A: Deoxy hemoglobin (deoxyHb)
In 1937 J. Brooks (109
) showed that deoxygenated hemoglobin (deoxyHb, Fe2+
Hb) reacts with nitrite to form equimolar concentrations of methemoglobin (metHb, Fe3+
Hb) and ferrous nitrosyl hemoglobin (NO-Fe2+
Hb). In 1981, Michael Doyle and colleagues investigated the mechanism and kinetics of this reaction (110
). From the pH dependence of the reaction they hypothesized that nitrous acid HNO2
be involved. They proposed the following set of reactions:
However, Doyle reported a complicated stoichiometery where the ratio metHb: NO-Fe2+
Hb was about 5:2. However, in 2005 it was demonstrated that traces of oxygen have a pronounced effect on the stoichiometry of the reaction, in particular on the balance between metHb and NO-Fe2+
Hb. A truly 1:1 ratio is only achieved with careful exclusion of all oxygen from the samples (111
). Remaining traces of oxygen lead to formation of oxyHb which reacts very rapidly with free NO to metHb and nitrate (see section 9.A). This additional pathway favors formation of metHb, and increases its yield over that of NO-Fe2+
Hb. The kinetics of the truly anoxic reactions was carefully studied (111
), and the results were quite surprising:
predicts pseudo-first order kinetics in [Fe2+
Hb] when nitrite is in excess. However, the experimental timecurves of [Fe2+
Hb] suggest zero-order kinetics and actually have sigmoidal character, with the rate being slowest at the beginning and end of the reaction (). The sigmoidal character of the kinetic trace is evident when the instantaneous rate is plotted as a function of time (). Interestingly, the reaction is fastest about halfway through the reaction. The surprising kinetics of this reaction are explained by the freedom of Hb tetramers to exist in two quaternary conformations (R or T geometry) that have differing binding affinities and reaction rates for small ligands like oxygen or nitrite. Unliganded ferrous heme in the relaxed (high oxygen affinity) R-state quaternary form reacts with nitrite approximately sixty times faster than heme in the tense form (low oxygen affinity, T-state) (113
). In absence of ligands, the T-state is more stable than R-state. Accordingly, the Hb tetramers exist in T-state with low reactivity just before the nitrite is added. Therefore, the nitrite consumption remains slow although the number of vacant ferrous heme sites for nitrite binding is highest. As the reaction proceeds, the number of vacant ferrous heme sites is depleted which slows the reaction down. However, some of the Hb tetramers are converted to the R-state due to formation of metHb and NO- Fe2+
Hb. This conversion to highly reactive R-state accelerates the reaction. The balance of these two counteracting influences results in a sigmoidal kinetics as shown in . We note that consumption of one nitrite molecule results in the formation of one ferric heme (metHb) and one NO-Fe2+
Hb, introducing an element of autocatalysis in the reaction, since one nitrite affects the conformation of two Hb tetramers.
Figure 1 Kinetics of the reaction between human deoxyHb (50 μM) and nitrite (10 mM) at pH 7.4 and 37° C. (A) UV/VIS absorption spectra were deconvoluted to determine the percentage of each species as a function of time. DeoxyHb is observed to form (more ...)
The hypothesis of allosteric autocatalysis predicts that the rate of the reaction between nitrite and hemoglobin be dependent on the oxygen tension. As the Hb oxygen saturation is increased, the number of available ferrous hemes decreases (slowing the reaction down), but the number of available ferrous hemes that are in the R-state increases (speeding the reaction up). This prediction was experimentally confirmed in , where the initial reaction rate shows a sigmoidal dependence on oxygen level. The figure confirms that the highest initial reaction rate occurs for oxygen levels near the half-loading point P50
of human Hb (111
This finding has three important implications for physiology:
- Hemoglobin functions as a mammalian nitrite reductase whose activity is controlled by the ambient oxygen level. The highest rate of reduction is reached for [O2] ~ 35 μM, corresponding to P50 of human adult Hb (cf ).
- Because hemoglobin deoxygenates under physiological conditions, hemoglobin shows nitrite reductase activity over a wide range of oxygen tensions that are higher than the oxygen tensions required to reduce nitrite by other enzymes, except XO (cf fig 10). illustrates the allosteric nature of the hemoglobin nitrite reductase activity.
Figure 2 Oxygen-dependence of nitrite reductase activity of hemoglobin. As oxygen tension increases the amount of deoxyHb (blue trace) decreases while the amount of R-state Hb (and free hemes in R-state Hb tetramers, red trace) increases. The nitrire reductase (more ...)
- Because the R state of hemoglobin is the fastest nitrite reductase (highest bimolecular rate constant for nitrite reduction is in the R-state tetramer) the formation of NO from the nitrite-hemoglobin reaction should be most efficient during rapid deoxygenation from artery to vein. Under these conditions the R-state (oxygenated) tetramer releases oxygen to expose deoxygenated heme sites on R-state molecules (R3 and R2 tetramers) (7).
The above describes the reaction of nitrite with deoxyHb. However, in presence of oxygen an alternative pathway exists: nitrite also reacts with oxyHb to form metHb and nitrate. Since Hb is partially saturated in vivo, Grubina et al (114
) studied the reaction of nitrite with Hb at varying oxygen tensions and found that the reactions with oxyHb and deoxyHb proceed simultaneously under these conditions. At the beginning of the reaction, deoxyHb is consumed much faster than oxyHb. In fact, the reaction of nitrite with deoxyHb partially inhibits the reaction of nitrite with oxyHb. The autocatalytic phase of the oxyHb reaction is inhibited by the presence of deoxyHb and products of the deoxyHb/nitrite reaction, namely NO-Fe2+
Hb. Interestingly, intermediates in the oxyHb/nitrite reaction, probably NO2•
, oxidize NO-Fe2+
Hb and release NO in a process called oxidative denitrosylation (114
). These results demonstrate that the reaction of nitrite with oxyHb is limited under physiological conditions, yet its occurrence can also facilitate NO release from NO-Fe2+
Hb, provided that there is sufficient compartmentalization of these chemistries within the RBC. More details on the reaction of nitrite with oxyHb will be given in section 9.A.
The above reactions were investigated in vitro
. The three implications for mammalian physiology were also tested in more biological models like aortic ring bioassays (22
). In this context a careful distinction should be made between the vasodilating effects of free Hb and intact RBC’s. Deoxygenation of RBC’s induces the release of ATP. This by itself may cause vasodilation by activation of purinergic P2γ
receptors and eNOS (22
). This alternative pathway is independent of nitrite.
