Nitrite reductase activity of hemoglobin as a systemic nitric oxide generator mechanism to detoxify plasma hemoglobin produced during hemolysis

Hemoglobin (Hb) potently inactivates the nitric oxide (NO) radical via a dioxygenation reaction forming nitrate (NO(3)(-)). This inactivation produces endothelial dysfunction during hemolytic conditions and may contribute to the vascular complications of Hb-based blood substitutes. Hb also functions as a nitrite (NO(2)(-)) reductase, converting nitrite into NO as it deoxygenates. We hypothesized that during intravascular hemolysis, nitrite infusions would limit the vasoconstrictive properties of plasma Hb. In a canine model of low- and high-intensity hypotonic intravascular hemolysis, we characterized hemodynamic responses to nitrite infusions. Hemolysis increased systemic and pulmonary arterial pressures and systemic vascular resistance. Hemolysis also inhibited NO-dependent pulmonary and systemic vasodilation by the NO donor sodium nitroprusside. Compared with nitroprusside, nitrite demonstrated unique effects by not only inhibiting hemolysis-associated vasoconstriction but also by potentiating vasodilation at plasma Hb concentrations of <25 muM. We also observed an interaction between plasma Hb levels and nitrite to augment nitroprusside-induced vasodilation of the pulmonary and systemic circulation. This nitrite reductase activity of Hb in vivo was recapitulated in vitro using a mitochondrial NO sensor system. Nitrite infusions may promote NO generation from Hb while maintaining oxygen delivery; this effect could be harnessed to treat hemolytic conditions and to detoxify Hb-based blood substitutes.     

Features of the metabolism of nitric oxide in normal state and inflammation

Brief analysis of the metabolism of nitric oxide in living cells in normal state and pathology and also the analysis of the causes that hampered the progress of these studies were carried out. It was established that most of physiological fluids, including blood, normally contain nitrite and non-thiolate nitroso compounds in concentration less than 100 nM. Literature data from different researchers on the normal range of nitrite concentration in plasma of healthy people from several hundreds of nM to several μM is probably the result of low selectivity of the methods used. But nitrite and non-thiolate nitroso compounds concentration in blood is dramatically increased in case of inflammatory diseases. It is proposed that the main mechanism for the production of these substances in blood is the nitrosyl iron complexes transformation by active oxygen species but not the activation of NO production as it was considered previously.     

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.     

Overexpression of the human inducible nitric oxide synthase gene enhances radiation-induced apoptosis in colorectal cancer cells via a caspase-dependent mechanism.

Nitric oxide (NO) has been reported to sensitize cancer cells to radiation. Since delivery of NO to tumors is limited in vivo by systemic toxicity of NO, we examined the potential of gene delivery of the human inducible nitric oxide synthase (iNOS) gene as a means of achieving high output NO production. We successfully transduced two colorectal cancer cell lines as evidenced by increased iNOS protein accumulation and nitrite production. We found that overexpression of iNOS enhanced the effects of radiation on apoptosis in both cell lines in a caspase-dependent fashion. Gene transfer of iNOS holds much promise as a potential radiosensitizer of cancer cells since it increases apoptosis in an additive manner with radiation.     

Low-dose intravenous nitrite improves hemodynamics in a canine model of acute pulmonary thromboembolism

Acute pulmonary thomboembolism (APT)-induced pulmonary hypertension can be counteracted by activating the nitric oxide (NO)-cGMP pathway. Recent studies have demonstrated that the naturally occurring anion nitrite (NO₂^-) is a bioactive storage reservoir for NO, and is reduced to NO under conditions of hypoxia and acidosis. We hypothesized that nitrite infused intravenously could attenuate the hemodynamic changes associated with APT. APT was induced with autologous blood clots injected into the right atrium in mongrel dogs. After APT (or saline), the dogs received an intravenous nitrite (or saline) infusion (6.75 μmol/kg over 15 min and then 0.28 μmol/kg/min) and hemodynamic evaluations were carried out for 2 h. Plasma nitrite concentrations were measured using ozone-based reductive chemiluminescence methodologies. APT decreased cardiac index (CI) and increased pulmonary vascular resistance index (PVRI); these effects were improved during infusions of sodium nitrite. Accordingly, nitrite infusion increased cardiac index by 28%, reduced the PVRI by 48%, and the systemic vascular resistance index (SVRI) by 21% in embolized dogs, suggesting a greater effect on the ischemic embolized vascular system than the systemic circulation following embolization. Interestingly, in nonembolized control dogs the same nitrite infusion decreased MAP and CI (all P < 0.05). The nitrite infusion increased plasma nitrite concentrations by approximately 2 μM, and produced dose-dependent effects on PVRI, MAP, and SVRI. Remarkably, blood levels of nitrite as low as 500 nM decreased PVRI and SVRI in this model, suggesting a potential role of nitrite in physiological blood flow regulation. These results suggest that a low-dose nitrite infusion produces beneficial hemodynamic effects in a dog model of APT. These findings suggest a new therapeutic application for nitrite and support emerging evidence for a surprisingly potent and potentially physiological vasoactivity of nitrite.     

