NO Generation From Nitrite and Its Role in
`Vascular Control
`
`Jon O. Lundberg, Eddie Weitzberg
`
`Abstract—NO generated from L-arginine by NO synthases (NOSs) in the endothelium and in other cells plays a central role
`in several aspects of vascular biology. The biological activity of NO is acutely terminated by oxidation to nitrite and
`nitrate, and these compounds have long been considered only as inert end-products of NO. However, this dogma is now
`being challenged because recent research convincingly has shown that the nitrite ion can be recycled back to bioactive
`NO again in blood and tissues. Nitrite reduction to NO can occur via several routes involving enzymes, proteins,
`vitamins, or even simple protons. This pathway may serve as a backup system for NO generation in conditions such as
`hypoxia, in which the NOS/L-arginine system is compromised, but detrimental effects can also be foreseen. With this
`new knowledge, nitrate and nitrite should probably be viewed as storage pools for NO rather than inert waste products.
`Here we discuss novel aspects of nitrite-dependent NO generation in vivo and its role in vascular control. (Arterioscler
`Thromb Vasc Biol. 2005;25:915-922.)
`
`Key Words: nitric oxide 䡲 nitrite 䡲 nitrate 䡲 S-nitrosothiol 䡲 superoxide 䡲 xanthine oxidase 䡲 hemoglobin
`
`NO is transiently released from endothelial cells whereby
`
`it serves to regulate important functions in the vessel
`itself and in circulating cells. When generated (eg, in re-
`sponse to shear stress), NO diffuses radially from the produc-
`tion site near the vessel lumen. Some of this NO will survive
`unaffected all the way to the underlying smooth muscle cells
`to promote vasorelaxation,1 and some will affect cells passing
`in the blood stream, for example platelets2,3 and leukocytes.4
`However, the major part of NO will be destroyed before it
`ever reaches a target cell because of rapid oxidation either by
`hemoglobin (Hb) in blood or in tissues. This ensures that the
`effects of NO are restricted to the close vicinity of its
`production site, which helps to control and precisely target
`the effects of this potent biological messenger. However,
`from an energetic point of view, such “waste” of NO is far
`from optimal because its generation is performed by complex
`enzymes in an energy-consuming reaction that requires nu-
`merous substrates and cofactors. So, is then the rapid oxida-
`tion and inactivation of NO really an irreversible process as
`long believed? Pathways designed for the reuse of important
`biological messengers are ubiquitous in biological systems.
`Here we discuss recent advances in NO biology pointing to
`the fact that also, NO metabolites can be recycled back to
`bioactive NO again. The physiological and pathophysiologi-
`cal aspects of this newly recognized salvage pathway are
`covered with focus on regulation of blood flow.
`
`Conservation of NO Bioactivity in Blood
`As stated above, the general view of NO has been that it can
`only act in a paracrine manner on neighboring cells because
`of its very short half life. However, more recent studies have
`suggested that the bioactivity of NO in blood in fact can be
`conserved, thereby allowing for more distal and sustained
`effects. As an example, inhaled NO can have vasodilatory
`effects not only locally in the pulmonary circulation but also
`in peripheral tissues.5–7 In addition, intravascular delivery of
`authentic NO results in vasodilation distal to the site of
`injection.8 How then is this NO transported in blood? Over
`the last years, several alternative explanations have emerged.
`First, it is possible that NO itself can remain intact in the
`blood vessel for a longer period than initially believed.
`During normal laminar flow, the red blood cells travel mainly
`near the center of a vessel, thereby leaving a cell-free zone
`near the endothelium.9,10 Similarly, a second effective diffu-
`sion barrier is created by the unstirred plasma layer surround-
`ing the erythrocyte11,12 and possibly also by the plasma
`membrane. It has been suggested that NO can survive
`unchanged in these zones away from the scavenging Hb
`inside the red blood cells, thereby allowing for transportation
`to more distal sites of action.
