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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:697.)
© 2006 American Heart Association, Inc.

Brief Reviews

Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and Therapeutics

Daniel B. Kim-Shapiro; Alan N. Schechter; Mark T. Gladwin

From the Department of Physics (D.B.K.-S.), Wake Forest University, Winston-Salem, NC; Laboratory of Chemical Biology (A.N.S.), NIDDK, National Institutes of Health, Bethesda, Md; Vascular Medicine Branch (M.T.G.), NHLBI and Critical Care Medicine Department Clinical Center, National Institutes of Health, Bethesda, Md.

Correspondence to Daniel Kim-Shapiro, Department of Physics, Wake Forest University, Winston-Salem, NC 27109. E-mail shapiro{at}wfu.edu

Series Editor: Joseph Loscalzo
ATVB In Focus Nitric Oxide Redux

	Abstract

Top Abstract Introduction Destruction of NO by... Preservation of NO Activity... Endocrine NO Transport as... The SNO-Hb Hypothesis Pathology and Therapeutics References

 
The ability of oxyhemoglobin to inhibit nitric oxide (NO)-dependent activation of soluble guanylate cyclase and vasodilation provided some of the earliest experimental evidence that NO was the endothelium-derived relaxing factor (EDRF). The chemical behavior of this dioxygenation reaction, producing nearly diffusion limited and irreversible NO scavenging, presents a major paradox in vascular biology: The proximity of large amounts of oxyhemoglobin (10 mmol/L) to the endothelium should severely limit paracrine NO diffusion from endothelium to smooth muscle. However, several physical factors are now known to mitigate NO scavenging by red blood cell encapsulated hemoglobin. These include diffusional boundaries around the erythrocyte and a red blood cell free zone along the endothelium in laminar flowing blood, which reduce reaction rates between NO and red cell hemoglobin by 100- to 600-fold. Beyond these mechanisms that reduce NO scavenging by hemoglobin within the red cell, 2 additional mechanisms have been proposed suggesting that NO can be stored in the red blood cell either as nitrite or as an S-nitrosothiol (S-nitroso-hemoglobin). The latter controversial hypothesis contends that NO is stabilized, transported, and delivered by intra-molecular NO group transfers between the heme iron and ß-93 cysteine to form S-nitroso-hemoglobin (SNO-Hb), followed by hypoxia-dependent delivery of the S-nitrosothiol in a process that links regional oxygen deficits with S-nitrosothiol–mediated vasodilation. Although this model has generated a field of research examining the potential endocrine properties of intravascular NO molecules, including S-nitrosothiols, nitrite, and nitrated lipids, a number of mechanistic elements of the theory have been challenged. Recent data from several groups suggest that the nitrite anion (NO₂^–) may represent the major intravascular NO storage molecule whose transduction to NO is made possible through an allosterically controlled nitrite reductase reaction with the heme moiety of hemoglobin. As subsequently understood, the hypoxic generation of NO from nitrite is likely to prove important in many aspects of physiology, pathophysiology, and therapeutics.

Several factors nitigate nitric oxide (NO) scavenging by red blood cell encapsulated hemoglobin (Hb). Recent data suggests that the nitrate amion is converted to NO by Hb under hyposia. The hypothesis is currently the subject of intense investigations in the areas of biochemistry, physiology, and therapatics.

Key Words: nitric oxide • nitrite • hemoglobin • vasodilation • red blood cell

	Introduction

Top Abstract Introduction Destruction of NO by... Preservation of NO Activity... Endocrine NO Transport as... The SNO-Hb Hypothesis Pathology and Therapeutics References

 
Nitric oxide (NO) is the endothelium-derived relaxing factor that modulates vascular tone by activating soluble guanylyl cyclase (sGC) in smooth muscle.^1–4 It is now appreciated that NO is produced in endothelial cells by the endothelial NO synthase enzyme and participates in many aspects of normal vascular physiology, including tonic vasodilation and inhibition of platelet activation and endothelial adhesion molecule expression. Endothelial dysfunction, characterized by reduced bioavailability of endothelial derived NO, is a central mechanistic feature of coronary artery disease and its risk factors, including diabetes, hypertension, smoking, and obesity. Excessive NO production from inducible NO synthase is a central mechanism of septic shock. Novel therapeutic strategies based on increasing or decreasing native concentrations of NO are undergoing investigation or have already translated into clinical practice.

