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.     

Low NO concentration dependence of reductive nitrosylation reaction of hemoglobin

The reductive nitrosylation of ferric (met)hemoglobin is of considerable interest and remains incompletely explained. We have previously observed that at low NO concentrations the reaction with tetrameric hemoglobin occurs with an observed rate constant that is at least 5 times faster than that observed at higher concentrations. This was ascribed to a faster reaction of NO with a methemoglobin-nitrite complex. We now report detailed studies of this reaction of low NO with methemoglobin. Nitric oxide paradoxically reacts with ferric hemoglobin with faster observed rate constants at the lower NO concentration in a manner that is not affected by changes in nitrite concentration, suggesting that it is not a competition between NO and nitrite, as we previously hypothesized. By evaluation of the fast reaction in the presence of allosteric effectors and isolated β- and α-chains of hemoglobin, it appears that NO reacts with a subpopulation of β-subunit ferric hemes whose population is influenced by quaternary state, redox potential, and hemoglobin dimerization. To further characterize the role of nitrite, we developed a system that oxidizes nitrite to nitrate to eliminate nitrite contamination. Removal of nitrite does not alter reaction kinetics, but modulates reaction products, with a decrease in the formation of S-nitrosothiols. These results are consistent with the formation of NO(2)/N(2)O(3) in the presence of nitrite. The observed fast reductive nitrosylation observed at low NO concentrations may function to preserve NO bioactivity via primary oxidation of NO to form nitrite or in the presence of nitrite to form N(2)O(3) and S-nitrosothiols.     

植物多铜氧化酶及其亚家族成员SKS蛋白

多铜氧化酶包括抗坏血酸氧化酶、漆酶、血浆铜蓝蛋白等多种类型,是植物体内非常重要的一类金属氧化酶,并在植物多种生理过程中发挥着举足轻重的作用。SKS(The skewed5simliar)蛋白是多铜氧化酶家族中一类缺乏铜离子连接所必需的组氨酸残基的特殊成员,由于缺失正常的多铜氧化酶酶活性中心,可能在植物发育中被赋予了新的功能。本文就多铜氧化酶铜离子连接位点、底物选择、演化过程以及植物SKS家族基因的研究进行了阐述。     

Nitrite-dependent vasodilation is facilitated by hypoxia and is independent of known NO-generating nitrite reductase activities

The reduction of circulating nitrite to nitric oxide (NO) has emerged as an important physiological reaction aimed to increase vasodilation during tissue hypoxia. Although hemoglobin, xanthine oxidase, endothelial NO synthase, and the bc(1) complex of the mitochondria are known to reduce nitrite anaerobically in vitro, their relative contribution to the hypoxic vasodilatory response has remained unsolved. Using a wire myograph, we have investigated how the nitrite-dependent vasodilation in rat aortic rings is controlled by oxygen tension, norepinephrine concentration, soluble guanylate cyclase (the target for vasoactive NO), and known nitrite reductase activities under hypoxia. Vasodilation followed overall first-order dependency on nitrite concentration and, at low oxygenation and norepinephrine levels, was induced by low-nitrite concentrations, comparable to those found in vivo. The vasoactive effect of nitrite during hypoxia was abolished on inhibition of soluble guanylate cyclase and was unaffected by removal of the endothelium or by inhibition of xanthine oxidase and of the mitochondrial bc(1) complex. In the presence of hemoglobin and inositol hexaphosphate (which increases the fraction of deoxygenated heme), the effect of nitrite was not different from that observed with inositol hexaphosphate alone, indicating that under the conditions investigated here deoxygenated hemoglobin did not enhance nitrite vasoactivity. Together, our results indicate that the mechanism for nitrite vasorelaxation is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation independently of the nitrite reductase activities investigated here. Possible reaction mechanisms for nitrite vasoactivity, including formation of S-nitrosothiols within the arterial smooth muscle, are discussed.     

NO synthesis and its regulation in the arachidonic-acid-stimulated rat polymorphonuclear leukocytes.