`A second theory that has received much attention is the
`notion that NO is transported in blood and tissues in the form
`of S-nitrosothiols (SNOs), which would then act as stable
`carriers of NO.13,14 SNOs are formed when thiol groups react
`
`Original received January 21, 2005; final version accepted February 21, 2005.
`From the Department Physiology and Pharmacology (J.O.L.), Karolinska Institutet, and the Department of Surgical Science (E.W.), Karolinska
`University Hospital, Stockholm, Sweden.
`The authors own shares in a biotech company, Aerocrine AB, Stockholm, Sweden. Aerocrine develops equipment for measurements of nitric oxide
`in exhaled air.
`Correspondence to Jon Lundberg, Professor, Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden. E-mail
`jon.lundberg@fyfa.ki.se
`© 2005 American Heart Association, Inc.
`Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
`
`DOI: 10.1161/01.ATV.0000161048.72004.c2
`
`915
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`with NO15 or chemically related species,16 and they can act as
`donors of NO. One example is S-nitrosylation of albumin,13
`but most recent focus has been on a thiol group in the Hb
`molecule.17–21 According to this theory, NO or a closely
`related species binds to a cysteine residue in the Hb (cys-
`␤93), thereby forming S-nitroso Hb (SNO-Hb). The binding
`and subsequent release of free NO is allosterically regulated
`by the oxygenation of Hb and the redox state so that SNO-Hb
`formation is favored in well-oxygenated conditions (in the
`lungs when Hb is in the R-form), and release of NO occurs
`mainly when oxygen leaves Hb (T-form).17,21 In this way, NO
`is delivered by the red blood cells preferably to less oxygen-
`ated tissues where it is most needed. This very attractive
`theory has gained great interest over the past decade, but
`more recently,
`its role in human biology has also been
`questioned.22–27 The main criticism has been the extremely
`low levels of SNO in human blood reported recently by some
`groups and the absence of an arteriovenous gradient for
`SNOs.
`More recently, a third theory has emerged, which is the main
`topic of this review. In this pathway, NO is generated from the
`nitrite ion by simple reduction, and this can occur in blood and
`in tissues. There are several different pathways for NO formation
`from nitrite, and each is discussed in detail below.
`
`Nitrite as a Vasodilator
`The knowledge that inorganic nitrite can dilate vessels is not
`new. Furchgott used acidified sodium nitrite to relax precon-
`stricted rabbit aortic strips in 1953.28 However, in these early
`experiments, the nitrite concentrations and the pH used were
`far outside physiological
`levels, and the mechanism of
`dilatation (NO generation) was unknown. Other studies have
`shown that ingestion of fairly large amounts of inorganic
`nitrite can reduce blood pressure in spontaneously hyperten-
`sive rats, which is suggestive of systemic NO generation.29 –31
`Very recently, Tsuchiya et al32,33 showed that ingestion of
`nitrite resulted in rapid appearance of nitrosylhemoglobin in
`blood, which is highly indicative of systemic NO formation
`from the nitrite. They also found that coadministration of nitrite
`with NG-nitro-L-arginine methyl ester for 3 weeks attenuated the
`hypertension induced by this NO synthase (NOS) inhibitor.
`Thus, nitrite could partly compensate for the loss of NOS-
`derived NO, thereby counteracting a rise in blood pressure.
`
`Nitrite Sources In Vivo
`There are at least 3 sources of nitrite in mammals. First, nitrite
`is an oxidation product of NOS-derived NO.34 Second, nitrite
`is present in some food stuffs, for example in processed meat,
`in which this anion is used to prevent botulism.35 Third,
`nitrite is generated from commensal bacteria in the digestive
`system as a result of nitrate reduction.35,36 The relative
`contribution from these different sources of nitrite during
`normal conditions is variable and therefore difficult to judge.
`During fasting conditions with a low previous intake of
`nitrate/nitrite, the NOS/L-arginine pathway probably domi-
`nates37 and even more so during systemic inflammation when
`much additional NO is generated from inducible NOS.