Historically, hemoglobin (Hb) was thought to interact with NO solely in a manner that would inactivate it. Given the rapid rate that NO is scavenged by hemoglobin, the amount of NO that could diffuse from endothelial cells to smooth muscle cells would seem insufficient to effect vasodilation.⁵ Thus, there is a paradox between NO working as the endothelium-derived relaxing factor and its proximity to hemoglobin, the NO destroyer.

A number of mechanistic solutions to this paradox have been proposed. The first invokes rate limitations caused by NO diffusion to the erythrocyte, through the red cell submembrane protein lattice, and from the endothelium in laminar flowing blood. The second solution to the paradox suggests that blood plasma and/or red cells may stabilize and transport NO species that generate NO, particularly in hypoxic or acidic tissues (hypoxic vasodilation).

Several NO-modified compounds have been proposed as species that function to preserve and subsequently transduce NO bioactivity in blood.^6–11 One set of compounds proposed to preserve NO activity are the nitrosothiols.^6,8,12,13 This mechanism was first advanced with the measurement and characterization of S-nitrosated serum albumin, found to be present in human blood and capable of stable intravascular transduction of NO-dependent vasodilation and inhibition of platelet aggregation.^12,13 This paradigm of preservation and endocrine delivery of NO in blood via S- nitrosation of a protein was subsequently extended to the S-nitrosation of hemoglobin to form S-nitrosated hemoglobin (SNO-Hb), which was proposed to allosterically deliver NO to hypoxic tissue.¹⁴

Recently, we and others have proposed that NO bioactivity may be transduced by plasma, erythrocyte, and tissue nitrite (NO₂^–).^{15,16,16a,16b} Under conditions of low oxygen and pH, nitrite can be reduced to NO by acidic disproportionation and by the reductase activity of xanthine oxidoreductase.^17–20 In addition, we have found that hemoglobin possesses a nitrite reductase activity that is allosterically regulated with maximal reductase activity at the hemoglobin P₅₀.^21,22 The recent appreciation of this mechanism and observations that nitrite vasodilates the human circulation at near-physiological concentrations supports a role for nitrite in hypoxic vasodilation.^21,22 Therapeutic use of nitrite is being tested for a variety of pathological conditions.^23–25

In this article, we review in detail the specific mechanisms invoked that limit the dominant inactivation reactions of hemoglobin and NO in an effort to clarify the critical role of hemoglobin and the red cell in modulating NO bioavailability within the vasculature.

	Destruction of NO by Hemoglobin: Dioxygenation and Iron-Nitrosylation Reactions

Top Abstract Introduction Destruction of NO by... Preservation of NO Activity... Endocrine NO Transport as... The SNO-Hb Hypothesis Pathology and Therapeutics References

 
The major way that Hb destroys NO is through the dioxygenation reaction in which NO reacts with oxygenated hemoglobin (OxyHb) to form methemoglobin (MetHb, in which the heme irons are ferric) and nitrate.

This reaction occurs at a rate of 6 to 8x10⁷ M^–1s^{–1 26–28} so that the half-life of NO in an oxygenated red blood cell (RBC) is &0.5 µs. During this time, intraerythrocytic NO could only diffuse &0.02 µm (assuming an intraerythrocytic diffusion constant of 1000 µm² s^–1). Without regard to other potential factors discussed, the RBC thus appears to NO as a black hole—there is no escape.