It has been noted that the combination of nitrite with RBC is particularly effective to elicit hypoxic vasodilation. RBCs in the presence of 2 μM nitrite resulted in vessel relaxation at much higher oxygen tensions than nitrite or RBCs alone (26
). These vasodilations do not require high nitrite levels: It has been shown that 200 nM nitrite could relax rat and rabbit thoracic aortic rings in the presence (but not in the absence) of 25 μM deoxyHb (22
). The vasodilating potency of nitrite infusions was assessed in humans in Ref (82
). It was found that enhancement of venous nitrite by only 300 nM enhanced the forearm blood flow significantly. Such values are in the normal physiological range of blood nitrite (cf section 2.).
A number of observations suggest that this vasodilation be mediated by free NO radicals: Firstly, Nitrite/RBC-dependent vasorelaxation was shown to coincide with increased cGMP levels, and could be inhibited by the NO scavenger C-PTIO (22
). Moreover, the efficiency of vasorelaxation was shown to have the same dependence on oxygen tension as the nitrite reductase activity of human Hb, as shown in and illustrated in (22
). A further indication of the release of free NO was the inhibition of mitochondrial respiration by nitrite in the presence, but not in the absence, of RBCs. These data strongly suggests that Hb in RBC release sufficient NO from nitrite to achieve vasodilation under physiological conditions.
A major conceptual obstacle to NO signalling via nitrite reduction by Hb in RBC is the low probability that NO escape from the intracellular compartment. Numerical simulations of the diffusion process suggest that NO cannot leave the RBC since the reaction with oxyHb is very fast (diffusion limited (110
). From the experimental lifetime and diffusion rate, it is estimated that the diffusion length of NO is of the order of 0.02 μm. This distance is far smaller than the cellular dimensions. Therefore, free NO radicals could not escape the cell (120
). Computer simulations predicted that even with a high therapeutic dose of nitrite (200 μM), only 0.1 picomolar of NO generated from the reaction of nitrite with Hb would reach the smooth muscle cells at steady state; this quantity is far below the activation threshold for vasodilation (121
The explanation for this paradox may lie in the nature of the species escaping from the RBC: it is possible that it is not NO per se
, but some more stable intermediate neutral species, such as N2
. This highly polar molecule easily reacts with water, but is thought to be a major agent for S-nitrosation of thiol residues in biological systems (cf chapter 1 of (33
)). It was proposed that this intermediate could diffuse out of the RBC release free NO in the extracellular space, possibly via S-nitrosated intermediates (121
). Interestingly, a mechanism for N2
formation from the nitrite/deoxyHb reaction was recently proposed (123
). This nitrite reductase/anhydrase activity of hemoglobin is illustrated in . A key player in this mechanism is nitrite bound metHb. Surprisingly, it was found that nitrite-metHb has no signal in electron paramagnetic resonance (EPR), indicating a peculiar electronic structure that, by density functional theory calculations, was shown to include some Fe2+
). This species reacts quickly with NO to form N2
. As suggested by the illustration in , metHb can be formed by the reaction of nitrite with deoxyHb and/or the reaction with oxyHb. NO is also formed by the deoxyHb/nitrite reaction as well as by oxidative denitrosylation at various levels of oxygen. Thus, initially, metHb could build up under high oxygen tension. Subsequently, as the oxygen tension is lowered, N2
would be produced. The mechanism is illustrated in . Together with the discussion above, it suggests that Hb is an allosterically controlled nitrite reductase/anhydrase and the overall stoichiometry of the set of reactions is
Figure 3 The N2O3 forming reaction of nitrite and hemoglobin may regulate the export of NO from the erythrocyte. Hemoglobin deoxygenation (purple) occurs preferentially at the submembrane of the red blood cell as it traverses the arteriole. Nitrite reacts with (more ...)
demonstrating the catalytic nature of the ferrous Hb protein.
In order for nitrite-metHb to react with NO, it must compete with reactions of oxyHb and deoxyHb. The relative yields of these three competing pathways depends on reaction rates, starting concentrations and cellular compartment. These kinetic challenges cannot be surmounted by the rate of the nitrite-metHb/NO reaction alone, however the reaction can be inefficient. Since NO has high potency as a vasodilator (EC50 ~ 5 nM), very little nitrite must be reduced and only small quantities of NO need to escape the red cell to exert physiological effects. Future work will elucidate the nature and extent of any intraerythrocytic compartmentalization that could promote the formation of N2O3.
The monomer myoglobin (Mb) is a small (17.6 kDa) but important intracellular oxygen binding heme protein. Under basal conditions the tissue is well oxygenated and Mb predominantly exists in the oxygenated state (cf ). Therefore, it has long been accepted as an intracellular oxygen store. In exercising skeletal muscle and in the beating heart, Mb serves as a short-term oxygen reservoir, tiding the muscle over from one contraction to the next. Similarly, Mb is expressed in high concentrations in skeletal muscle of mammals and humans adapted to high altitudes (126
). More controversially, a potential role of Mb in intracellular oxygen diffusion within muscle cells has been considered (124
The Mb concentration various between mammalian species (124
) and between tissue type: Mb is highly expressed in type I and IIa skeletal muscle fibers, in cardiac and tongue muscles, and to a lesser extent in smooth muscle cells (124
). In human cardiac tissue, Mb levels are ca 200 – 300 μmol/kg wet tissue, whereas skeletal muscles reach concentrations of ca 400 – 500 μmol/kg wet tissue (124
). The Mb contents of different skeletal muscles in man and rats are tabulated in ref (129
). In diving mammals, the Mb concentrations reach ca 2 mmol/kg wet tissue. This value is about tenfold higher than in terrestrial mammals and serves as an O2
store contributing to the extension of diving time (125
The role of Mb in vivo
has been investigated by comparison of wild type mice with Mb deficient mutants (Mb−/−
). In these mutants, multiple compensatory mechanisms were activated to compensate for the loss of the oxygen-storage and –transport function of Mb. These included increased capillary density, elevated hemoglobin levels, increased coronary flow, and a switch in cardiac substrate utilization from fatty acid to glucose (132
). In addition, oxyMb is a highly effective scavenger of free NO radicals. Therefore, the NO status of cardiac tissue was investigated in this mutant.
Whereas the role of endogenous NO for myocardial function is still a subject of significant controversy, a it was postulated (133
) that the effect of NO depends on its concentration: A positive inotropic effect at low concentrations and a negative one at higher concentrations. Similar concentration-dependent effects of NO play a role in the modulation of transduction of the parasympathetic effects of cholinergic stimulation, in attenuation of oxygen consumption, and in apoptosis of cardiomyocytes.