`However, with a high intake of nitrate, the plasma levels of
`nitrite increase greatly as discussed below.38
`
`Basal Blood/Plasma Levels and Half Lives of Some NO-Related
`Compounds
`
`Nitrate
`Nitrite
`NO
`Hb-NO*
`S-nitroso-Hb*
`S-nitroso-albumin*
`
`Blood/Plasma
`Levels, nmol/L
`
`20 000–50 000
`100–500
`⬍1
`⬍1–200
`⬍1–200
`1–200
`
`T1/2
`5–8 hours
`1–5 minutes
`1–2 milliseconds
`15 minutes
`—
`—
`
`Values are approximated from recent studies in humans.
`*For Hb-NO, SNO-Hb, and S-nitroso-albumin, no firm agreement about
`normal values has been reached, and reported values vary greatly.
`T1/2 for Hb-NO is from pig experiments, whereas while T1/2 for SNO-Hb and
`S-nitroso-albumin is unknown.
`
`Plasma Nitrate/Nitrite as an Index of
`Endothelial NOS Activity
`A common and simple way of estimating body formation of
`NO is to measure its more stable oxidation products nitrate
`and nitrite in plasma or other biological fluids. This is
`convenient because direct in vivo measurements of NO can
`be very difficult because of the extremely low levels and its
`short half life. When combined measurements of nitrate/
`nitrite are made, this is usually denoted by the term NOx. In
`simple aqueous solution, NO is oxidized almost exclusively
`to nitrite, whereas in whole blood in the presence of oxygen-
`ated Hb, nitrite is rapidly further oxidized to nitrate.34,39
`Therefore, plasma nitrate is normally much higher than
`nitrite, and as a consequence, NOx is almost identical to
`nitrate. Plasma nitrite levels are usually in the range 0.1 to
`0.5 ␮mol/L38,40 – 43 (Table), whereas tissue concentrations are
`typically 1 to 2 orders of magnitude higher.44 – 46
`The nitrate content in food can be very high, for example,
`in green leafy vegetables and sometimes in drinking water.35
`In addition, the plasma half life of nitrate is fairly long (⬇6
`hours).35 Because of this, one cannot rely on nitrate measure-
`ment as an adequate index of systemic NO generation. On the
`other hand, nitrite measurements are thought to much better
`reflect ongoing endothelial NOS (eNOS) activity.37,40,41 In a
`study by Kleinbongard et al, it was concluded that ⬇70% of
`plasma nitrite is derived from eNOS in the endothelium.40
`However, recent studies show that ingestion of nitrate can
`influence not only plasma nitrate but also nitrite.38 This is
`very surprising because it requires reduction of nitrate to
`nitrite, a reaction that cannot be catalyzed by mammalian
`enzymes. The answer lies in the enterosalivary circulation of
`nitrate. When nitrate is ingested, as much as 25% is actively
`taken up by the salivary glands and secreted into saliva.35 In
`the oral cavity, much of this nitrate is reduced to nitrite by
`commensal bacteria,35,36 and this nitrite enters the circulation
`when saliva is swallowed.38
`
`Nitrite Reduction to NO In Vivo
`In 1994, 2 independent groups reported that nitrite can be a
`substrate for NOS-independent generation of NO in vivo.47,48
`This was first demonstrated in the stomach, where nitrite-derived
`NO seems to play an important role in host defense49,50 and in
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`Lundberg and Weitzberg
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`NOS-Independent NO Generation From Nitrite
`
`917
`
`⫺) in
`Figure 1. The fate of nitrite (NO2
`blood and tissue. In well-oxygenated tis-
`sues, NOS generates NO from L-arginine
`and oxygen. Some of this NO reaches
`the smooth muscle cells to promote
`vasodilation, whereas much is oxidized
`⫺; in oxygenated blood)
`to nitrate (NO3
`and nitrite (in tissues). A second source
`of nitrate and nitrite is the diet, and
`ingestion of nitrate results in great
`increases in plasma levels of nitrate and
`nitrite. Recent studies have shown that
`nitrite can be recycled to bioactive NO
`again, and the different suggested path-
`ways for this reaction are outlined in the
`figure. Under hypoxic conditions, nitrite
`in blood can react with deoxy-Hb to
`generate vasodilatory NO. In hypoxic tis-
`sues, nitrite is reduced to NO by XO,
`simple protons (H⫹), or by mitochondrial
`cytochromes (mtC). Bioactivation of the
`vasodilatory drug nitroglycerine (GTN)
`also occurs via intracellular formation of
`nitrite. A mitochondrial aldehyde dehy-
`drogenase has been suggested to cata-
`lyze nitrite formation from GTN in smooth
`muscle cells, and then nitrite is further
`reduced to vasodilatory NO, possibly via
`one of the pathways outlined in the fig-
`ure. HbO2 indicates oxyhemoglobin.