Nitric oxide (NO) synthesis and free radical generation from polymorphonuclear leukocytes (PMNs) play an important role in several pathological conditions. In the present study, regulation of NO synthesis has been investigated in the unstimulated and arachidonic-acid (AA)-stimulated rat PMNs. L-Citrulline formation or nitrite content was used as a marker of NO synthesis, while AA-induced free radical generation was assessed by flow cytometry using a dye, 2('),7(')-dichlorofluoreseindiacetate. L-Citrulline formation in the unstimulated PMNs increased in a time-dependent manner for up to 120 min. The increase was significantly less (25-55%) in AA-stimulated PMNs at all the time points. AA-induced free radical generation was maximum during the first 15 min followed by a time-dependent decrease. Interestingly, similar experiments under hyperoxic conditions did not exhibit any decrease in L-citrulline and nitrite formation after AA stimulation even though the free radical generation further increased. Scavenging or inhibition of free radicals by several types of interventions increased NO generation from AA-stimulated PMNs. The results of the present study suggest that the availability of oxygen, a common substrate for both NADPH oxidase and NOS, can inversely affect the synthesis of NO and PMNs seem to prefer oxygen utilization over NO synthesis for free radical generation.     

The catabolic fate of nitric oxide: the nitric oxide oxidase and peroxynitrite reductase activities of cytochrome oxidase

Stimulation of cardiomyocytes to endogenously evolve nitric oxide is shown by microsensor measurements on single cells to lead to transient nitric oxide concentrations of a few hundred nanomolar. At these submicromolar concentrations, no evidence could be found for the expected reaction between nitric oxide generated and the oxymyoglobin present in the cells: nitric oxide + oxymyoglobin --> nitrate + metmyoglobin. No metmyoglobin formation was detected by electron paramagnetic resonance spectroscopy, and microsensor measurements revealed near quantitative conversion of the nitric oxide to nitrite rather than nitrate ion. Moreover, the rate of nitrite formation is shown to be too rapid to be accounted for by non-enzymatic means. The essentially quantitative and rapid catabolism of nitric oxide to nitrite ion can plausibly be explained on the basis of a cycle of reactions catalyzed by cytochrome c oxidase. It is demonstrated with the purified hemoproteins in vitro that the terminal oxidase can outcompete oxymyoglobin for available nitric oxide. It is proposed that under normal physiological and most pathological (non-inflammatory) conditions, reaction with cytochrome c oxidase is the major route by which NO is removed from mitochondria-rich cells.     

Thiols enhance NO formation from nitrate photolysis

Nitrate is generally considered an inert oxidative breakdown product of nitric oxide (NO). Whereas it has been shown that limited amounts of NO are produced during the photolysis of nitrate in aqueous solution, the photochemistry of nitrate in biological matrices such as plasma is unknown. We hypothesized that thiols, which are ubiquitously present in biological systems, may significantly enhance NO-quantum yields from nitrate photolysis. Exposure of fresh human plasma to high-intensity UV-light resulted in NO-formation (19 +/- 3 nmol/l/min) as measured by gas phase chemiluminescence, and this signal was almost completely abolished by the removal of plasma N-oxides (2 +/- 1 nmol/l/min). Reconstitution of NOx-depleted plasma samples with a physiological concentration of nitrate, but not nitrite, restored photolytic NO-generation to values comparable to na?ve plasma. Addition of the thiol-reducing agent, dithiothreitol or the sulfhydryl-bearing amino acid, L-cysteine increased NO-formation above control levels. Thiol-blockade by either N-ethylmaleimide (NEM) or mercuric chloride (HgCl2) reduced basal NO formation from 19 +/- 3 to 7 +/- 2 and 4 +/- 1 nmol/l/min, respectively. Exposure of plasma to UV-light increased NO-adduct concentrations from 18 +/- 5 to 1662 +/- 658 nmol/l. Collectively, our results show that thiols facilitate photolytic conversion of nitrate to NO and NO-adducts such as S-nitrosothiols. This may lead to substantial overestimation of the latter when photolysis-based methodologies are used for their determination. Whether this novel reaction channel also has in vivo relevance remains to be investigated.     