Depending on ambient oxygen level, Mb acts either as an NO-scavenger under normoxic conditions or as a nitrite-reductase under conditions of hypoxia and ischemia (134
) (cf. ). Because Mb must be at least partially deoxygenated to act as nitrite-reductase, the latter reaction pathway can become significant only when the oxygen level falls below the P50
of myoglobin (ca 3 Torr, equivalent to a free oxygen concentration of ca 4 μM, cf ). In contrast, the detrimental effects of overproduction of NO radicals in heart tissue may be prevented by the dioxygenase function of oxygenated myoglobin (135
). Mb efficiently protects the respiratory chain against nitrosative stress from NO radicals (137
). Experiments in Mb knockout mice confirmed (134
) that the presence of Mb has a significant effect on NO levels in cardiac tissue: In hearts from Mb−/−
mice, endogenous and exogenous NO were more effective in the regulation of coronary tone and myocardial contractility. It suggests that the hearts of Mb−/−
mutants have higher NO levels than the hearts from WT mice.
Figure 4 Depending on ambient oxygen, myoglobin acts as a dioxygenase or as a nitrite-reductase. Under normoxia, oxymyoglobin acts as an NO-scavenger, protecting the mitochondria from inhibition by NO) (left). Under hypoxia, myoglobin changes its function from (more ...)
The oxygen levels in the left ventricle of beating dog hearts were measured electrochemically (74
) and show a wide distribution of values between zero and venous levels (cf ). The distribution suggests that certain regions of ventricular tissue have very low oxygen pressures below 5 Torr, offering the physiological prerequisite for the role of Mb as a functionally relevant nitrite-reductase. The oxygen binding curve of Mb is a hyperbolic curve with a half-loading pressure of P50
~2.75 Torr (cf ). This value is an order of magnitude lower than P50
of the sigmoid-shaped binding curve of Hb. It allows Mb to take up oxygen from Hb and to load and unload oxygen in the range of the pO2
values that occur within the cell. Although oxyMb limits NO bioavailability in tissues due to its rapid reaction with NO, under hypoxic conditions, the myoglobin-dependent nitrite reduction may provide a mechanism by which NO is generated to regulate the physiological functions under conditions where the arginine to citrulline conversion by NOS is oxygen-limited. The redox potential of Mb is lower than R-state Hb and deoxyMb was found to reduce nitrite and generate NO at a faster rate than deoxyHb (112
) with a bimolecular rate constant of 12 (Ms)−1
at 37°C (24
). In isolated cardiomyocytes the nitrite reductase activity of deoxyMb releases NO in proximity to mitochondria and regulates mitochondrial respiration through cytochrome c oxidase (24
Recent studies showed (134
) that this NO interacts reversibly with myocytic cytochromes and down-regulates cardiac mitochondrial activity. This leads to a reduction in oxygen consumption and consecutively also of cardiac contractility (134
). Cardiac contractile function and energy metabolism are actively downregulated, when coronary blood supply is critically reduced. On acute restriction of coronary artery inflow, the contractile function of the ischemic region is rapidly decreased and the oxygen consumption is reduced. This dampens the fall in high energy phosphates and over time even can restore myocardial energy balance. This adaptive response is referred to as “short-term hibernation” (138
A very similar response of the metabolic system was observed upon nitrite infusion in mice. These infusions led to a marked decrease in phosphocreatine (PCr), together with an increase in inorganic phosphate and a reduction of the available driving force for all energy-consuming processes (ΔGATP
). Simultaneously, the infusion of nitrite reduced the synthesis and utilization of ATP. By implication,, the reduction of endogenous nitrite to NO by deoxyMb may be significant for such “short-term hibernation” as observed upon restriction of coronary arterial flow. The presented experiments were carried out under hypoxic perfusion conditions which cause Mb to be deoxygenated by about 50%. Severe low-flow ischemia certainly can lower tissue pO2
even further, thereby further augmenting the ability of deoxyMb to form NO from nitrite (138
These data suggest that the mechanisms may be relevant under physiologic conditions and at physiological nitrite levels. Typical endogenous nitrite levels in Wistar rats are ca 0.3 μM in plasma, ca 0.8 μM in cardiac tissue, and up to 20 μM in aortic tissue (48
) (for a more comprehensive discussion of endogenous nitrite levels, cf section 2.). Although high extracellular concentrations of nitrite (10 to 100 μmol/L) were required to elicit the biological response, it is the intracellular concentration of nitrite which is of critical importance for the reaction with deoxyMb. Pretreatment of animals with the NOS-inhibitor L-NIO decreased cytosolic nitrite by appromimately 70%, and perfusion with concentrations ≥ 10μmol/L nitrite was required to replenish the myocytic levels to the range of untreated controls (138
). Obviously, comparatively high extracellular nitrite concentrations have to be applied under our experimental conditions to mimic the in vivo conditions with unrestricted activity of NOS and unlimited availability of its substrate L-arginine (the latter was deliberately not supplemented with the perfusion buffer). Together, these data suggest that endogenous levels of intracellular nitrite be sufficient to affect cardiac function upon imposition of hypoxia.
Recent results (139
) showed that deoxyMb acted as a functional nitrite reductase that generatedf NO and downregulated cellular respiration. This beneficial cascade is a cytoprotective response to cardiac ischemia-reperfusion (I/R) injury. Myoglobin was found responsible for nitrite-dependent NO generation and cardiomyocyte protein iron-nitrosylation. Nitrite reduction to NO by myoglobin dynamically inhibits cellular respiration and limits reactive oxygen species generation and mitochondrial enzyme oxidative inactivation after I/R injury. In vivo
administration of nitrite reduced myocardial infarction by 60 % in myoglobin+/+
mice, whereas similar administration of nitrite had no protective effects in myoglobin−/−
mutants. These data support an emerging paradigm that myoglobin subserves a critical function as an intrinsic nitrite reductase that regulates responses to cellular hypoxia and reoxygenation.
The preceding discussion considered the relevance of deoxyMb for cardiac tissue. The same mechanism could also contribute to hypoxic vasodilation described for the human circulation (26
). The oxygenation state of Mb has been studied in vivo
H NMR spectroscopy. The deoxy fraction of Mb in skeletal muscle of healthy humans was found to be 9 % at rest (140
). Upon exercise (50–60 % of maximum work rate) the deoxyMb fraction increased to about 50% corresponding to an intracellular pO2
less than 5 Torr (141
). According to our definitions of , this oxygen pressure represents deep hypoxia.
The exact role and the potential impact of deoxyMb as a nitrite-reductase in physiology and pathophysiology remain an important area for future studies.