`
`already at a moderately low pH and within nitrite concentra-
`tions normally present in vivo. In 1995, Zweier et al demon-
`strated systemic generation of NO from nitrite.46 They found
`that N15-labeled nitrite was reduced to NO in ischemic rat
`hearts, and this production was not blocked by NO synthase
`inhibitors. During global ischemia in the heart, pH fell to
`⬇5.5, and under these conditions, reduction of nitrite to NO
`was greatly accelerated. Modin et al showed that physiolog-
`ical levels of nitrite relaxed rat aortic rings when the acidity
`of the buffer solution was adjusted to pH just ⬍7, which is
`commonly seen in tissues during ischemia (Figure 2).44 This
`relaxation was blocked by an inhibitor of soluble guanylyl
`cyclase (ODQ) and paralleled by NO generation in head
`space gas, supporting that NO was mediating the effects.
`Interestingly, NO generation from nitrite and relaxation was
`further increased by vitamin C.44 This antioxidant is known to
`enhance NO-mediated vasorelaxation, and several alternative
`explanations for this effect have been put forward. Scaveng-
`ing of superoxide (an NO destroyer) has been suggested,56 –58
`as well as direct stimulation of eNOS activity.59 However,
`Jackson et al showed that effective scavenging of superoxide
`occurred only at very high concentrations of vitamin C,60 so
`the mechanism of action still remains incompletely under-
`stood. It is possible that some of the effect is related to
`enhancement of NO generation from nitrite.44
`
`Reduction by Xanthine Oxidase
`Xanthine oxidase (XO) is a ubiquitous enzyme involved in a
`variety of physiological and pathophysiological processes. It
`has a critical role in purine and pyrimidine catabolism, but it
`also reduces oxygen to superoxide and hydrogen peroxide,
`thereby contributing to oxidative cellular injury. The ability
`of XO to generate reactive oxygen species has led to
`widespread interest in the pathogenic role of this enzyme in
`
`regulation of gastric mucosal integrity.51 It is now clear that
`reduction of nitrite can occur not only locally on epithelial
`surfaces such as in the stomach,47,48 in the oral cavity,49,52 on the
`skin,53 or in urine,54 but also systemically in blood and tissues
`(Figure 1). Several alternative pathways for NO generation from
`nitrite have been described, which are discussed below.
