Inorganic nitrate is a possible source for systemic generation of nitric oxide

Nitrate and nitrite have been considered stable inactive end products of nitric oxide (NO). While several recent studies now imply that nitrite can be reduced to bioactive NO again, the more stable anion nitrate is still considered to be biologically inert. Nitrate is concentrated in saliva, where a part of it is reduced to nitrite by bacterial nitrate reductases. We tested if ingestion of inorganic nitrate would affect the salivary and systemic levels of nitrite and S-nitrosothiols, both considered to be circulating storage pools for NO. Levels of nitrate, nitrite, and S-nitrosothiols were measured in plasma, saliva, and urine before and after ingestion of sodium nitrate (10 mg/kg). Nitrate levels increased greatly in saliva, plasma, and urine after the nitrate load. Salivary S-nitrosothiols also increased, but plasma levels remained unchanged. A 4-fold increase in plasma nitrite was observed after nitrate ingestion. If, however, the test persons avoided swallowing after the nitrate load, the increase in plasma nitrite was prevented, thereby illustrating its salivary origin. We show that nitrate is a substrate for systemic generation of nitrite. There are several pathways to further reduce this nitrite to NO. These results challenge the dogma that nitrate is biologically inert and instead suggest that a complete reverse pathway for generation of NO from nitrate exists.     

Xanthine oxidase catalyzes anaerobic transformation of organic nitrates to nitric oxide and nitrosothiols: characterization of this mechanism and the link between organic nitrate and guanylyl cyclase activation

Organic nitrates have been used clinically in the treatment of ischemic heart disease for more than a century. Recently, xanthine oxidase (XO) has been reported to catalyze organic nitrate reduction under anaerobic conditions, but questions remain regarding the initial precursor of nitric oxide (NO) and the link of organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of XO-mediated biotransformation of organic nitrate, studies using electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, NO electrode, and immunoassay were performed. The XO reducing substrates xanthine, NADH, and 2,3-dihydroxybenz-aldehyde triggered the reduction of organic nitrate to nitrite anion (NO2-). Studies of the pH dependence of nitrite formation indicated that XO-mediated organic nitrate reduction occurred via an acid-catalyzed mechanism. In the absence of thiols or ascorbate, no NO generation was detected from XO-mediated organic nitrate reduction; however, addition of L-cysteine or ascorbate triggered prominent NO generation. Studies suggested that organic nitrite (R-O-NO) is produced from XO-mediated organic nitrate reduction. Further reaction of organic nitrite with thiols or ascorbate leads to the generation of NO or nitrosothiols and thus stimulates the activation of sGC. Only flavin site XO inhibitors such as diphenyleneiodonium inhibited XO-mediated organic nitrate reduction and sGC activation, indicating that organic nitrate reduction occurs at the flavin site. Thus, organic nitrite is the initial product in the process of XO-mediated organic nitrate biotransformation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.     

A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis

Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.     

Decreased leukocyte recruitment by inorganic nitrate and nitrite in microvascular inflammation and NSAID-induced intestinal injury

Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues.     

Dietary inorganic nitrate mobilizes circulating angiogenic cells

Nitric oxide (NO) was implicated in the regulation of mobilization and function of circulating angiogenic cells (CACs). The supposedly inert anion nitrate, abundant in vegetables, can be stepwise reduced in vivo to form nitrite, and consecutively NO, representing an alternative to endogenous NO formation by NO synthases. This study investigated whether inorganic dietary nitrate influences mobilization of CACs. In a randomized double-blind fashion, healthy volunteers ingested 150 ml water with 0.15 mmol/kg (12.7 mg/kg) of sodium nitrate, an amount corresponding to 100-300 g of a nitrate-rich vegetable, or water alone as control. Mobilization of CACs was determined by the number of CD34(+)/KDR(+) and CD133(+)/KDR(+) cells using flow cytometry and the mobilization markers stem cell factor (SCF) and stromal cell-derived factor-1a (SDF-1α) were determined in plasma via ELISA. Nitrite and nitrate were measured using high-performance liquid chromatography and reductive gas-phase chemiluminescence, respectively. NOS-dependent vasodilation was measured as flow-mediated vasodilation. Further mechanistic studies were performed in mice after intravenous application of nitrite together with an NO scavenger to identify the role of nitrite and NO in CAC mobilization. Nitrate ingestion led to a rise in plasma nitrite together with an acute increase in CD34(+)/KDR(+) and CD133(+)/KDR(+)-CACs along with increased NOS-dependent vasodilation. This was paralleled by an increase in SCF and SDF-1α and the maximal increase in plasma nitrite correlated with CD133(+)/KDR(+)-CACs (r=0.73, P=0.016). In mice, nitrate given per gavage and direct intravenous injection of nitrite led to CAC mobilization, which was abolished by the NO scavenger cPTIO, suggesting that nitrite mediated its effect via formation of NO. Dietary inorganic nitrate acutely mobilizes CACs via serial reduction to nitrite and NO. The nitrate-nitrite-NO pathway could offer a novel nutritional approach for regulation of vascular regenerative processes.     