Adaptation of the Griess reaction for detection of nitrite in human plasma

The determination of nitrite in human plasma or serum has been most frequently used as a marker of nitric oxide (NO) production. In addition, it has recently been suggested that nitrite could act as a vasodilating agent at physiological concentrations by NO delivery. Therefore, nitrite determination in biological fluids is becoming increasingly important. The most frequently used method to measure nitrite is based on the spectrophotometric analysis of the azo dye obtained after reaction with the Griess reagent. This method has some limitations regarding detection limit and sensitivity, thus resulting unsuitable for nitrite detection in plasma. We have identified some drawbacks and modified the original procedure to overcome these problems. By the use of the newly developed method, we measured 221±72 nM nitrite in human plasma from healthy donors.     

The role of nitrite in nitric oxide homeostasis: A comparative perspective

Nitrite is endogenously produced as an oxidative metabolite of nitric oxide, but it also functions as a NO donor that can be activated by a number of cellular proteins under hypoxic conditions. This article discusses the physiological role of nitrite and nitrite-derived NO in blood flow regulation and cytoprotection from a comparative viewpoint, with focus on mammals and fish. Constitutive nitric oxide synthase activity results in similar plasma nitrite levels in mammals and fish, but nitrite can also be taken up across the gills in freshwater fish, which has implications for nitrite/NO levels and nitrite utilization in hypoxia. The nitrite reductase activity of deoxyhemoglobin is a major mechanism of NO generation from nitrite and may be involved in hypoxic vasodilation. Nitrite is readily transported across the erythrocyte membrane, and the transport is enhanced at low O₂ saturation in some species. Also, nitrite preferentially reacts with deoxyhemoglobin rather than oxyhemoglobin at intermediate O₂ saturations. The hemoglobin nitrite reductase activity depends on heme O₂ affinity and redox potential and shows species differences within mammals and fish. The NO forming capacity is elevated in hypoxia-tolerant species. Nitrite-induced vasodilation is well documented, and many studies support a role of erythrocyte/hemoglobin-derived NO. Vasodilation can, however, also originate from nitrite reduction within the vessel wall, and at present there is no consensus regarding the relative importance of competing mechanisms. Nitrite reduction to NO provides cytoprotection in tissues during ischemia-reperfusion events by inhibiting mitochondrial respiration and limiting reactive oxygen species. It is argued that the study of hypoxia-tolerant lower vertebrates and diving mammals may help evaluate mechanisms and a full understanding of the physiological role of nitrite.     

eNOS gene T-786C polymorphism modulates atorvastatin-induced increase in blood nitrite

Statins inhibit cholesterol synthesis and produce pleiotropic, cholesterol-independent effects including endothelial NO synthase (eNOS) stimulation and increased expression. However, a functional polymorphism in the promoter of the eNOS gene (T-786C) reduces its activity and could modulate the response to statins. Here, we examined whether this polymorphism modulates the effects of atorvastatin on the plasma levels of markers of NO formation and oxidative stress. We genotyped 200 healthy subjects for this polymorphism, and 15 subjects with the TT genotype and 15 with the CC genotype were selected to receive placebo or atorvastatin 10 mg/day po for 14 days. To assess NO bioavailability, the plasma concentrations of nitrate, nitrite, and cGMP and the whole blood nitrite concentrations were determined after placebo or atorvastatin using an ozone-based chemiluminescence assay and an enzyme immunoassay. Thiobarbituric acid-reactive species (TBA-RS) were measured in the plasma to assess oxidative stress. Atorvastatin decreased cholesterol concentrations independent of genotype. Whereas atorvastatin produced no significant changes in plasma nitrite, nitrate, or cGMP concentrations in both genotype groups, atorvastatin increased whole blood nitrite concentrations and decreased plasma TBA-RS concentrations in the CC (but not in the TT) genotype group. These findings suggest that the T-786C polymorphism modulates the effects of atorvastatin on NO bioavailability and oxidative stress.     

The anoxic plant mitochondrion as a nitrite: NO reductase

Under the conditions of oxygen deprivation, accumulating nitrite can be reduced in the mitochondrial electron transport chain forming free radical nitric oxide (NO). By reducing nitrite to NO, plant mitochondria preserve the capacity to oxidize external NADH and NADPH and retain a limited power for ATP synthesis complementing glycolytic ATP production. NO participates in O(2) balance in mitochondria by competitively inhibiting cytochrome c oxidase which can oxidize it to nitrite when oxygen concentration increases. Some of the NO escapes to the cytosol, where the efficient scavenging system involving non-symbiotic hemoglobin oxygenates NO to nitrate and supports continuous anaerobic turnover of nitrogen species.     