Evidence for a physiological role of intracellularly occurring photolabile nitrogen oxides in human skin fibroblasts

Nitric oxide (NO) plays a pivotal role in human skin biology. Cutaneous NO can be produced enzymatically by NO synthases (NOS) as well as enzyme independently via photodecomposition of photolabile nitrogen oxides (PNOs) such as nitrite or nitroso compounds, both found in human skin tissue in comparably high concentrations. Although the physiological role of NOS-produced NO in human skin is well defined, nothing is known about the biological relevance or the chemical origin of intracellularly occurring PNOs. We here, for the first time, give evidence that in human skin fibroblasts (FB) PNOs represent the oxidation products of NOS-produced NO and that in human skin fibroblasts intracellularly occurring PNOs effectively protect against the injurious effects of UVA radiation by a NO-dependent mechanism. In contrast, in PNO-depleted FB cultures an increased susceptibility to UVA-induced lipid peroxidation and cell death is observed, whereas supplementation of PNO-depleted FB cultures with physiological nitrite concentrations (10 microM) or with exogenously applied NO completely restores UVA-increased injuries. Thus, intracellular PNOs are biologically relevant and represent an important initial shield functioning in human skin physiology against UVA radiation. Consequently, nonphysiological low PNO concentrations might promote known UVA-related skin injuries such as premature aging and carcinogenesis.     

Low-dose intravenous nitrite improves hemodynamics in a canine model of acute pulmonary thromboembolism

Acute pulmonary thomboembolism (APT)-induced pulmonary hypertension can be counteracted by activating the nitric oxide (NO)-cGMP pathway. Recent studies have demonstrated that the naturally occurring anion nitrite (NO₂^-) is a bioactive storage reservoir for NO, and is reduced to NO under conditions of hypoxia and acidosis. We hypothesized that nitrite infused intravenously could attenuate the hemodynamic changes associated with APT. APT was induced with autologous blood clots injected into the right atrium in mongrel dogs. After APT (or saline), the dogs received an intravenous nitrite (or saline) infusion (6.75 μmol/kg over 15 min and then 0.28 μmol/kg/min) and hemodynamic evaluations were carried out for 2 h. Plasma nitrite concentrations were measured using ozone-based reductive chemiluminescence methodologies. APT decreased cardiac index (CI) and increased pulmonary vascular resistance index (PVRI); these effects were improved during infusions of sodium nitrite. Accordingly, nitrite infusion increased cardiac index by 28%, reduced the PVRI by 48%, and the systemic vascular resistance index (SVRI) by 21% in embolized dogs, suggesting a greater effect on the ischemic embolized vascular system than the systemic circulation following embolization. Interestingly, in nonembolized control dogs the same nitrite infusion decreased MAP and CI (all P < 0.05). The nitrite infusion increased plasma nitrite concentrations by approximately 2 μM, and produced dose-dependent effects on PVRI, MAP, and SVRI. Remarkably, blood levels of nitrite as low as 500 nM decreased PVRI and SVRI in this model, suggesting a potential role of nitrite in physiological blood flow regulation. These results suggest that a low-dose nitrite infusion produces beneficial hemodynamic effects in a dog model of APT. These findings suggest a new therapeutic application for nitrite and support emerging evidence for a surprisingly potent and potentially physiological vasoactivity of nitrite.     

Hemodynamic effect of the nitroxide superoxide dismutase mimics.