`
`(1)
`
`NO2
`
`(2)
`
`(3)
`
`N2O3
`
`Acidic Reduction
`⫺) is acidified, it yields nitrous acid (HNO2,
`When nitrite (NO2
`reaction 1), which spontaneously decomposes to NO and
`other nitrogen oxides (reactions 2 and 3):
`⫺⫹H⫹7HNO2( pKa3.2 to 3.4)
`3N2O3⫹H2O
`2HNO2
`3NO⫹NO2
`The chemistry of acidified nitrite is very complex, and the
`amounts of NO generated from nitrite is dependent not only
`on pH and nitrite concentrations but also on the presence of
`other reducing agents (eg, vitamin C and thiocyanate), prox-
`imity to heme groups, proteins,
`thiols, and the oxygen
`tension.55 Reduction of nitrite is quantitatively most signifi-
`cant in the stomach, where the extremely low pH in combi-
`nation with a very high nitrite content (0.1 to 1 mmol/L from
`saliva) results in NO levels sometimes exceeding 100 parts
`per million (⬇4 ␮mol/L; ie, ⬎4 orders of magnitude higher
`than the levels required for vasodilation).35 These extremely
`high NO levels (and possibly other nitrogen oxides generated
`from acidified nitrite) help to kill luminal bacteria48 and even
`diffuse through the entire mucus layer to enhance mucus
`generation and blood flow in the gastric mucosa.51 Interest-
`ingly, studies have indicated that acid-catalyzed nitrite reduc-
`tion to NO can also take place in blood vessels and tissues
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`ischemia-reperfusion injury.61 The expression and activity of
`XO is induced by hypoxia and proinflammatory cytokines,
`whereas hyperoxia has the opposite effect.62 Interestingly
`XO, is structurally related to bacterial nitrate and nitrite
`reductases, and early studies have indeed showed that this
`enzyme can reduce nitrate and nitrite.63 More recently, it has
`been shown that NO is formed by XO-catalyzed reduction of
`nitrite64 – 67 or by a 2-step reduction of nitrate to NO.68 Nitrite
`reduction by XO is greatly enhanced at low oxygen tensions
`and acidic conditions such as those seen during ischemia. It
`has been shown that under conditions of severe ischemia,
`myocardial XO and nitrite levels are sufficient to generate
`NO at
`levels exceeding those from maximally activated
`NOS.69 Webb et al recently studied XO-dependent NO
`generation from nitrite in perfused rat hearts subjected to
`ischemia.70 They found that
`large amounts of NO were
`generated from nitrite by XO during ischemia, and this NO
`was strongly cardioprotective, with a marked reduction in
`infarct size. In the presence of an NO scavenger, the protec-
`tive effect was completely blocked. A likely location of the
`XO is the endothelium of blood vessels in the heart, as
`demonstrated by functional70 and histochemical studies.71
`These new findings challenge the notion that XO is only
`damaging in ischemia-reperfusion. The factors that determine
`whether the net effect of a certain enzymatic pathway is
`beneficial or harmful may in part be related to the substrate
`supply. If XO is presented with nitrite as an alternative
`substrate, the protective effects of the NO generated seems to
`dominate,70 whereas in other situations, more superoxide is
`generated with possible harmful effects. Also, when NO and
`superoxide combine another potentially harmful product,
`peroxynitrite is formed.72 Interestingly, it is now clear that
`XO and NOS can generate either NO or superoxide, depend-
`ing on the substrate supply and the oxygen status (Figure 3).
`Although NOS requires oxygen to generate NO from
`L-arginine,34 XO-catalyzed nitrite reduction to NO is greatly
`enhanced by hypoxia.66,67 Superoxide generation from NOS
`is enhanced when the supply of arginine or cofactors is
`scarce,73,74 whereas XO generates superoxide, for example,
`during reperfusion after ischemia.61 The balance between
`substrate supply and tissue oxygenation may determine
`whether the net effects of these combined enzyme systems
`are beneficial or harmful in a particular situation. Thus, the
`
`Figure 2. Vasorelaxation by nitrite. Origi-
`nal recordings of isolated segments of
`rat aorta in organ bath containing modi-
`fied Krebs solution of neutral pH (top
`curve) and low pH (bottom curve) when
`contracted with phenylephrine (phe;
`10 ⫺6 M), followed by addition of sodium
`nitrite in increasing concentrations. Time
`scale is indicated for 60 seconds. Repro-
`duced from Modin et al44 with
`permission.
`
`dominating species generated could be NO, oxygen radicals,
`or their reaction products.