Nitrite reductase activity of cytochrome c

Small increases in physiological nitrite concentrations have now been shown to mediate a number of biological responses, including hypoxic vasodilation, cytoprotection after ischemia/reperfusion, and regulation of gene and protein expression. Thus, while nitrite was until recently believed to be biologically inert, it is now recognized as a potentially important hypoxic signaling molecule and therapeutic agent. Nitrite mediates signaling through its reduction to nitric oxide, via reactions with several heme-containing proteins. In this report, we show for the first time that the mitochondrial electron carrier cytochrome c can also effectively reduce nitrite to NO. This nitrite reductase activity is highly regulated as it is dependent on pentacoordination of the heme iron in the protein and occurs under anoxic and acidic conditions. Further, we demonstrate that in the presence of nitrite, pentacoordinate cytochrome c generates bioavailable NO that is able to inhibit mitochondrial respiration. These data suggest an additional role for cytochrome c as a nitrite reductase that may play an important role in regulating mitochondrial function and contributing to hypoxic, redox, and apoptotic signaling within the cell.     

Reaction of nitric oxide with the turnover intermediates of cytochrome c oxidase: reaction pathway and functional effects

The reactions of nitric oxide (NO) with the turnover intermediates of cytochrome c oxidase were investigated by combining amperometric and spectroscopic techniques. We show that the complex of nitrite with the oxidized enzyme (O) is obtained by reaction of both the "peroxy" (P) and "ferryl" (F) intermediates with stoichiometric NO, following a common reaction pathway consistent with P being an oxo-ferryl adduct. Similarly to chloride-free O, NO reacted with P and F more slowly [k approximately (2-8) x 10(4) M(-1) s(-1)] than with the reduced enzyme (k approximately 1 x 10(8) M(-1) s(-1)). Recovery of activity of the nitrite-inhibited oxidase, either during turnover or after a reduction-oxygenation cycle, was much more rapid than nitrite dissociation from the fully oxidized enzyme (t(1/2) approximately 80 min). The anaerobic reduction of nitrite-inhibited oxidase produced the fully reduced but uncomplexed enzyme, suggesting that reversal of inhibition occurs in turnover via nitrite dissociation from the cytochrome a(3)-Cu(B) site: this finding supports the hypothesis that oxidase may have a physiological role in the degradation of NO into nitrite. Kinetic simulations suggest that the probability for NO to be transformed into nitrite is greater at low electron flux through oxidase, while at high flux the fully reduced (photosensitive) NO-bound oxidase is formed; this is fully consistent with our recent finding that light releases the inhibition of oxidase by NO only at higher reductant pressure [Sarti, P., et al. (2000) Biochem. Biophys. Res. Commun. 274, 183].     

Human neuroglobin functions as a redox-regulated nitrite reductase

Neuroglobin is a highly conserved hemoprotein of uncertain physiological function that evolved from a common ancestor to hemoglobin and myoglobin. It possesses a six-coordinate heme geometry with proximal and distal histidines directly bound to the heme iron, although coordination of the sixth ligand is reversible. We show that deoxygenated human neuroglobin reacts with nitrite to form nitric oxide (NO). This reaction is regulated by redox-sensitive surface thiols, cysteine 55 and 46, which regulate the fraction of the five-coordinated heme, nitrite binding, and NO formation. Replacement of the distal histidine by leucine or glutamine leads to a stable five-coordinated geometry; these neuroglobin mutants reduce nitrite to NO ～2000 times faster than the wild type, whereas mutation of either Cys-55 or Cys-46 to alanine stabilizes the six-coordinate structure and slows the reaction. Using lentivirus expression systems, we show that the nitrite reductase activity of neuroglobin inhibits cellular respiration via NO binding to cytochrome c oxidase and confirm that the six-to-five-coordinate status of neuroglobin regulates intracellular hypoxic NO-signaling pathways. These studies suggest that neuroglobin may function as a physiological oxidative stress sensor and a post-translationally redox-regulated nitrite reductase that generates NO under six-to-five-coordinate heme pocket control. We hypothesize that the six-coordinate heme globin superfamily may subserve a function as primordial hypoxic and redox-regulated NO-signaling proteins.     