If one had 10 mmol/L cell-free rather than RBC encapsulated Hb in the blood, taking a diffusion rate of 3300 µm² s^–1 in the plasma, NO would only have a half life of &1 µs and could only diffuse &0.1 µm. The effectiveness of the hemoglobin sink is only slightly diminished by the fact that the smooth muscle cells are on one side of the endothelium and the blood is on the other.²⁹ Diffusion of individual NO molecules has no preferred direction and the blood can be viewed as a sink for NO from which there is no return. The net flux of NO is always defined by the spatial gradient in its concentration so that the presence of Hb on one side of the endothelium decreases the concentration of NO on the other side, as well. Based on this efficient scavenging of Hb in the blood, the ability of NO to diffuse from endothelial cells to smooth muscle cells to activate sGC should be limited.⁵ However, we know of course that this sGC activation and NO-dependent vasodilation does indeed occur. The reason that endothelial-derived NO does not undergo the dioxygenation reaction (see equation) to the extent predicted, based purely on kinetic calculations, is caused by the fact that RBC encapsulated Hb in the blood reacts with NO much slower than does cell-free Hb.^30–40 Three mechanisms have been identified that contribute to reduced NO scavenging by RBCs (Figure 1): (1) to a large extent, the rate of the reaction is limited by the time it takes NO to diffuse to the red cell (2) there is a physical barrier to diffusion of NO across the RBC membrane; and (3) RBC encapsulated Hb is efficiently compartmentalized in the lumen—there is no extravasation of RBC encapsulated Hb into endothelium and the interstitium.

View larger version (61K): [in this window] [in a new window]   Figure 1. Factors involved in mitigating NO scavenging by RBCs. NO is produced in endothelial cells (EC) by the enzyme NO synthase (NOS), which then must diffuse to smooth muscle cells (shown at the very top and bottom of the illustration) without getting scavenged by red cells in the blood. Velocity gradients result in differential pressures that push the red cells inward and create the cell-free zone, which reduces NO scavenging by RBCs. Nitric oxide is depleted around the red cell creating an inhomogeneous NO concentration called the unstrirred layer. NO must diffuse across the unstirred layer, which slows down NO uptake, particularly in stopped-flow experiments. An intrinsic membrane barrier has been purported to exist at the red cell surface that may involve the protein scaffold and diminish NO uptake. Hemolysis results in release of cell-free hemoglobin into the plasma with enhanced NO scavenging potential caused by the fact that the cell-free Hb enters the cell-free zone and can even extravasate into the interstitial tissue. This illustration is not drawn to scale.

External Diffusion Limitations: The Cell-Free Zone Along Endothelium and the Unstirred Layer Around the Erythrocyte
The first external diffusion barrier that reduces NO scavenging occurs secondary to a red cell-free zone (and thus hemoglobin free zone) next to the endothelium, which is created by blood flow velocity gradients.^32–34 Faster relative blood flow velocities in the center of the blood vessel result in lower relative pressures (Bernoulli’s Principle) and concentration of the red cells. When you turn on your shower, the air in the bath moves faster, the pressure is reduced, and the shower curtain moves inward. Likewise, the velocity of blood is slowest near the vessel wall, so there is a pressure gradient pushing the red cells inward. This creates the cell-free zone. With a cell-free zone of 5 µm (a reasonable conservative estimate for many arterioles^32,41), the lifetime of NO would be &7.5 ms before it would reach the red cell-rich zone and be rapidly scavenged (ignoring scavenging by other species within the cell-free zone). Note that the estimated lifetime of the NO increases by a factor of almost 10 000 because of the cell-free zone.

In 1999, Liao et al conducted seminal experiments demonstrating the importance of flow on NO scavenging by examining vasoconstriction in a microvessel bioassay.³⁴ In addition to the cell-free zone that develops during flow, mechanotransduction may also have contributed to the effects of flow observed by Liao et al.³⁴ Mechanotransduction is the process by which cells convert mechanical stimuli into biochemical signals. Flow of red blood cells produces shear stress, which augments endothelial NO production.^42–46 It is possible that when flow decreased in the bioassay, mechanotransduction also decreased.