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.     

Characterization of the magnitude and kinetics of xanthine oxidase-catalyzed nitrate reduction: evaluation of its role in nitrite and nitric oxide generation in anoxic tissues

In addition to nitric oxide (NO) generation from specific NO synthases, NO is also formed during anoxia from nitrite reduction, and xanthine oxidase (XO) catalyzes this process. While in tissues and blood high nitrate levels are present, questions remain regarding whether nitrate is also a source of NO and if XO-mediated nitrate reduction can be an important source of NO in biological systems. To characterize the kinetics, magnitude, and mechanism of XO-mediated nitrate reduction under anaerobic conditions, EPR, chemiluminescence NO-analyzer, and NO-electrode studies were performed. Typical XO reducing substrates, xanthine, NADH, and 2,3-dihydroxybenz-aldehyde, triggered nitrate reduction to nitrite and NO. The rate of nitrite production followed Michaelis-Menten kinetics, while NO generation rates increased linearly following the accumulation of nitrite, suggesting stepwise-reduction of nitrate to nitrite then to NO. The molybdenum-binding XO inhibitor, oxypurinol, inhibited both nitrite and NO production, indicating that nitrate reduction occurs at the molybdenum site. At higher xanthine concentrations, partial inhibition was seen, suggesting formation of a substrate-bound reduced enzyme complex with xanthine blocking the molybdenum site. The pH dependence of nitrite and NO formation indicate that XO-mediated nitrate reduction occurs via an acid-catalyzed mechanism. With conditions occurring during ischemia, myocardial xanthine oxidoreductase and nitrate levels were determined to generate up to 20 microM nitrite within 10-20 min that can be further reduced to NO with rates comparable to those of maximally activated NOS. Thus, XOR catalyzed nitrate reduction to nitrite and NO occurs and can be an important source of NO production in ischemic tissues.     

Nitrate Reduction to Nitrite,Nitric Oxide and Ammonia by Gut Bacteria under Physiological Conditions

The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. Neither process has been thought to have relevance to mammalian physiology; however in recent years the salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration; however, the importance of this pathway in bacterial species colonizing the human intestine has been little studied. We measured nitrite, nitric oxide (NO) and ammonia formation in cultures of Escherichia coli, Lactobacillus and Bifidobacterium species grown at different sodium nitrate concentrations and oxygen levels. We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract. Nitrite and ammonia accumulated in the growth medium when at least 2.5 mM nitrate was present. Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia. Strains of L.rhamnosus, L.acidophilus and B.longum infantis grown with nitrate produced minor changes in nitrite or ammonia levels in the cultures. However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate. Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction. In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH. We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.     

The increase in plasma nitrite after a dietary nitrate load is markedly attenuated by an antibacterial mouthwash

Recent studies surprisingly show that dietary inorganic nitrate, abundant in vegetables, can be metabolized in vivo to form nitrite and then bioactive nitric oxide. A reduction in blood pressure was recently noted in healthy volunteers after dietary supplementation with nitrate; an effect consistent with formation of vasodilatory nitric oxide. Oral bacteria have been suggested to play a role in bioactivation of nitrate by first reducing it to the more reactive anion nitrite. In a cross-over designed study in seven healthy volunteers we examined the effects of a commercially available chlorhexidine-containing antibacterial mouthwash on salivary and plasma levels of nitrite measured after an oral intake of sodium nitrate (10 mg/kg dissolved in water). In the control situation the salivary and plasma levels of nitrate and nitrite increased greatly after the nitrate load. Rinsing the mouth with the antibacterial mouthwash prior to the nitrate load had no effect on nitrate accumulation in saliva or plasma but abolished its conversion to nitrite in saliva and markedly attenuated the rise in plasma nitrite. We conclude that the acute increase in plasma nitrite seen after a nitrate load is critically dependent on nitrate reduction in the oral cavity by commensal bacteria. The removal of these bacteria with an antibacterial mouthwash will very likely attenuate the NO-dependent biological effects of dietary nitrate.     