Nitric oxide production from nitrite occurs primarily in tissues not in the blood: critical role of xanthine oxidase and aldehyde oxidase

Recent studies have shown that nitrite is an important storage form and source of NO in biological systems. Controversy remains, however, regarding whether NO formation from nitrite occurs primarily in tissues or in blood. Questions also remain regarding the mechanism, magnitude, and contributions of several alternative pathways of nitrite-dependent NO generation in biological systems. To characterize the mechanism and magnitude of NO generation from nitrite, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The addition of nitrite triggered a large amount of NO generation in tissues such as heart and liver, but only trace NO production in blood. Carbon monoxide increased NO release from blood, suggesting that hemoglobin acts to scavenge NO not to generate it. Administration of the xanthine oxidase (XO) inhibitor oxypurinol or aldehyde oxidase (AO) inhibitor raloxifene significantly decreased NO generation from nitrite in heart or liver. NO formation rates increased dramatically with decreasing pH or with decreased oxygen tension. Isolated enzyme studies further confirm that XO and AO, but not hemoglobin, are critical nitrite reductases. Overall, NO generation from nitrite mainly occurs in tissues not in the blood, with XO and AO playing critical roles in nitrite reduction, and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations.     

Recent methodological advances in the analysis of nitrite in the human circulation: nitrite as a biochemical parameter of the L-arginine/NO pathway

Nitric oxide (NO) plays a pivotal role in the modulation of multiple physiological processes. It acts as a messenger molecule within the cardiovascular system. NO is a highly unstable free radical in circulating blood and is oxidized rapidly to nitrite and nitrate. Recent studies suggest that nitrite has the potential to function as a surrogate of NO production under physiological and pathophysiological conditions and could therefore be of high relevance as a biochemical parameter in experimental and clinical studies. Under hypoxic conditions nitrite is reduced to bioactive NO by deoxyhemoglobin. This mechanism may represent a dynamic cycle of NO generation to adapt the demand and supply for the vascular system. Because of these potential biological functions the concentration of nitrite in blood is thought to be of particular importance. The determination of nitrite in biological matrices represents a considerable analytical challenge. Methodological problems often arise from pre-analytical sample preparation, sample contamination due to the ubiquity of nitrite, and from lack of selectivity and sensitivity. These analytical difficulties may be a plausible explanation for reported highly diverging concentrations of nitrite in the human circulation. The aim of this article is to review the methods of quantitative analysis of nitrite in the human circulation, notably in plasma and blood, and to discuss pre-analytical and analytical factors potentially affecting accurate quantification of nitrite in these human fluids.     

Bound NO in human red blood cells: fact or artifact?

There has been considerable debate over the nature and chemistry of the interaction between nitric oxide (NO) and red blood cells (RBCs), in particular whether hemoglobin consumes or conserves NO bioactivity. Given the vast range of nitrosation levels reported for human RBCs in the literature, we sought to investigate whether there was a common denominator that could account for such discrepancies across different methodologies and reaction conditions and if such a pathway may exist in physiology. Here, we show that there are marked differences in reactivity toward NO between human and rat hemoglobin, which offers a mechanistic explanation for why basal levels of NO-adducts in primate RBCs are considerably lower than those in rodents. We further demonstrate that the inadvertent introduction of trace amounts of nitrite and incomplete thiol alkylation lead to rapid heme and thiol nitros(yl)ation, with generation of nitrosylhemoglobin (NOHb) and S-nitrosohemoglobin (SNOHb), while neither species is detectable in human RBCs at physiological nitrite concentrations. Thus, caution should be exercised in interpreting experimental results on SNOHb/NOHb levels that were obtained in the absence of knowledge about the degree of nitrite contamination, in particular when a physiological role for such species is implicated.     

Interaction of nitric oxide with ceruloplasmin lacking an EPR-detectable type 2 copper.