C: Xanthine Oxidase (XO)
Xanthine oxidase (XO) is a ubiquitous enzyme in mammalian cells that is involved in the catabolism of purine and pyrimidines, oxidizing hypoxanthine to xanthine and xanthine to uric acid. XO also reduces oxygen to superoxide (O2•−
) and hydrogen peroxide (H2
) and is one of the key enzymes responsible for superoxide-mediated cellular injury. Interestingly, XO has structural similarity to some bacterial nitrate or nitrite reductases (142
It has been established that XO can reduce nitrite to NO (17
). It was shown that NADH (17
) and xanthine (144
) can donate electrons to XO and catalyze the reduction of nitrite. The kinetics of the anaerobic reaction were subsequently studied with EPR spectroscopy, chemiluminescence NO analyzers, and NO electrodes (31
). Each of the typical reducing substrates for xanthine, 2,3-dihydroxybenz-aldehyde (DBA), and NADH can act as electron donors to support this XO-mediated nitrite reduction. Moreover, the reaction was inhibited by oxypurinol, a specific ligand for reduced Mo4+
as in the catalytic site of XO. It suggests that reduced XO was the direct electron donor to nitrite, with nitrite binding and reduction occurring at the molybdenum site (31
). Whereas NADH-stimulated NO generation was inhibited by the flavin modifier DPI, NO generation stimulated by xanthine or DBA was unaffected. Thus, whereas xanthine or DBA directly reduce the molybdenum center, NADH initially reduces the flavin, which subsequently transfers electrons to the molybdenum.
The binding constant of nitrite was found as Km
= 2.4 ± 0.2 mM, and did not depend on the substrate (NADH, xanthine, or 2,3-dihydroxybenz-aldehyde). The three substrates were distinguished by markedly different binding constants: The Km
= 878 μM for NADH, 1.5 μM for xanthine, 35 μM for DBA (all in the presence of 1 mM nitrite (31
)). Although xanthine was the most efficient substrate for XO-catalyzed nitrite reduction, excessive xanthine inhibited the release of NO (31
Nitrite reduction to NO occurs at the molybdenum site, with either NADH or xanthine serving as reducing substrates. Diphenyleneiodonium (DPI), which acts at the FAD site, inhibited XO dependent nitrite reduction by NADH but not from xanthine. This suggests that NADH donates electrons to FAD, and then electrons are transported back to reduce the Mo that in turn reduces nitrite to NO. When xanthine or aldehydes are the electron donors, both XO reduction (by xanthine or aldehydes) and oxidation (by nitrite) takes place at the Mo site of the enzyme. This explains why only oxypurinol could inhibit XO dependent NO formation.
It has been reported that hydroxylation of the purine and aldehyde substrates takes place via a base-catalyzed mechanism and that substrate must be protonated for hydroxylation (147
). The rate of XO reduction by purine and aldehydes greatly increases when the pH is increased from 6.0 to 8.0, and this increased rate of XO reduction will lead to an increased rate of nitrite reduction. However, acidification from pH 8.0 to 6.0 accelerated XO-catalyzed nitrite reduction (cf ). It suggests that nitrite reduction takes place via an acid-catalyzed mechanism, presumably due to nitrite protonation. HNO2
concentration increases when the pH decreases, and it could be the direct binding substrate of XO. Although lowering pH would decrease the rate of XO reduction by reducing substrates, it would greatly accelerate the oxidation of XO by nitrite/HNO2
Effect of pH and substrate on the release of NO by 0.02 mg/ml XO. The nitrite concentration is fixed at 1 mM. The rates of release are in nmol·mg−1·s−1
The levels of tissue nitrite and enzyme reducing substrates have a critical role in controlling the reaction. Nitrite is the limiting substrate, given the high value of Km
~2.5 mM. This number exceeds typical tissue levels of nitrite by at least 2 orders of magnitude (cf section 2). Therefore, any enhancement of tissue nitrite by, for example, activation of constitutive or inducible NOS in inflammatory conditions, dietary sources, pharmacological sources, or bacterial sources, could all modulate this pathway of NO generation (148
). This pathway also requires a reducing substrate, such as NADH or xanthine. Xanthine was the most effective substrate, triggering NO generation under anaerobic conditions with a Vmax
4-fold higher than that of NADH (31
) and Km
~ 1.5 μM (31
). Excess xanthine, above 20 μM, results in prominent inhibition. If particularly high levels of xanthine accumulate, this pathway would be inhibited, and perhaps this may serve a regulatory role to prevent overproduction of NO. Under anaerobic conditions, XO reduces nitrite to NO at the molybdenum site of the enzyme with xanthine, NADH, or aldehyde providing the necessary electrons. It makes XO an alternative source of NO under ischemic conditions when NO production from NOS is impaired.
While XO-mediated reduction of nitrite and nitrate occurs under conditions of limited tissue perfusion and resulting acidosis, questions remain regarding whether XO-mediated NO generation also occurs in the presence of oxygen. In mammalian organs under normoxic resting conditions, the O2
concentration ranges from ca 130 μM) in arterial blood to ca 50 μM in the myocardium (cf and ). During mild hypoxia, myocardial O2
levels drop below 20 μM (cf ). Therefore, studies have been performed to measure the magnitude and kinetics of XO-mediated NO formation under different oxygen tensions (156
All three typical reducing substrates of XO induced release of NO under hypoxia; however, their kinetics are quite different in the presence of molybdenum-site binding substrates xanthine or DBA, compared with that of the FAD-site binding substrate NADH. With xanthine or DBA as reducing substrates, the rate of NO production followed typical Michaelis-Menten kinetics, with oxygen acting as a strong competitive inhibitor. Under aerobic conditions, with xanthine or DBA as reducing substrates, XO-mediated NO production is less than the 10% of NO production under anaerobic conditions (156
). With the FAD site binding reducing substrate, NADH, as electron donor, XO-mediated NO production is maintained at more than 70% of anaerobic levels. With NADH, under aerobic conditions, XO-mediated nitrite reduction did not follow Michaelis-Menten kinetics. NADH serves as electron donor to XO at the FAD site, the same site as that for oxygen binding, whereas nitrite reduction takes place at the molybdenum site of the enzyme (156
). With NADH as reducing substrate, XO-mediated NO generation may occur through two processes as shown in .
Depending on the substrate, nitrite may be reduced to free NO at the Molybdenum site of the XO enzyme. Process I is progressively inhibited by oxygen. Process II continues to operate even under normoxia.