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N^ω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.     

Nitrite anion stimulates ischemic arteriogenesis involving NO metabolism

Nitric oxide (NO) is a potential regulator of ischemic vascular remodeling, and as such therapies augmenting its bioavailability may be useful for the treatment of ischemic tissue diseases. Here we examine the effect of administering the NO prodrug sodium nitrite on arteriogenesis activity during established tissue ischemia. Chronic hindlimb ischemia was induced by permanent unilateral femoral artery and vein ligation. Five days postligation; animals were randomized to control PBS or sodium nitrite (165 μg/kg) therapy twice daily. In situ vascular remodeling was measured longitudinally using SPY angiography and Microfil vascular casting. Delayed sodium nitrite therapy rapidly increased ischemic limb arterial vessel diameter and branching in a NO-dependent manner. SPY imaging angiography over time showed that nitrite therapy enhanced ischemic gracillis collateral vessel formation from the profunda femoris to the saphenous artery. Immunofluorescent staining of smooth muscle cell actin also confirmed that sodium nitrite therapy increased arteriogenesis in a NO-dependent manner. The NO prodrug sodium nitrite significantly increases arteriogenesis and reperfusion of established severe chronic tissue ischemia.     

Endothelial dysfunction is induced by proinflammatory oxidant hypochlorous acid

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl) plays a role in tissue injury under inflammatory conditions. The present study tests the hypothesis that HOCl decreases nitric oxide (NO) bioavailability in the vasculature of Sprague-Dawley rats. Aortic ring segments were pretreated with HOCl (1-50 microM) followed by extensive washing. Endothelium-dependent relaxation was then assessed by cumulative addition of acetylcholine (ACh) or the calcium ionophore A23187. HOCl treatment significantly impaired both ACh- and A23187-mediated relaxation. In contrast, endothelium-independent relaxation induced by sodium nitroprusside was unaffected. The inhibitory effect of HOCl on ACh-induced relaxation was reversed by exposure of ring segments to L-arginine but not D-arginine. In cellular studies, HOCl did not alter endothelial NO synthase (NOS III) protein or activity, but inhibited formation of the NO metabolites nitrate (NO3(-) and nitrite (NO2(-). The reduction in total NO metabolite production in bovine aortic endothelial cells was also reversed by addition of L-arginine. These data suggest that HOCl induces endothelial dysfunction via modification of L-arginine.     

Antioxidant and anti-inflammatory effects of products from Croton celtidifolius Bailon on carrageenan-induced pleurisy in rats

Croton celtidifolius Bailon, commonly known as Sangue-de-Adáve or Pau-Andrade, is a tree found in the Atlantic forest of southern Brazil. It has been popularly used for the treatment of inflammatory and ulcerative disorders. Phytochemical analysis demonstrated the presence of flavonoids and proanthocyanidins in an ethyl acetate fraction (EAF) from C. celtidifolius Bailon. In this study, we have evaluated the effects of EAF and its sub-fractions (35 and 63, catechin) on inflammatory (cell migration and plasma extravasation) and oxidative (lipid peroxidation, superoxide dismutase (SOD) activity and superoxide anion production) parameters in carrageenan-induced pleurisy in rats. NO production was also measured by nitrite/nitrate levels. EAF and sub-fraction 63 (63SF) showed anti-inflammatory activity, as indicated by a reduction in plasma extravasation and cell migration (mainly polymorphonuclear leukocytes) to the pleural cavity. Furthermore, EAF treatment decreased the production of superoxide radical anion by cells isolated from the pleural cavity, while it did not affect the nitrite/nitrate levels in exudates. The results show that C. celtidifolius contains substances with antioxidant and anti-inflammatory activity that, at least in part, act by a modulation of oxidative stress by phenolic compounds.     

