological response. These two groups are not related to sex but show significant different nitrite uptake rates.     

Energy conservation in Nitrobacter

Abstract The generation of ATP and NADH in total cells of Nitrobacter was measured under aerobic and anaerobic conditions. NADH synthesis was driven by an ATP independent reaction with nitrite or nitric oxide as electron donors. The rate of NADH formation was about 200 times higher, if nitric oxide instead of nitrite served as electron donor. Approximately 2 mol nitric oxide were needed for reduction of 1 mol NAD⁺. Nitrite caused an end-product inhibition of the nitric oxide induced NADH synthesis. ATP was synthesized by NADH oxidation with oxygen and nitrate as terminal electron acceptors.     

Electron paramagnetic resonance spectroscopic investigation of the inhibition of the phosphoroclastic system of Clostridium sporogenes by nitrite

The proposal that nitrite exerts its inhibitory effect on anaerobic bacteria by direct interaction with the iron-sulphur proteins of the phosphoroclastic system was investigated. The effects of nitrate, nitrite with or without ascorbate, and nitric oxide on the growth of Clostridium sporogenes in liquid cultures at pH 7.4, on the rates of hydrogen production, and on the activities of the enzymes pyruvate-ferredoxin oxidoreductase and hydrogenase, and of ferredoxin were investigated. In agreement with previous studies, nitrate was the least effective inhibitor of cell growth, and nitric oxide the most effective. Nitrite reductase activity was very low in C. sporogenes, indicating that the presence of external reducing agents would be necessary for the reduction of nitrite to nitric oxide. Inhibition by nitrite was enhanced by ascorbate; 0.5 mM-nitrite with 10 mM-ascorbate stopped growth completely. In partially-purified preparations 4.1 mM-NaNO2 and equimolar ascorbate caused complete inactivation of hydrogenase activity but only partial (up to 78%) inactivation of pyruvate-ferredoxin oxidoreductase. This agreed with the loss of hydrogen production observed with nitrite in vivo. Inhibition occurred within 5 min, and was irreversible in each case. Electron paramagnetic resonance (EPR) spectroscopy showed that paramagnetic [Fe(NO)2(SR)2] species were formed during growth in the presence of nitrite, and were associated with cells. However, the intensity of these EPR signals did not correlate with the inhibition of cell growth. The [4Fe-4S] clusters in ferredoxin were shown by EPR spectroscopy to be resistant to treatment with 3.6 mM-NaNO2 and 3.6 mM-ascorbate. It is concluded that the effects of nitrite on pre-formed iron-sulphur proteins are not convincing as a basis for the lethal effects on bacterial cells.     

Rat liver-mediated metabolism of hydroxyurea to nitric oxide

Hydroxyurea is an approved treatment for sickle cell disease. Oxidation of hydroxyurea results in the formation of nitric oxide (NO), which also has drawn considerable interest as a sickle cell disease therapy. Although patients on hydroxyurea demonstrate elevated levels of nitric oxide-derived metabolites, little information regarding the site or mechanism of the in vivo conversion of hydroxyurea to nitric oxide exists. Chemiluminescence detection experiments show the ability of crude rat liver homogenate to convert hydroxyurea to nitrite/nitrate, evidence for NO production. Nitrite/nitrate form at therapeutic concentrations of hydroxyurea in a clinically relevant time frame. Electron paramagnetic resonance (EPR) studies show the formation of iron nitrosyl complexes during this incubation and experiments with labeled hydroxyurea show the NO derives from the drug. Gas chromatography-mass spectrometry measurements indicate the hydrolysis of hydroxyurea to hydroxylamine in this system. Incubation of hydroxylamine with crude rat liver homogenate also generates nitrite/nitrate and iron nitrosyl complexes. A line of evidence including inhibitor studies, EPR spectroscopy, and nitrite/nitrate detection identifies catalase as a possible oxidant for the conversion of hydroxyurea to NO. These results reveal the ability of liver tissue to convert hydroxyurea to nitric oxide and provide insight into the metabolism of this drug.     

Dietary nitrate increases gastric mucosal blood flow and mucosal defense

Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria. In the acidic stomach, nitrite is further reduced to bioactive nitrogen oxides, including nitric oxide (NO). In this study, we investigated the gastroprotective role of nitrate intake and of luminally applied nitrite against provocation with diclofenac and taurocholate. Mucosal permeability ((51)Cr-EDTA clearance) and gastric mucosal blood flow (laser-Doppler flowmetry) were measured in anesthetized rats, either pretreated with nitrate in the drinking water or given acidified nitrite luminally. Diclofenac was given intravenously and taurocholate luminally to challenge the gastric mucosa. Luminal NO content and nitrite content in the gastric mucus were determined by chemiluminescence. The effect of luminal administration of acidified nitrite on the mucosal blood flow was also investigated in endothelial nitric oxide synthase-deficient mice. Rats pretreated with nitrate or given nitrite luminally had higher gastric mucosal blood flow than controls. Permeability increased more during the provocation in the controls than in the nitrate- and nitrite-treated animals. Dietary nitrate increased luminal NO levels 50 times compared with controls. Nitrate intake also resulted in nitrite accumulation in the loosely adherent mucous layer; after removal of this mucous layer, blood flow was reduced. Nitrite administrated luminally in endothelial nitric oxide synthase-deficient mice increased mucosal blood flow. We conclude that dietary nitrate and direct luminal application of acidified nitrite decrease diclofenac- and taurocholate-induced mucosal damage. The gastroprotective effect likely involves a higher mucosal blood flow caused by nonenzymatic NO production. These data suggest an important physiological role of nitrate in the diet.     