`
`Reduction by Deoxyhemoglobin
`Hb can interact with NO and related compounds in many
`ways (Figure 4). The traditional view is that Hb in the red
`blood cells is an extremely effective NO scavenger, and in
`many ways, this notion still holds true.27 Oxygenated Hb
`reacts rapidly with NO to form nitrate and methemoglobin
`(met-Hb), and this reaction is near– diffusion limited. How-
`ever, other interactions between NO and Hb have been
`characterized, including formation of nitrosyl-Hb and SNO-
`Hb, as discussed above. In 1981, Doyle et al described a
`specific reaction between nitrite and deoxyhemoglobin
`(deoxy-Hb) by which met-Hb and NO is formed (Figure 4).75
`However, these observations were made before the biological
`significance of NO was revealed. More recently, several
`authors have expanded on the role of this reaction in NO
`physiology.76 –78 It is suggested that nitrite is recycled back
`into bioactive NO by this mechanism and that this ensures an
`autoregulated NO generation in regions of poor oxygenation
`where deoxy-Hb predominates. Cosby et al showed that
`
`Figure 3. XO and NOS are capable of generating either NO or
`⫺) depending on the conditions. When the supply
`superoxide (O2
`of L-arginine and oxygen is good, NOS makes NO, whereas the
`same enzyme may generate considerable amounts of superox-
`ide when arginine or cofactors are limited. XO generates super-
`oxide, for example, during reperfusion after ischemia, whereas
`nitrite reduction to NO occurs preferentially during hypoxia. NO
`generation from XO can be beneficial and work as a backup
`system to supply NO during hypoxia when NO synthesis from
`NOS is compromised. Detrimental effects of these 2 enzyme
`systems can also be foreseen, for example, in a situation in
`which NO and superoxide are generated simultaneously and
`react to form potentially harmful peroxynitrite.
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`NOS-Independent NO Generation From Nitrite
`
`919
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`reaction.76 Using inhibitors of the respiratory chain for
`chemical sequestration of respiratory segments, Kozlov et al
`identified the ubiquinone/cyt bc1 couple as another reductant
`site where nitrite is recycled.83,84 Recycling of NO from
`nitrite requires respiring mitochondria under conditions es-
`tablished during ischemia, when electron carriers are highly
`reduced.83
`The magnitude of NO generated from nitrite by mitochon-
`drial enzymes in vivo and its physiological or pathophysio-
`logical role needs to be clarified. The high affinity of NO to
`the heme-iron of cytochrome oxidase may support a detri-
`mental role because NO has been shown to impede the
`energy-linked respiration and to trigger mitochondrial gener-
`ation of superoxide radicals.84,86 – 89 On the other hand, ben-
`eficial effects of NO on mitochondrial function have also
`been described. For example, NO stimulates mitochondrial
`biogenesis in vitro and in vivo, which results in increased
`mitochondrial function and enhanced ATP formation.88 –90
`
`Nitrate and Nitrite Reduction by
`Commensal Bacteria
`Some bacteria are highly effective in reducing nitrate and
`nitrite.35 They use these substrates as alternative electron
`donors during oxygen starvation or in protein synthesis for
`incorporation in biomass.91 Nitrite reduction to NO is a part
`of the nitrogen cycle in nature and is performed by denitri-
`fying anaerobic bacteria in soil and sediments.35 NO gener-
`ation also involving commensal bacteria was reported re-
`cently by Sobko et al. Luminal levels of NO were measured
`along the gastrointestinal tract of rats and were found to be
`high in conventional rats but almost absent in germ-free
`animals.92 NO generation by gut commensals is likely a
`combination of nitrite reduction by bacterial nitrite reductases
`and acidification (eg, by lactic acid–producing bacteria re-
`sulting in nonenzymatic reduction of nitrite; J.O. Lundberg,
`unpublished observation, 2005). Interestingly, bacteria may
`participate not only in local generation of NO in the gastro-
`intestinal tract but also systemically. In fact, the oral micro-
`flora is required to obtain an increase in plasma nitrite after
`ingestion of nitrate.38 This is because ingested nitrate has to
`pass the salivary glands into saliva before it is reduced to
`nitrite by oral bacterial enzymes. Indeed, if a test person
`avoids swallowing after ingestion of nitrate, the increase in
`plasma nitrite is abolished.38 With the recently described
`identification of systemic nitrite generation from inorganic
`nitrate, a reverse pathway for regeneration of NO from nitrate
`is completed. Thus, the nitrate ion could be considered a
`major vascular storage pool for NO rather than an inert waste
`product. In an extension, this implies that generation and
`activity of NO in the cardiovascular system can be influenced
`by dietary intake of nitrate.