In Process I, XO starts in reduced state. With FAD site free, XO can pass its electron to either oxygen or nitrite. Thus, under aerobic conditions, oxygen is a strong competitive inhibitor to reduction of nitrite. Therefore, Process I is inhibited by the presence of oxygen. Process II is different in that the FAD site is occupied by the NADH, and remains inaccessible to oxygen. Meanwhile, at the molybdenum site, XO-mediated nitrite reduction is unaffected. Process II should not be strongly affected by the presence of oxygen. In Process II, under aerobic conditions, less than 30% of the nitrite reductase activity of XO is inhibited, which suggests that most nitrite reduction happens while the FAD site is occupied by NADH.
NADH is necessary for many biochemical reactions within the body and is found in every living cell. Typical concentrations are 50 μg NADH/gram brain tissue (ca 75 μM), and 90 μg NADH/gram heart (ca 135 μM). With molybdenum-site binding electron donors xanthine or DBA, nitrite reduction is greatly inhibited by the presence of oxygen, whereas with NADH, XO-mediated NO generation remains at more than 70% of anaerobic levels. This makes NADH the major electron donor for XO-catalyzed NO production under aerobic conditions.
Interestingly, DPI, the inhibitor of FAD site-related function, greatly increased NO generation under aerobic conditions with xanthine or DBA as reducing substrate. It is known that oxypurinol blocks the binding of xanthine, DBA, and nitrite, whereas DPI inhibits the reduction of XO by NADH. With xanthine or DBA as reducing substrates, the presence of DPI inhibits XO-mediated oxygen reduction at the FAD side and thus increases the capability of the enzyme for nitrite reduction at the molybdenum site. Both the reduction of nitrite and the oxidation of xanthine and DBA take place on the molybdenum site of XO. The potential effects of DPI in stimulating NO generation from XO should be taken into account when DPI is used in biological systems, especially when high concentrations of nitrite are present.
Normoxic superoxide generation from XO depends on pH (147
), and is maximized at alkaline conditions (pH 8 – 9). In contrast, anaerobic XO-mediated NO generation accelerates tenfold when pH values fall from 8.0 to 6.0. With lower pH, a more rapid increase of XO-mediated NO generation rate was observed under aerobic conditions than under anaerobic conditions. This would be expected, because under aerobic conditions, the acidification would significantly increase XO-mediated nitrite reduction and simultaneously decelerate the competitive reaction of oxygen reduction (147
), thus facilitating NO generation under aerobic conditions.
Above, it was noted that XO may simultaneously release NO and superoxide. These radicals react rapidly to the potent oxidant peroxynitrite. The risk from peroxynitrite seems acute when nitrite levels are enhanced as, for example, under inflammation or pharmacological treatment with organic nitrates or NO-donors. However, several in vivo
reaction pathways provide protection: First, most peroxynitrite should be removed by the rapid reaction with CO2
) or by ubiquitous physiological scavengers like urate or NADH (160
). In addition, superoxide levels are kept low by superoxide dismutase (SOD), that efficiently catalyzes the dismutation of superoxide radicals to oxygen and hydrogen peroxide. Therefore, the availability of free NO is dependent upon the local activity of SOD (162
These results suggest that in presence of oxygen, only NADH can significantly sustain XO-catalyzed NO production. During ischemia, the myocardial NADH/NAD+
concentration ratio can increase more than 10-fold (163
), xanthine levels rise to the 10–100 μM, with nitrite levels of about 10 μM (154
), and the low oxygen pressure and acidosis greatly facilitate XO-mediated NO generation and limit superoxide production. The magnitude of XO-mediated NO generation can approach that of the maximal NO production from NOS (31
). Even with mild to moderate levels of hypoxia, as can occur with subtotal coronary lesions or regional ischemia in the presence of collateral flow, this process would be stimulated. Indeed, XO activity is up-regulated during hypoxia (164
) with increasing acidosis (147
), and with atherosclerosis. In patients with coronary artery disease, endothelium-bound XO activity is increased twofold (167
In summary, XO-mediated NO generation may be supported by a range of reducing substrates. Interestingly, the NADH consuming reaction is not blocked by admission of oxygen. The NO release from XO is modulated by oxygen tension, pH, and the local concentrations of nitrite and reducing substrate.
Aldehyde oxidase is another molybdenum containing flavoenzyme with high sequential homology to xanthine oxidase. This enzyme is expressed in many mammalian tissues, and was recently shown to contribute significantly to the anoxic reduction of nitrite in rat tissue homogenates (168
The preceding discussion considered the role of xanthine oxidase under hypoxia. Interestingly, it was recently shown that xanthine oxidase also contributes to a slow reduction of nitrate in normoxic tissues (32
). Intraperitoneal injection of nitrate enhanced circulating nitrite levels in normoxic mice on a slow timescale of an hour. Applications of selective inhibitors implied that xanthine oxidase contributes significantly to this reaction. In accordance, pretreatment with nitrate attenuated the increase in systemic blood pressure caused by NOS inhibitors and enhanced blood flow during post-ischemic reperfusion. It suggests that mammalian xanthine oxidase mediates nitrate reduction in regulation of nitrite and NO homeostasis (32
D: Cytochrome P450 (CYP)
(CYP) refers to a very large superfamily of heme proteins with over 7800 different members currently known. They are found in all eukaryotes, and most prokaryotes (169
). CYP’s catalyze a vast variety of different reactions, but all share the characteristic catalytic site in the form of a heme with an axial thiolate ligand derived from a nearby cysteine residue. Because of the vast variety of reactions catalyzed by CYPs, the activities and properties of the many CYPs differ in many aspects. The resting state of the protein is ferric Fe3+
. For the catalytic cycle, the heme is reduced by electrons supplied by a variety of other proteins like cytochrome P450
reductase (CPR), ferredoxins, or cytochrome b5. Electron transfer from the redox partner to CYP is a key step in the CYP catalytic cycle. Bacterial and mitochondrial CYP receive electrons from a small soluble iron-sulfur protein, whereas the redox partner for mammalian microsomal CYP is a FAD/FMN-dependent NADPH-CPR (170
). In CPR, FAD serves as an electron acceptor from NADPH, whereas FAD serves as an electron acceptor from NADPH (171
The most common reaction catalyzed by CYP is that of a monooxygenase. This reaction is unselective and accepts a wide range of target substrates:
Denitrification was long believed to be restricted to the bacteria (172
), according to the reaction
However, in 1989, Shoun and co-workers observed that CYP from the fungus Fusarium oxysporum
was specifically induced upon exposure to nitrate and nitrite (173
). This observation led to the finding of denitrifying activity in the fungus. With NADH as the direct electron donor, CYP can catalyze a chain of reduction: from nitrate to nitrite, nitrite to nitric oxide, and nitric oxide to dinitrogen oxide (N2
Mammalian CYP’s are involved in the metabolism of many drugs and dietary substances, and in the synthesis of steroid hormones and other extracellular signaling lipids. CYP from mammalian liver can reduce nitrite as first demonstrated more than thirty years ago (177
). EPR spectroscopy confirmed CYP-mediated nitrite reduction by detection of paramagnetic ferrous nitrosyl-heme complexes (179
). Subsequent studies detected release of free NO from rat liver CYP or human recombinant CYP (171
Although it has been reported that the vascular biotransformation of nitroglycerin (GTN) is mediated by CYP (180
), research (171
) on GTN biotransformation indicated that rat liver microsomal CYP can not catalyze the reduction of GTN per se
. Instead, rat liver CYP can serve as a nitrite reductase and generate NO radicals from NO2−
). In the presence of NADPH (100 μM), addition of 20 μM or 40 μM nitrite triggered the release of ~ 2.8 nM.min−1
or ~ 4.5 nM.min−1
NO from microsomes (2 mg/ml). The CYP inhibitor clotrimazole (5 μM) greatly inhibited the generation of NO from nitrite. These results confirm that CYP reduces nitrite to NO in rat liver microsomes. Kinetic studies revealed that NO generation from nitrite reduction contributed less than 10% of total NO generated in the decomposition of GTN in vivo
All studies to date of NO generation from CYP-mediated nitrite reduction have been performed under anaerobic conditions. This leaves a need to further characterize this process in the presence of small quantities of oxygen. Under aerobic conditions, molecular oxygen will be bound and split by the reduced heme iron of CYP; furthermore, many CYP substrates such as steroids, fatty acids and xenobiotics will compete with nitrite for reaction with CYP under aerobic conditions. Thus, at this time while there is an absence of published data, one would expect that CYP-mediated nitrite reduction would be inhibited by oxygen under aerobic conditions and also by CYP substrates. Future studies will be needed to characterize the precise effects of oxygen on the process of CYP-mediated nitrite reduction.