Reactive oxygen species play critical roles in a number of physiologic and pathologic processes. Nitroxides are stable free radical compounds that possess superoxide dismutase (SOD) mimetic activity and have been shown to protect against the toxicity of reactive oxygen species in vitro and in vivo. Tempol, a cell-permeable hydrophilic nitroxide, protects against oxidative stress and also is an in vitro and in vivo radioprotector. In the course of evaluating the pharmacology and toxicity of the nitroxides, Tempol and another nitroxide, 3-carbamoyl-PROXYL (3-CP), were administered intravenously in various concentrations to miniature swine. Tempol caused dose-related hypotension accompanied by reflex tachycardia and increased skin temperature. Invasive hemodynamic monitoring with Swan Ganz catheterization (SGC) confirmed the potent vasodilative effect of Tempol. However, 3-CP had no effect on porcine blood pressure. The hemodynamic effects of Tempol and 3-CP are discussed in the context of differential catalytic rate constants for superoxide disumation that may impact systemic nitric oxide (NO) levels and lead to vasodilation. These findings are consistent with a role for the superoxide ion in the modulation of blood pressure and have potential implications for the systemic use of nitroxides.     

Intragastric generation of antimicrobial nitrogen oxides from saliva--physiological and therapeutic considerations

Salivary nitrite is suggested to enhance the antimicrobial properties of gastric juice by conversion to nitric oxide (NO) and other reactive nitrogen intermediates in the stomach. Intubated patients exhibit extremely low gastric levels of NO, because they do not swallow their saliva. The present investigation was designed to examine the antibacterial effects of human saliva and gastric juice. Furthermore, we studied a new mode of NO delivery, involving formation from acidified nitrite, which could prevent bacterial growth in the gastric juice of intubated patients in intensive care units. The growth of Escherichia coli ATCC 25922 and the formation of NO and nitroso/nitrosyl species were determined after incubation of gastric juice with saliva from healthy volunteers that was rich (nitrate ingestion) or poor (overnight fasting) in nitrite. In a stomach model containing gastric juice from intubated patients, we inserted a catheter with a silicone retention cuff filled with ascorbic acid and nitrite and determined the resulting antibacterial effects on E. coli and Candida albicans. Saliva enhanced the bactericidal effect of gastric juice, especially saliva rich in nitrite. Formation of NO and nitroso/nitrosyl species by nitrite-rich saliva was 10-fold greater than that by saliva poor in nitrite. In our stomach model, E. coli and C. albicans were killed after exposure to ascorbic acid and nitrite. In conclusion, saliva rich in nitrite enhances the bactericidal effects of gastric juice, possibly through the generation of reactive nitrogen intermediates, including NO. Acidified nitrite inside a gas-permeable retention cuff may be useful for restoring gastric NO levels and host defense in critically ill patients.     

The role of nitric oxide in regulation of the cardiovascular system in reptiles

The roles that nitric oxide (NO) plays in the cardiovascular system of reptiles are reviewed, with particular emphasis on its effects on central vascular blood flows in the systemic and pulmonary circulations. New data is presented that describes the effects on hemodynamic variables in varanid lizards of exogenously administered NO via the nitric oxide donor sodium nitroprusside (SNP) and inhibition of nitric oxide synthase (NOS) by l-nitroarginine methyl ester (l-NAME). Furthermore, preliminary data on the effects of SNP on hemodynamic variables in the tegu lizard are presented. The findings are compared with previously published data from our laboratory on three other species of reptiles: pythons (), rattlesnakes () and turtles (). These five species of reptiles possess different combinations of division of the heart and structural complexity of the lungs. Comparison of their responses to NO donors and NOS inhibitors may reveal whether the potential contribution of NO to vascular tone correlates with pulmonary complexity and/or with blood pressure. All existing studies on reptiles have clearly established a potential role for NO in regulating vascular tone in the systemic circulation and NO may be important for maintaining basal systemic vascular tone in varanid lizards, pythons and turtles, through a continuous release of NO. In contrast, the pulmonary circulation is less responsive to NO donors or NOS inhibitors, and it was only in pythons and varanid lizards that the lungs responded to SNP. Both species have a functionally separated heart, so it is possible that NO may exert a larger role in species with low pulmonary blood pressures, irrespective of lung complexity.     

Nitrosative stress in an animal model of necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a disease of newborns characterized by gut barrier failure. We reasoned that upregulation of inducible nitric oxide synthase (iNOS) may result in nitrosative stress and accumulation of nitroso species in the intestine. Newborn rats were either breast-fed (BF), or formula-fed and additionally subjected to hypoxia (FFH). At Day 4 after birth, the distal ilea were harvested and processed for Western blot analysis and measurement of NO-related metabolites. While BF neonates showed normal morphology, FFH neonates developed signs of NEC by Day 4. These pathological changes correlated with upregulation of iNOS and increases in tissue nitrite, nitrosothiol, and nitrosamine concentrations. Enhanced nitroso levels were most prominent in the mucosal layers of the ileum and iNOS inhibition resulted in a significant decrease in both nitroso species and incidence of NEC. In contrast, increased nitrite levels were distributed evenly throughout the ileum and remained unchanged following iNOS inhibition. Similarly, specimens from NEC patients had higher intestinal levels of NO-related metabolites compared to non-NEC controls. This is the first report of tissue levels of nitroso species in the gut of an animal model of NEC and of human specimens. The results suggest that local nitrosative stress contributes to the pathology associated with NEC. Unexpectedly, the NO breakdown product nitrite, previously considered biologically inert, was found to be present throughout the ileal wall, suggesting that cellular NO metabolism is altered significantly in NEC. Whether nitrite plays a protective or deleterious role remains to be investigated.     