`
`Nitrite and Nitroglycerine
`The mechanism behind the vasodilatory action of organic
`nitrates (eg, nitroglycerine, glyceryl
`trinitrate [GTN]) re-
`mained a mystery for almost 100 years before Murad et al
`showed in 1978 that they act through release of NO via
`activation of soluble guanylyl cyclase.93,94 However, still
`today, the exact way by which NO is formed in vivo from
`
`Figure 4. Simplified overview of some reactions of NO and
`nitrite with Hb. Depending on conditions, NO can be generated,
`bound, or consumed by these reactions. Hb-Fe(II) indicates
`deoxyhemoglobin.
`
`intra-arterial infusion of nitrite at near-physiological levels
`caused vasodilation in healthy people.77 This was an exten-
`sion of previous research from the same group and others,
`showing the existence of an arteriovenous gradient for ni-
`trite.23,41,79 In many ways, nitrite is an ideal vascular storage
`pool for NO. It is present in blood at fairly high levels (0.1 to
`0.5 ␮mol/L), and it is much more stable than NO or SNOs.
`When oxygen is sufficient, the predominant reaction product
`from nitrite and Hb would be nitrate, whereas NO formation
`is favored when oxygen levels fall. Therefore, an autoregu-
`lated system for optimal NO delivery along the entire oxygen
`gradient is created similar to the original theory involving
`SNO-Hb formation described above. As with the SNO-Hb
`concept, deoxy-Hb/nitrite– dependent vasorelaxation is also
`debated, and some authors have failed to see any vasodilatory
`effects of physiological amounts of nitrite.41,80 A remaining
`key question is by which mechanism nitrite is taken up by the
`red blood cell and how NO is exported without being trapped
`by the abundant heme. A compartmentalized NO production
`at the red blood cell membrane, coupled with the extremely
`low levels of NO needed for vasodilation, has been suggest-
`ed.77 Others have suggested the nitrite effects are caused by
`SNO-Hb formation.81 Another criticism that has evolved is
`related to the site of maximum NO release.82 According to the
`nitrite/deoxy-Hb theory, NO generation in blood is expected
`to be greatest
`in the veins where deoxy-Hb levels are
`maximal, but
`these vessels are not actively involved in
`control of blood flow. On the other hand, only minute
`concentrations of NO are needed for vasorelaxation, so the
`amounts released earlier
`in the vascular
`tree may be
`sufficient.
`
`Reduction by Mitochondrial Enzymes
`Components of the respiratory chain in mitochondria are
`theoretically well suited for NO generation from nitrite
`because they could act as electron carriers when nitrite is
`reduced. In addition, nitrite-reducing bacteria use periplas-
`matic cytochrome complexes for effective reduction of ni-
`trite.35 Indeed, several studies have now convincingly shown
`that respiring mitochondria in mammalian cells76,83,84 and in
`plants85 can generate NO from nitrite. The idea of nitrite
`reduction by mitochondria was first put forward by Reutov
`and Sorokina, who suggested that cytochrome c oxidase was
`the enzyme most likely responsible for carrying out this
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`these agents remains controversial. Interestingly, several
`observations suggest that the major obligate intermediate in
`this process is in fact nitrite (Figure 1).86,95,96 Chen et al
`showed that a mitochondrial aldehyde dehydrogenase cata-
`lyzed formation of nitrite from GTN in smooth muscle cells.96
`In this study, the further reduction to NO from nitrite was not
`characterized. Hepatocytes generate nitrite from GTN by
`glutathione–S-transferase, and in these cells, nitrite is further
`reduced to NO by an enzyme of the cytochrome P-450
`family.86 Other candidates for the final step in GTN-derived
`nitrite reduction to NO are the mitochondrial enzymes and the
`XO discussed above. Considering the potent vasorelaxatory
`effects of organic nitrates, this again illustrates that highly
`efficient intracellular pathways exist to generate NO from
`nitrite. Because the nitrite ion itself is charged, it traverses
`biological membranes more slowly and less effectively than
`organic compounds (eg, nitroglycerine and amyl nitrite), and
`this probably explain why inorganic nitrite is a much less
`potent vasodilator than organic nitrates/nitrites.