The reaction between nitrite and oxyhemoglobin: a mechanistic study

The nitrite anion (NO(-)(2)) has recently received much attention as an endogenous nitric oxide source that has the potential to be supplemented for therapeutic benefit. One major mechanism of nitrite reduction is the direct reaction between this anion and the ferrous heme group of deoxygenated hemoglobin. However, the reaction of nitrite with oxyhemoglobin (oxyHb) is well established and generates nitrate and methemoglobin (metHb). Several mechanisms have been proposed that involve the intermediacy of protein-free radicals, ferryl heme, nitrogen dioxide (NO(2)), and hydrogen peroxide (H(2)O(2)) in an autocatalytic free radical chain reaction, which could potentially limit the usefulness of nitrite therapy. In this study we show that none of the previously published mechanisms is sufficient to fully explain the kinetics of the reaction of nitrite with oxyHb. Based on experimental data and kinetic simulation, we have modified previous models for this reaction mechanism and show that the new model proposed here is consistent with experimental data. The important feature of this model is that, whereas previously both H(2)O(2) and NO(2) were thought to be integral to both the initiation and propagation steps, H(2)O(2) now only plays a role as an initiator species, and NO(2) only plays a role as an autocatalytic propagatory species. The consequences of uncoupling the roles of H(2)O(2) and NO(2) in the reaction mechanism for the in vivo reactivity of nitrite are discussed.     

Inhaled nebulized nitrite is a hypoxia-sensitive NO-dependent selective pulmonary vasodilator

The blood anion nitrite contributes to hypoxic vasodilation through a heme-based, nitric oxide (NO)-generating reaction with deoxyhemoglobin and potentially other heme proteins. We hypothesized that this biochemical reaction could be harnessed for the treatment of neonatal pulmonary hypertension, an NO-deficient state characterized by pulmonary vasoconstriction, right-to-left shunt pathophysiology and systemic hypoxemia. To test this, we delivered inhaled sodium nitrite by aerosol to newborn lambs with hypoxic and normoxic pulmonary hypertension. Inhaled nitrite elicited a rapid and sustained reduction ( approximately 65%) in hypoxia-induced pulmonary hypertension, with a magnitude approaching that of the effects of 20 p.p.m. NO gas inhalation. This reduction was associated with the immediate appearance of NO in expiratory gas. Pulmonary vasodilation elicited by aerosolized nitrite was deoxyhemoglobin- and pH-dependent and was associated with increased blood levels of iron-nitrosyl-hemoglobin. Notably, from a therapeutic standpoint, short-term delivery of nitrite dissolved in saline through nebulization produced selective, sustained pulmonary vasodilation with no clinically significant increase in blood methemoglobin levels. These data support the concept that nitrite is a vasodilator acting through conversion to NO, a process coupled to hemoglobin deoxygenation and protonation, and evince a new, simple and inexpensive potential therapy for neonatal pulmonary hypertension.     

Sodium nitrite downregulates vascular NADPH oxidase and exerts antihypertensive effects in hypertension

Dietary nitrite and nitrate are important sources of nitric oxide (NO). However, the use of nitrite as an antihypertensive drug may be limited by increased oxidative stress associated with hypertension. We evaluated the antihypertensive effects of sodium nitrite given in drinking water for 4 weeks in two-kidney one-clip (2K1C) hypertensive rats and the effects induced by nitrite on NO bioavailability and oxidative stress. We found that, even under the increased oxidative stress conditions present in 2K1C hypertension, nitrite reduced systolic blood pressure in a dose-dependent manner. Whereas treatment with nitrite did not significantly change plasma nitrite concentrations in 2K1C rats, it increased plasma nitrate levels significantly. Surprisingly, nitrite treatment exerted antioxidant effects in both hypertensive and sham-normotensive control rats. A series of in vitro experiments was carried out to show that the antioxidant effects induced by nitrite do not involve direct antioxidant effects or xanthine oxidase activity inhibition. Conversely, nitrite decreased vascular NADPH oxidase activity. Taken together, our results show for the first time that nitrite has antihypertensive effects in 2K1C hypertensive rats, which may be due to its antioxidant properties resulting from vascular NADPH oxidase activity inhibition.     