Nitric oxide (NO) has previously been reported to modify the EPR spectrum of multicopper blue oxidases, disclosing a pure type 2 copper and inducing half-field transitions at g = 4. In the present work the reactivity of NO was reinvestigated with respect to ceruloplasmins having an apparently EPR-silent type 2 copper in their native state. The optical properties of NO-treated ceruloplasmin were independent of the initial redox state of the metal sites. Addition of NO caused the absorption at 600 nm to decrease in the case of oxidized ceruloplasmin and to increase when starting from the reduced proteins. In this latter case the absorbance at 330 nm was also restored, indicating that NO was able to reoxidize the reduced protein. In all cases the band at 600 nm leveled to ca. 60% of the intensity of the native untreated protein, and new bands below 500 nm appeared in the spectra. While the blue absorption band was restored by removal of NO, the absorbance below 500 nm remained higher even after dialysis. The EPR spectrum resulting from reaction of NO with either oxidized, partially reduced, or fully reduced ceruloplasmin consisted in all cases of a broad, structureless resonance around g = 2. NO caused the reversible disappearance of the type 1 copper EPR spectrum in oxidized ceruloplasmin. Also, the transient novel copper signal that arises during the anaerobic reduction process by ascorbate completely disappeared in the presence of NO and did not reappear upon removal of the gas.(ABSTRACT TRUNCATED AT 250 WORDS)     

Iron catalyzed conversion of NO into nitrosonium (NO+) and nitroxyl (HNO/NO-) species.

The conversion of NO into its congeners, nitrosonium (NO+) and nitroxyl (HNO/NO-) species, has important consequences in NO metabolism. Dinitrosyl iron complex (DNIC) combined with thiol ligands was shown to catalyze the conversion of NO into NO+, resulting in the synthesis of S-nitrosothiols (RSNO) both in vitro and in vivo. The formation mechanism of DNIC was proposed to involve the intermediate release of nitroxyl. Since the detection of hydroxylamine (as the product of a rapid reaction of HNO/NO- with thiols) is taken as the evidence for nitroxyl generation, we examined the formation of hydroxylamine, RSNO, and nitrite (the product of a rapid reaction of NO+ with water) in neutral solutions containing iron ions and thiols exposed to NO under anaerobic conditions. Hydroxylamine was detected in NO treated solutions of iron ions in the presence of cysteine, but not glutathione (GSH). The addition of urate, a major "free" iron-binding agent in humans, to solutions of GSH and iron ions, and the subsequent treatment of these solutions with NO increased the synthesis of GSNO and resulted in the formation of hydroxylamine. This caused a loss of urate and yielded a novel nitrosative/nitration product. GSH attenuated the urate decomposition to such a degree that it could be reflected as the function of GSH:urate. Results described here contribute to the understanding of the role of iron ions in catalyzing the conversion of NO into HNO/NO- and point to the role of uric acid not previously described.     

Nitric oxide and mitochondrial complex IV

Micromolar nitric oxide (NO) rapidly (ms) inhibits cytochrome c oxidase in turnover with physiological substrates. Two reaction mechanisms have been identified leading, respectively, to formation of a nitrosyl- [a3(2+) -NO] or a nitrite- [a3(3+) -NO2-] derivative of the enzyme. In the presence of O2, the nitrosyl adduct recovers activity slowly, following NO displacement at k' approximately equal to 0.01 s(-1) (37 degrees C); the recovery of the nitrite adduct is much faster. Relevant to pathophysiology, the enzyme does not degrade NO by following the first mechanism, whereas by following the second one it promotes NO oxidation and disposal as nitrite/nitrate. The reaction between NO and cytochrome c oxidase has been investigated at different integration levels of the enzyme, including the in situ state, such as in mouse liver mitochondria or cultured human SY5Y neuroblastoma cells. The respiratory chain is inhibited by NO, either supplied exogenously or produced endogenously via the NO synthase activation. Inhibition of respiration is reversible, although it remains to be clarified whether reversibility is always full and how it depends on concentration of and time of exposure to NO. Oxygraphic measurements show that cultured cells or isolated state 4 mitochondria exposed to micromolar (or less) NO recover from NO inhibition rapidly, as if the nitrite reaction was predominant. Mitochondria in state 3 display a slightly more persistent inhibition than in state 4, possibly due to a higher accumulation of the nitrosyl adduct. Among a number of parameters that appear to control the switch over between the two mechanisms, the concentration of reductants (reduced cytochrome c) at the cytochrome c oxidase site has been proved to be the most relevant one.