Generation of NO from nitrite by human recombinant CYP was also studied. With the presence of 100 μM NADPH, addition of 100 μM nitrite triggered an NO release of ~ 7 nM NO/min from 0.1 mg/ml CYP 2B4. NO generation from nitrite was increased with increasing nitrite concentrations or CYP concentration in the reaction mixture. Furthermore, this NO generation derived from nitrite was strongly inhibited by the heme inhibitor cyanide.
It was proposed (160
) that nitrite reacts with CYP in its reduced ferrous form resulting in formation of NO-(Fe3+
) heme. The nitrosyl ligand is rather weakly bound and may be released as free NO or re-trapped forming the far more stable NO-(Fe2+
) heme. The proposed reaction sequence is as shown below.
The final step in this scheme is the reduction to ferrous state by CPR. In the presence of excess nitrite, significant quantities of free NO have been detected. This suggests that CYP-mediated nitrite reduction might be a source of NO in vivo
Under physiologic conditions CPR was proposed to cycle between the 1- and 3-electron reduced states with NADPH or NADH as electron donor (184
). CPR prefers NADPH as electron donor and its affinity for NADPH is more than ten times higher than NADH (171
). Electron transfer occurs from CPR to CYP and thus completes the catalytic cycle.
The resting state of CYP is oxidized ferric Fe3+. Under ischemic conditions most CYP would be reduced to the ferrous state with the increased NADPH in the tissues and much lower oxygen levels. This ferrous-CYP can be a source of NO in the tissues with a generation rate = [NO-Fe2+ CYP]·Koff
However, ferrous CYP can also bind free NO as a ligand to the heme. The first order binding rate is Kon. · [Fe2+CYP] · [NO].
Whether CYP acts as a sink or source of free NO depends on Kon, Koff, CYP redox-state, NO, and nitrite concentrations. The kinetics and pathophysiological roles of CYP reaction with nitrite remains unclear and needs further investigation.
In conclusion, CYP can reduce nitrite and produce NO. Although most previous studies have been performed under anaerobic conditions, the reaction would also proceed in the presence of small quantities of oxygen. Spectrophotometric studies have also shown that NO binds to ferric and ferrous CYP and can inhibit the normal catalytic cycle of CYP (186
). Similarly, reaction of nitrite with CYP leads to formation of paramagnetic ferrous mononitrosyl complexes (NO-Fe2+
CYP) complexes as shown by EPR spectroscopy (179
). Such ferrous mononitrosyl complexes are quite stable (33
) and thus cause reversible inhibition of CYP. Thus, CYP can be a significant source of nitrite reduction to NO and in addition nitrite also inhibits CYP function in drug metabolism. Further studies will be required to provide a more detailed characterization of the role and importance of CYP in the process of nitrite reduction under aerobic conditions and in vivo.
E: Endothelial nitric oxide synthase (eNOS)
The endothelial isoform of NOS releases NO radicals from L-arginine with the consumption of 1.5 NADPH equivalents and two oxygen molecules per NO formed. This aerobic catalysis requires the presence of the cofactors Ca2+
-Calmodulin and tetrahydrobiopterin (BH4
), and is tightly regulated via a combination of mechanisms (cytosolic Ca2+
-Calmodulin, (de)phosphorylation, (de)palmitoylation, and intracellular relocalization between the Golgi system and membrane calveolae within the endothelial cells (188
)). Deficiency of arginine or BH4
causes “uncoupling” (189
) where the oxygenase domain of eNOS releases superoxide radicals (O2•−
) instead of NO (190
). Oxygen deficiency is known to halt the L-arginine cycle if the oxygen levels fall below a threshold level of ca [O2
] ~ 10 μM (63
). However, eNOS is not wholly inactivated by the absence of oxygen. Instead, in the presence of nitrite, it switches to a novel nitrite reductase activity which releases NO (18
The formation of free NO radicals was observed with three independent techniques carried out simultaneously in the same argon-purged optical cell: First, UV/VIS absorption of the heme group showed reduction of the heme to ferrous state by addition of NADPH, and subsequently nitrosylation of a significant fraction of the available heme. Second, electrochemical detection with an NO sensitive electrode showed the release of significant quantities of free NO radicals into the anoxic solution. Third, using EPR spectroscopy and iron-dithiocarbamate complexes as NO traps (33
), significant quantities of gaseous NO were detected in the purging argon flow leaving the reaction vessel. The ferrous mononitrosyl iron-dithiocarbamate complex NO-Fe2+
is paramagnetic (192
) and the shape of its EPR spectrum is sensitive to isotopic labeling of the nitrogen with the stable 15
N isotope (cf chapter 18 of (33
)). In this way it was proven that the observed 15
NO was released from the 15
N-nitrite anions and not from the reaction of 14
N-arginine with residual traces of oxygen (18
). In absence of eNOS, no NO was detected, and the pH was stabilized at neutral 7.4 so that the release of NO from acidic reduction of nitrite could be ruled out.