`
`Summary and Future Directions
`Oxidation of NO to nitrite and nitrate acutely terminates its
`biological activity. However, as discussed here, several dis-
`tinct pathways exist in vivo whereby nitrite can be reduced
`back into bioactive NO. Interestingly, these reactions are all
`greatly enhanced during ischemia with low oxygen tension, a
`situation in which NO synthesis by NOS is repressed. The
`different pathways work in concert to ensure sufficient NO
`synthesis along the entire oxygen gradient. When oxygen
`supply is sufficient, NO generation from NOS will dominate.
`However, with falling oxygen tensions and ischemia, the
`nitrite-dependent pathway comes into play.
`In blood,
`deoxy-Hb seems to be the predominant catalyzer of nitrite
`reduction to NO, whereas in a situation of tissue hypoxia,
`XO-derived NO also becomes important. Finally, in situa-
`tions of global ischemia when pH drops dramatically in the
`tissues, NO generation can occur via simple nonenzymatic
`acidic reduction of nitrite, a reaction that is accelerated by
`vitamin C and other reducing agents. One could view this as
`a salvage pathway for NO, where much of the spill over from
`NOS-derived NO is being recycled when most needed. A
`major future challenge for the researchers working in this
`field will be to finally settle the exact significance of
`nitrite-dependent NO in normal physiology as well as patho-
`logical states for example during tissue hypoxia. Because diet
`can have a great influence on systemic levels of nitrate and
`nitrite, it will be very interesting to study the long-term
`effects of nitrate-rich food (eg, vegetables) on systemic
`nitrite-derived NO generation. Indeed,
`it
`is tempting to
`speculate that the well-known cardioprotective effects of a
`vegetarian diet in part could be attributed to a continuous
`low-grade generation of NO from nitrate in the diet via
`formation of nitrite. A study by Richardson et al actually
`supports the notion that inorganic nitrate can have systemic
`effects in humans.97 They found that ingestion of sodium
`nitrate in an amount equivalent to 300 g of spinach inhibited
`platelet function, an effect that might be attributed to NO.
`It
`is possible that
`increasing knowledge about nitrite-
`derived NO can result in development of new drugs to be
`
`used in cardiovascular medicine. Nitrite-based pharmaceuti-
`cals that selectively deliver NO only to ischemic areas are
`indeed an interesting alternative approach. Theoretical advan-
`tages with such compounds in relation to traditional organic
`nitrates could be fewer side effects (eg, headache) and
`possibly less development of “nitrate tolerance.” As dis-
`cussed here, several recent studies suggest a protective role of
`nitrite-derived NO, but in certain situations (eg, ischemia-
`reperfusion), a massive NO generation from NOS-
`independent sources could instead be harmful, for example, if
`it coincides with generation of superoxide, thereby promoting
`formation of oxidizing radicals.
`Besides nitrite-derived NO, which is the main focus of this
`review, other means of preserving NO activity in blood have
`been suggested, including SNO formation and transport of
`free NO, and there is an intense ongoing debate as to which
`pathway is the most significant in vascular biology. These
`controversies aside, today, there is no longer any doubt that
`NO activity in blood can be preserved for much longer than
`originally believed. In addition, nitrite, the oxidation product
`of NO, can be recycled into bioactive NO again in blood and
`tissues. These novel aspects of NO biology will provide
`further important insights into the mechanisms of blood flow
`regulation, which could lead to novel strategies for treatment
`and prevention of cardiovascular pathologies.
`
`Acknowledgments
`The authors acknowledge support from the Swedish Research
`Council,
`the Swedish Heart and Lung Foundation,
`the Ekhaga
`Foundation, and the EU 6TH Framework Program.
`
`References
`1. Furchgott RF, Zawadzki JV. The obligatory role of th