Methods of quantitative analysis of the nitric oxide metabolites nitrite and nitrate in human biological fluids

In human organism, the gaseous radical molecule nitric oxide (NO) is produced in various cells from L-arginine by the catalytic action of NO synthases (NOS). The metabolic fate of NO includes oxidation to nitrate by oxyhaemoglobin in red blood cells and autoxidation in haemoglobin-free media to nitrite. Nitrate and nitrite circulate in blood and are excreted in urine. The concentration of these NO metabolites in the circulation and in the urine can be used to measure NO synthesis in vivo under standardized low-nitrate diet. Circulating nitrite reflects constitutive endothelial NOS activity, whereas excretory nitrate indicates systemic NO production. Today, nitrite and nitrate can be measured in plasma, serum and urine of humans by various analytical methods based on different analytical principles, such as colorimetry, spectrophotometry, fluorescence, chemiluminescence, gas and liquid chromatography, electrophoresis and mass spectrometry. The aim of the present article is to give an overview of the most significant currently used quantitative methods of analysis of nitrite and nitrate in human biological fluids, namely plasma and urine. With minor exception, measurement of nitrite and nitrate by these methods requires method-dependent chemical conversion of these anions. Therefore, the underlying mechanisms and principles of these methods are also discussed. Despite the chemical simplicity of nitrite and nitrate, accurate and interference-free quantification of nitrite and nitrate in biological fluids as indicators of NO synthesis may be difficult. Thus, problems associated with dietary and laboratory ubiquity of these anions and other preanalytical and analytical factors are addressed. Eventually, the important issue of quality control, the use of commercially available assay kits, and the value of the mass spectrometry methodology in this area are outlined.     

7-nitroindazole reduces nitrite concentration in rat brain after intrahippocampal kainate-induced seizure

Kainic acid is an endogenous excitotoxin acting on glutamate receptors, that leads to neurotoxic damage resembling the alterations observed in some neurological disorders. Stimulation of glutamate receptors induces neuronal nitric oxide (NO) release, which in turn modulates glutamate transmission. NO may be a key mediator of excitotoxic neuronal injury in the central nervous system. We investigated the effects of 7-nitroindazole, a selective neuronal nitric oxide synthase inhibitor in vivo, on nitrite concentration after kainic acid injection (0.6 mg/ml, pH 7.2) unilaterally into the CA3 region of the rat hippocampus. The accumulation of nitrite, the stable metabolite of NO, was measured by the Griess reaction at different times (5 min, 15 min, 2 h, 48 h and 7 days) following kainate injection in the ipsilateral and contralateral hippocampus, forebrain cortex, striatum and cerebellum homogenates. 7-Nitroindazole (100 microM) can effectively inhibit NO synthesis in rat brain after kainate-induced intrahippocampal neurotoxicity and suppressed nitrite accumulation. The present results suggest that neuronal NO synthase inhibitors may be useful in the treatment of neurological diseases where excitotoxic mechanisms play a role.     

Nitric oxide bioactivity of traditional Chinese medicines used for cardiovascular indications

Traditional Chinese medicine (TCM) has been used for centuries to treat and prevent certain ailments and diseases. Although TCM has served as mainstream medical care throughout Asia for many generations, it is considered an alternative medical system in much of the Western world. Because many TCMs are used primarily for cardiovascular indications characterized by a nitric oxide (NO) insufficiency, we hypothesized that some, if not all, of these TCMs have a robust NO bioactivity that may act to restore NO homeostasis. We tested a group of convenience samples of TCMs obtained in the United States for endogenous nitrite, nitrate, nitroso, and nitrite reductase activity as well as their ability to relax isolated aortic rings. The results from this study reveal that all of the TCMs tested reveal NO bioactivity through their inherent nitrite and nitrate content and their ability to reduce nitrite to NO. Many of the TCM extracts contain a nitrite reductase activity greater by 1000 times that of biological tissues. Repletion of biological nitrite and nitrate by these extracts and providing a natural system for NO generation in both endothelium-dependent and -independent mechanisms may account for some of the therapeutic effects of TCMs.     