The anoxic reaction was initiated by administration of NADPH into the buffered solution of eNOS repleted with necessary cofactors and nitrite. The new reaction has some similarities to the aerobic arginine pathway in that it also requires electron injection from NADPH to the flavin domain of the enzyme, and the presence of Ca2+
/calmodulin to afford intramolecular electron transport towards the heme (190
). Other aspects are very distinct, however: the reduction of nitrite is slowed but not halted by the removal of BH4
and it consumes neither arginine nor oxygen. Comparison of purified WT eNOS and its isolated oxygenase domain shows that nitrite reduction is achieved at the oxygenase domain of the protein (18
). Upon readmission of oxygen, eNOS reverts to the normal L-arginine pathway, and the enzyme appeared fully functional after several such argon/oxygen cycles. Such repeated regeneration of the nitrite reductase capacity by cyclic admission of oxygen provides eNOS with an “emergency” NO release in acute anoxia..
The effect of anoxia was studied further in cultured BEND3 cells (19
). These immortalized murine brain microvascular endothelial cells express only the endothelial isoform of NOS (194
). These cells were grown to confluence corresponding to ca 7.5 ± 0.5 × 106
cells in a monolayer. Confluence and cell count are important parameters for eNOS activity (188
) and were verified by inspection with a stereomicroscope. The NO production in these cell cultures was studied using NO trapping with iron-dithiocarbamate complexes. In this accumulating method, the yield of paramagnetic NO-Fe2+
-DETC adducts (MNIC) increases with time and is determined with EPR spectroscopy after termination of the experiment. The yields from trapping for 20 min are collected in . The basal (i.e. unstimulated) NO production was measured by trapping for 20 min under the controlled atmosphere with 5 % CO2
and 20 % O2
. From , the corresponding free oxygen concentration in the medium was ca 200 μM. This value is normally used for experiments on cultured cells but corresponds to hyperoxia in our classification of .
Table VII Yields of MNIC adducts (in pmol) in the cellular fractions of a single 75 cm2 flask of cultured confluent endothelial cells. Trapping proceeded for 20 min at 37° C. The second row gives the preincubation times τinc of the supplements. (more ...)
As expected, the basal yield of ca 110 pmol MNIC per flask may be cancelled by coincubation with NOS inhibitors like L-arginine-methyl-ester (L-NAME) or Nω-nitro-L-arginine (NLA), thereby proving that the NO results from enzymatic activity of eNOS. Upon stimulation with the calcium ionophore A23187, the yield was enhanced nearly fourfold to 400 pmol MNIC. These cell cultures were then subjected to acute anoxia by replacing the culture medium with deoxygenated medium and flushing the flasks with argon before closing the top. The imposition of anoxia reduced the oxygen concentration of the medium to a value below the detection limit of ca 2 μM of a Clark electrode. This limit is comfortably lower than the oxygen threshold of the L-arginine pathway of eNOS cf ). Although the L-arginine pathway was halted, significant quantities of NO were being released from these anoxic cell cultures. The anoxic yield of ca 160 pmol MNIC was significantly higher than the basal yield of 110 pmol in presence of oxygen. Therefore, deprivation of oxygen actually enhances the release of NO from this endothelial cell line. However, the anoxic NO yields remain in the normoxic regulatory range between basal and fully stimulated yield (110 resp 400 pmol MNIC). shows the kinetics of the anoxic reaction as a function of time. After imposition of anoxia, the MNIC yield increased roughly proportional with time for twenty minutes, and levelled off at an asymptotic value of ca 200 pmol MNIC per flask. The samples could be kept at 37°C for over an hour without significant loss of MNIC adducts. Trypan blue staining did not show enhanced mortality of cells after exposure to anoxia up to 30 min.
Figure 6 Kinetics of NO formation in a single flask of cultured BEND3 cells under anoxia and normoxia. The MNIC yields are given as function of incubation time of the trapping experiment. The anoxic values (solid squares) were taken from (19) with permission. (more ...)
When considering these yields, five potential sources of the NO should be considered: intracellular nitrite, extracellular nitrite, nitrate, arginine or endogenous S-nitrosothiols. Extracellular nitrite could be ruled out since the yields were unaffected by addition of 250 μM nitrite to the medium. Arginine is ruled out as a substrate since its oxidation requires oxygen. Although some residual oxygen may still be found after imposition of anoxia, it is inconceivable that the arginine pathway could remain functional at greatly increased rate for over twenty minutes. Hypoxic reduction of nitrate to nitrite and subsequently to NO by xanthine oxidase enzymes (17
) could be ruled out because the yields were unaffected by addition of the inhibitor oxypurinol. These results show that in this particular model the xanthine oxidase enzyme does not significantly contribute to the observed NO release. S-nitrosothiols were also ruled out because the anoxic NO production could be inhibited with NOS inhibitors L-NAME and LNA. This leaves intracellular nitrite as the probable source of NO. The intracellular nitrite levels were estimated by the Griess colorimetric assay on cell lysates. The data confirmed that the intracellular nitrite levels rapidly fell after imposition of anoxia from an initial concentration of ca 20 μM to below the detection threshold of ca 6 μM and suggests that depletion of intracellular nitrite causes the cessation of NO release after ca 30 min (cf ). Significantly, the magnitude of anoxic NO release was intermediate between the basal and fully stimulated yields and remained in the benign range of normoxic physiological regulation, far below the cytotoxic levels found in septic shock or allograft rejection.
It was recently found that eNOS is the only NOS isoform capable of reducing nitrite and releasing free NO radicals (18
). This gives eNOS a special significance when considering the effects of ischemia on various tissues. Anoxic NO release from reduction of nitrite by eNOS may be a plausible explanation for the plethora of animal and clinical studies showing a protective role of eNOS in the early stages of ischemia (196
F: Mitochondrial respiratory chain
Several years ago, mitochondrial cell fractions were found to release NO radicals from nitrite (179
). The activity involved two distinct components of the mitochondrial respiratory chain, namely complex III (198
) and cytochtome c oxidase (complex IV) (179
). Published literature shows that the results obtained for complexes III and IV were carried out at different ranges of external nitrite concentrations. At [NO2−
] < 50 μM, complex III reduced nitrite. At [NO2−
] > 300 μM, cytochrome oxidase was found to be the main source of nitrite reductase activity in the mitochondria (21
). A critical question is how nitrite enters the cells and gains access to mitochondria since the NO2−
anion carries charge and should not diffuse freely through membranes. It was recently pointed out that a small fraction of nitrite anions is protonated even at physiological pH, and may cross model lipid membranes as neutral molecule HNO2
). The situation is less clear for actual physiological membranes. Castello and coauthors have shown that only some 10% of nitrite added to isolated yeast mitochondria is internalized into the mitochondrial matrix (21
). Thus, equilibrium concentrations of nitrite available for mitochondrial nitrite reductase activity may be significantly lower than cytoplasmic nitrite concentrations. This may explain that nitrite transport to mitochondrial nitrite reductase seems facilitated by low pH.