Characterization of the mechanism of cytochrome P450 reductase-cytochrome P450-mediated nitric oxide and nitrosothiol generation from organic nitrates

Mammalian cytochrome P450 reductase (CPR) and cytochrome P450 (CP) play important roles in organic nitrate bioactivation; however, the mechanism by which they convert organic nitrate to NO remains unknown. Questions remain regarding the initial precursor of NO that serves to link organic nitrate to the activation of soluble guanylyl cyclase (sGC). To characterize the mechanism of CPR-CP-mediated organic nitrate bioactivation, EPR, chemiluminescence NO analyzer, NO electrode, and immunoassay studies were performed. With rat hepatic microsomes or purified CPR, the presence of NADPH triggered organic nitrate reduction to NO2(-). The CPR flavin site inhibitor diphenyleneiodonium inhibited this NO2(-) generation, whereas the CP inhibitor clotrimazole did not. However, clotrimazole greatly inhibited NO2(-)-dependent NO generation. Therefore, CPR catalyzes organic nitrate reduction, producing nitrite, whereas CP can mediate further nitrite reduction to NO. Nitrite-dependent NO generation contributed <10% of the CPR-CP-mediated NO generation from organic nitrates; thus, NO2(-) is not the main precursor of NO. CPR-CP-mediated NO generation was largely thiol-dependent. Studies suggested that organic nitrite (R-O-NO) was produced from organic nitrate reduction by CPR. Further reaction of organic nitrite with free or microsome-associated thiols led to NO or nitrosothiol generation and thus stimulated the activation of sGC. Thus, organic nitrite is the initial product in the process of CRP-CP-mediated organic nitrate activation and is the precursor of NO and nitrosothiols, serving as the link between organic nitrate and sGC activation.     

Denitrification by fungi

Many fungi in the centre of the group of Fusarium and its teleomorphs were shown to be capable of reducing nitrite anaerobically to form nitric oxide (NO), nitrous oxide (N2O), and/or dinitrogen (N2). Several strains could reduce nitrate as well. Nitrous oxide was the major product of the reduction of nitrate or nitrite. Several fungi could also form N2. When [15]nitrite was used as substrate for the N2-forming denitrification, 15N2O, 15NO, and 14N15N were obtained as the products. These results demonstrated that, unexpectedly, many fungi have denitrifying abilities. It was also shown that the fungal system contains a unique reaction, formation of a hybrid dinitrogen.     

Mechanisms of Human Erythrocytic Bioactivation of Nitrite

Nitrite signaling likely occurs through its reduction to nitric oxide (NO). Several reports support a role of erythrocytes and hemoglobin in nitrite reduction, but this remains controversial, and alternative reductive pathways have been proposed. In this work we determined whether the primary human erythrocytic nitrite reductase is hemoglobin as opposed to other erythrocytic proteins that have been suggested to be the major source of nitrite reduction. We employed several different assays to determine NO production from nitrite in erythrocytes including electron paramagnetic resonance detection of nitrosyl hemoglobin, chemiluminescent detection of NO, and inhibition of platelet activation and aggregation. Our studies show that NO is formed by red blood cells and inhibits platelet activation. Nitric oxide formation and signaling can be recapitulated with isolated deoxyhemoglobin. Importantly, there is limited NO production from erythrocytic xanthine oxidoreductase and nitric-oxide synthase. Under certain conditions we find dorzolamide (an inhibitor of carbonic anhydrase) results in diminished nitrite bioactivation, but the role of carbonic anhydrase is abrogated when physiological concentrations of CO₂ are present. Importantly, carbon monoxide, which inhibits hemoglobin function as a nitrite reductase, abolishes nitrite bioactivation. Overall our data suggest that deoxyhemoglobin is the primary erythrocytic nitrite reductase operating under physiological conditions and accounts for nitrite-mediated NO signaling in blood.     

A microscopic study of the deoxyhemoglobin-catalyzed generation of nitric oxide from nitrite anion

There is recent evidence suggesting that nitrite anion (NO 2 (-)) represents the major intravascular NO storage molecule whose transduction to NO is facilitated by a reduction mechanism catalyzed by deoxygenated hemoglobin (deoxy-Hb). In this work, we provide a detailed microscopic study of deoxy-Hb nitrite reductase (NIR) activity by combining classical molecular dynamics and hybrid quantum mechanical-molecular mechanical simulations. Our results point out that two alternative mechanisms could be operative and suggest that the most energetic barriers should stem from either reprotonation of the distal histidine or NO dissociation from the ferric heme. In the first proposed mechanism, which is similar to that proposed for bacterial NIRs, nitrite anion or nitrous acid coordinates to the heme through the N atom. This pathway involves HisE7 in a one or two proton transfer process, depending on whether the active species is nitrite anion or nitrous acid, to yield an intermediate Fe(III)NO species which eventually dissociates leading to NO and methemoglobin. In the second mechanism, the nitrite anion coordinates to the heme through the O atom. This pathway requires only one proton transfer from HisE7 and leads directly to the formation of a hydroxo Fe(III) complex and NO.