Circulating antibodies directed against nitrosylated antigens in trypanosome-infected mice

Nitric oxide has been implicated as an effector cytotoxic molecule in trypanosomiasis. In this work, we investigated the presence of circulating antibodies directed against nitrosylated epitopes as biological indicators for nitric oxide (NO) production in the sera of trypanosome-infected mice. We tested these sera with synthetic antigens, such as S-nitrosated protein or nitrosylated conjugates of amino acids that possess a high affinity to NO, by an immunoenzymatic assay. We detected antibodies directed against nitroso epitopes in the sera of infected mice, as compared to non-infected control mice. The antibody response was linked to the IgM isotype. Our results indicate the production of NO and its derivatives in trypanosomiasis. This production may potentially induce the synthesis of nitroso epitopes in vivo and favor the development of a humoral immune response.     

Nitrite as a vascular endocrine nitric oxide reservoir that contributes to hypoxic signaling, cytoprotection, and vasodilation

Accumulating evidence suggests that the simple and ubiquitous anion salt, nitrite (NO(2)(-)), is a physiological signaling molecule with potential roles in intravascular endocrine nitric oxide (NO) transport, hypoxic vasodilation, signaling, and cytoprotection after ischemia-reperfusion. Human and animal studies of nitrite treatment and NO gas inhalation provide evidence that nitrite mediates many of the systemic therapeutic effects of NO gas inhalation, including peripheral vasodilation and prevention of ischemia-reperfusion-mediated tissue infarction. With regard to nitrite-dependent hypoxic signaling, biochemical and physiological studies suggest that hemoglobin possesses an allosterically regulated nitrite reductase activity that reduces nitrite to NO along the physiological oxygen gradient, potentially contributing to hypoxic vasodilation. An expanded consideration of nitrite as a hypoxia-dependent intrinsic signaling molecule has opened up a new field of research and therapeutic opportunities for diseases associated with regional hypoxia and vasoconstriction.     

Nitrosation of uric acid induced by nitric oxide under aerobic conditions.

Uric acid is a well-established scavenger of reactive oxygen and nitrogen species such as hydroxyl radical and peroxynitrite. However, little attention has been paid to the relationship between uric acid and nitric oxide. This paper reports the identification and characterization of a reaction product of uric acid induced by nitric oxide. When uric acid was treated with nitric oxide gas in a neutral solution under aerobic conditions, uric acid was consumed, yielding an unknown product. The product was identified as nitrosated uric acid from mass spectrometric data, although the position of the nitroso group on the molecule was not determined. The nitrosated uric acid decomposed to several compounds including uric acid with a half-life of 2.2 min at pH 7.4 and 37 degrees C. The incubation of nitrosated uric acid with glutathione resulted in the formation of S-nitrosoglutathione. Nitrosated uric acid was also formed in the reaction with nitric oxide donors, but not with peroxynitrite. Nitrosated uric acid was detected in human serum and urine by in vitro treatment with a nitric oxide donor. In the reaction of glutathione with the nitric oxide donor, the addition of uric acid caused an increase in the yield of S-nitrosoglutathione. These results indicate that under aerobic conditions nitric oxide can convert uric acid into its nitroso derivative, which can give a nitroso group to glutathione. Uric acid may act as a vehicle of nitric oxide in humans.     

Requirement of argininosuccinate lyase for systemic nitric oxide production

Nitric oxide (NO) is crucial in diverse physiological and pathological processes. We show that a hypomorphic mouse model of argininosuccinate lyase (encoded by Asl) deficiency has a distinct phenotype of multiorgan dysfunction and NO deficiency. Loss of Asl in both humans and mice leads to reduced NO synthesis, owing to both decreased endogenous arginine synthesis and an impaired ability to use extracellular arginine for NO production. Administration of nitrite, which can be converted into NO in vivo, rescued the manifestations of NO deficiency in hypomorphic Asl mice, and a nitric oxide synthase (NOS)-independent NO donor restored NO-dependent vascular reactivity in humans with ASL deficiency. Mechanistic studies showed that ASL has a structural function in addition to its catalytic activity, by which it contributes to the formation of a multiprotein complex required for NO production. Our data demonstrate a previously unappreciated role for ASL in NOS function and NO homeostasis. Hence, ASL may serve as a target for manipulating NO production in experimental models, as well as for the treatment of NO-related diseases.