The sites where nitrite reduction occurs were identified by applying selective inhibitors of the respiratory chain (198
). Rotenone inhibits the electron transfer from complex I to the Q-cycle. Application of rotenone to mitochondrial fractions inhibited NO generation from nitrite by 60%. Thenoyltrifluoroacetone (TTFA) is an inhibitor of complex II. In the presence of succinate, the substrate of complex II, TTFA inhibited NO formation by 40%. In this experiment, reversed electron flow to complex I was suppressed by the presence of rotenone (198
). Myxothiazol is an inhibitor of electron transfer from the Q-cycle to complex III. Irrespective of the substrate supplying reducing equivalents to the respiratory chain, myxothiazol completely inhibited release of NO. In contrast, antimycin A, an inhibitor of electron transfer at the oxidant site of the bc1
complex, did not influence the formation of NO. Thus it was determined that the site of reduction is localized between myxothiazol sensitive and antimycine A sensitive components of the respiratory chain (). This part of the respiratory chain includes cytochromes b562, b566, and the ubisemiquinone-radical bound to complex III (). Myxothiazol is highly specific for mitochondria and completely inhibits nitrite reduction. Therefore, it can be used to estimate the contribution of complex III to the total nitrite reductase activity in different organs. A recent study (86
) confirmed inhibition of nitrite reduction by myxothiazol. The inhibition was complete in heart and intestinal homogenates, but only partial in liver homogenates. It shows that liver tissue contains an additional source of nitrite reductase activity. After separating the various fractions of liver tissue, significant nitrite reductase activity was found in the microsomal fractions as well (200
). Within these microsomal fractions, the nitrite reductase activity was attributed to cytochrome P450
enzymes in the endoplasmatic reticulum (cf section 7.D.).
Figure 7 Possible sites of nitrite reduction in mitochondria at complexes III and IV of the respiratory chain. Abbreviations: R - Rotenone, M - Myxothiazol, A - Antimycin A, T - Thenoyltrifluoroacetone, CN – Cyanide, UQ – Ubiquinone, UQH2 – (more ...)
It has been shown that at millimolar concentrations nitrite reacts with the oxygen binding site of cytochrome c oxidase (complex IV) rather than with the cytochrome c binding site of the enzyme (201
). The NO is a product of this reaction. It requires the presence of the substrates of both complexes I and II and is inhibited by antimycin A, myxothiazol, KCN, and carbon monoxide (21
). It has been suggested that Cytochromes aa3 (complex IV) catalyse the reduction of NO2−
to NO (21
) under anaerobic conditions. Under normal conditions this site reduces oxygen to water.
Under normal physiological conditions, the mitochondrial complex III releases a significant quantity of superoxide radicals. It is known that NO reacts very rapidly with superoxide to form peroxynitrite (ONOO−
). It seems that NO and superoxide are generated at different sites of complex III. The release of superoxide radicals is dramatically increased by antimycin A (202
), but antimycin A does not influence the reduction of NO2−
to NO (198
). One can expect that nitrite reductase activity of complex III is deleterious yielding peroxynitrite if NO and superoxide are generated simultaneously in the same segment of respiratory chain. However, significant release of NO occurs only under hypoxic conditions and the generation of superoxide requires oxygen. In addition, it has been shown that nitrite can also modulate the mitochondrial respiratory chain during anoxia by S-nitrosation of complex I. This modification decreases its activity and attenuates the release of reactive oxygen species from the mitochondrial chain (100
). Therefore it is unlikely that nitrite reductase activity significantly contributes to the formation of peroxynitrite. However, this point has not yet been addressed in detail.
It is important to consider the levels of oxygen and pH values in tissues. The physiological oxygen levels in resting tissues remain slightly below those of venous blood (cf and ). At these oxygen levels, the release of NO from nitrite must be very small or even zero as the quantity of nitrosyl-hemoglobin (NO-Fe2+
Hb) remains below the detection threshold of EPR (204
). The effect of pH was studied in homogenates prepared from rat intestine. At low pH values the nitrite reductase activity of intestinal homogenate was elevated due a slight increase in mitochondrial nitrite reductase activity and a far stronger increase in the rate of nitrite reduction by low molecular weight reducing agents (86
). This is in line with publications confirming that acidic reduction of nitrite become more significant at low pH values (16
The NO released by the mitochondria cannot be distinguished from that provided by other sources in the tissue. Therefore, the mitochondrial nitrite reductase activity is difficult to separate from other mechanisms such as deoxyHb or deoxyMb (see above). To avoid direct contact with blood the nitrite was administered into intestinal lumen (86
). Nitrite infusion in intestinal lumen results in the formation of NO-Fe2+
Hb complexes in blood and a simultaneous drop in blood pressure at a threshold concentration of 10 μM of NO-Fe2+
Hb complexes (86
). This mechanism is expected to operate independently of the route of nitrite administration. Recently, it has also been suggested that the mechanism for nitrite-induced vasorelaxation is largely intrinsic to the vessel and that under hypoxia physiological nitrite concentrations are sufficient to induce NO-mediated vasodilation (205
). These data suggest the existence of additional mechanisms releasing NO from nitrite, possibly mediated by eNOS (see section 7.E.).
Once released from the mitochondrial respiratory chain, NO can act in a variety of ways: NO and its metabolites like peroxynitrite (206
) are known to inhibit mitochondrial respiration via binding to the enzymes of the respiratory chain (207
). Protracted inhibition of the respiratory chain by NO has been shown to trigger apoptosis and, ultimately, cell death in rat thymocytes (209
). Since mitochondria reduce nitrite to NO one can expect that nitrite should inhibit mitochondrial respiration as well. It has long been known that well-oxygenated mitochondrial enzymes are inhibited by very high nitrite concentrations exceeding 0.3 mM (210
). In contrast, at low oxygen pressures, mitochondrial respiration is inhibited by far lower concentrations of nitrite (211
). This observation once again confirms that hypoxia significantly promotes the release of NO from nitrite in biological systems.