Epigallocatechin gallate preserves endothelial function by reducing the endogenous nitric oxide synthase inhibitor level

Asymmetric dimethylarginine (ADMA), the endogenous nitric oxide synthase inhibitor, is thought to be a key factor contributing to endothelial dysfunction. Tea catechins can cause an endothelium-dependent vasorelaxation. The present study examined the effect of epigallocatechin gallate (EGCG), the major component of tea catechins, on endothelial dysfunction induced by native low density lipoprotein (LDL) in rats and oxidized LDL (ox-LDL) in cultured endothelial cells, and whether the protective effect of EGCG is related to reduction of ADMA level. A single injection of LDL (4 mg x kg(-1), i.v.) markedly reduced endothelium-dependent relaxation and the serum nitrite/nitrate (NO) level, and increased serum concentrations of ADMA, malondialdehyde (MDA), and tumor necrosis factor-alpha (TNF-alpha). EGCG (10 or 50 mg x kg(-1), i.p.) significantly attenuated the inhibition of vasodilator response to acetylcholine and the decreased serum nitrite/nitrate level, and reduced the elevated levels of ADMA, MDA, and TNF-alpha. Exposure of endothelial cells to ox-LDL (100 microg x mL(-1)) for 24 h markedly increased the medium levels of lactate dehydrogenase (LDH), ADMA, TNF-alpha, and MDA, and decreased the level of nitrite/nitrate in the medium and the activity of dimethylarginine dimethylaminohydrolase (DDAH) in the endothelial cells. EGCG (10 and 100 microg x mL(-1)) significantly decreased the levels of LDH, ADMA, TNF-alpha, and MDA, and increased the level of nitrite/nitrate and the activity of DDAH. These results suggest that EGCG protects endothelial dysfunction induced by native LDL in vivo or by ox-LDL in endothelial cells, and the protective effect of EGCG on the endothelium is related to decrease in ADMA level via increasing of DDAH activity.     

Oxidation of iron-nitrosyl-hemoglobin by dehydroascorbic acid releases nitric oxide to form nitrite in human erythrocytes

The reaction of deoxyhemoglobin with nitric oxide (NO) or nitrite ions (NO 2 (-)) produces iron-nitrosyl-hemoglobin (HbNO) in contrast to the reaction with oxyhemoglobin, which produces methemoglobin and nitrate (NO 3 (-)). HbNO has not been associated with the known bioactivities of NO. We hypothesized that HbNO in erythrocytes could be an important source of bioactive NO/nitrite if its oxidation was coupled to the ascorbic acid (ASC) cycle. Studied by absorption and electron paramagnetic resonance (EPR) spectroscopy, DHA oxidized HbNO to methemoglobin and liberated NO from HbNO as determined by chemiluminescence. Both DHA and ascorbate free radical (AFR), the intermediate between ASC and DHA, enhanced NO oxidation to nitrite, but not nitrate; nor did either oxidize nitrite to nitrate. DHA increased the basal levels of nitrite in erythrocytes, while the reactions of nitrite with hemoglobin are slow. In erythrocytes loaded with HbNO, HbNO disappeared after DHA addition, and the AFR signal was detected by EPR. We suggest that the ASC-AFR-DHA cycle may be coupled to that of HbNO-nitrite and provide a mechanism for the endocrine transport of NO via hemoglobin within erythrocytes, resulting in the production of intracellular nitrite. Additionally, intracellular nitrite and nitrate seem to be largely generated by independent pathways within the erythrocyte. These data provide a physiologically robust mechanism for erythrocytic transport of NO bioactivity allowing for hormone-like properties.     

Ruhemann's purple from ninhydrin,ascorbate, and nitrite

Ruhemann's purple is formed from nitrite, ascorbate, and ninhydrin with the formation of variable amounts of ammonia. The reaction begins with the nitrosation of ninhydrin to form nitrosoninhydrin, which will release nitric oxide. The nitrosoninhydrin is reduced by ascorbate to diketohydrindamine, which then couples with a second molecule of ninhydrin to form the pigment. The concentration of pigment is linear with nitrite over the range 1 to 10 mm, but ammonia production is not stoichiometric with initial nitrite.     

Ascorbate inhibits iNOS expression and preserves vasoconstrictor responsiveness in skeletal muscle of septic mice

Inducible nitric oxide synthase (iNOS) expression in blood vessels contributes to the vascular hyporeactivity characteristic of sepsis. Our previous work demonstrated in vitro that ascorbate inhibits iNOS expression in lipopolysaccharide- and interferon-gamma-stimulated skeletal muscle endothelial cells (ECs) through an antioxidant mechanism. The present study evaluated in vivo the hypothesis that administration of ascorbate decreases oxidative stress, prevents endothelial iNOS expression, and improves vascular reactivity in septic skeletal muscle. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Plasma nitrite and nitrate (NOx) levels were elevated by 6 h after CLP. Prior ascorbate bolus injection (200 mg/kg body wt iv) blocked the elevation of plasma NOx and abolished the expression of iNOS protein and activity in the septic skeletal muscle. We also demonstrated that iNOS mRNA determined by RT-PCR was induced in the microvascular ECs of the muscle at 3 h after CLP. This induction was attenuated by prior ascorbate administration. Ascorbate inhibition of iNOS expression was associated with decreased oxidant levels in the septic muscle. Moreover, ascorbate administration restored partially the baseline arterial pressure and preserved completely the microvascular constriction and arterial pressure responses to norepinephrine in CLP mice. These results suggest that early administration of ascorbate may be a valuable adjunct treatment of sepsis.