Liao et al also demonstrated that even in the absence of flow, 1000-times more RBC-encapsulated Hb than cell-free Hb was required to produce equivalent vasoconstriction in the bioassay.³⁴ These results demonstrated an intrinsically slower rate of NO consumption by RBCs, as had been measured earlier³⁹ and also demonstrated for oxygen uptake.^31,47 These experiments suggested that kinetic limitations for NO uptake exist because of the unstirred layer around the red blood cell and possibly due to an intrinsic barrier in the submembrane space. The notion of an unstirred layer stems largely from experiments in which RBCs are rapidly mixed with NO (or oxygen) using a stopped-flow device.^30,31,47,48 Generally, even when the bulk solution is stirred, there is a region surrounding a cell membrane that is static.⁴⁸ NO that is adjacent to the RBCs is rapidly taken up so that the concentration of NO is made inhomogeneous. NO uptake by the RBCs is then rate-limited by external diffusion of NO through the unstirred layer, similar to the rate limiting diffusion of NO in a cell-free zone.

Intrinsic Membrane Barrier
In addition to the role of the unstirred layer in limiting the rate that the RBCs take up NO, a role for a physical barrier to diffusion across the red cell membrane has been proposed.^35,38,49 The notion that there is a physical barrier to diffusion within the red cell membrane or submembrane protein lattice is difficult to accept given that NO is expected to have similar properties in traversing membranes as oxygen. However, strong evidence for a role of the RBC membrane in limiting NO uptake has been provided by direct experiments in which chemical or physical modification of the RBC membrane resulted in a significant change in the RBC NO uptake rates.^38,49 The nature of the intrinsically slower rate of NO uptake by RBCs compared with cell-free Hb has been the subject of some controversy. Lancaster and others have argued that the unstirred layer that forms around the red blood cell is the major factor that limits NO diffusion.^37,39,50 Liao et al have argued that a physical membrane barrier within and beneath the red cell membrane is primarily responsible for the relatively slow NO consumption by RBCs.^35,38,49 The extent of the contribution of each factor, external diffusion versus intrinsic membrane barrier, is the subject of continuing investigation.

Hemoglobin Extravasation
Whereas red cells are too large to extravasate out of the blood vessel lumen, cell-free Hb can. Extravasation of cell-free Hb into the subendothelial space could increase NO consumption by allowing the Hb to come closer to the source of NO. Consequently, extravasation has been proposed to play a major role in the hypertensive effect of hemoglobin-based blood substitutes.^39,51–55 The hypertensive effect of various hemoglobins is partially dependent on their size.^52,53 It is possible that in addition to rate limitations caused by diffusion, extravasation of hemoglobin into the interstitial space may have contributed to the 1000-fold difference in scavenging between cell-free and RBC-encapsulated Hb observed by Liao et al in the absence of flow.³⁴

	Preservation of NO Activity by Hb

Top Abstract Introduction Destruction of NO by... Preservation of NO Activity... Endocrine NO Transport as... The SNO-Hb Hypothesis Pathology and Therapeutics References

 
One method that Hb could, in principle, preserve NO bioactivity is through the reaction of NO with deoxygenated hemes to form iron-nitrosyl Hb (HbNO):

This reaction, which can occur at any one of the 4 hemes of the hemoglobin tetramer when they are deoxygenated, does so at a rate of 2 to 6x10⁷ M^–1s^–1.^56–59 However, the rate of NO release is extremely slow (&10^–3-10^–5 s^–160–63). This slow off-rate argues that the capture of NO by deoxygenated hemes is not an effective way to subsequently transduce NO bioactivity. Moreover, once the NO is released from the deoxygenated hemes, it must once again contend with the large concentrations of oxyHb or deoxyHb in the RBC that can oxidize NO to nitrate (first equation) or re-bind the NO (second equation).

With the proposal that albumin and hemoglobin could stabilize and transport NO as an S-nitrosothiol (this bond requires a 1- electron oxidation of either NO or the sulfhydryl), Loscalzo and Stamler challenged the existing paradigm that blood could only react with and destroy NO.^12,13 Whereas there is increasing acceptance that endocrine NO transport in blood does occur, a heated controversy surrounds the nature of the endocrine NO species in blood, the relative contribution of these species to basal and hypoxic blood flow, and the mechanisms of formation and subsequent NO release. In addition to SNO-albumin and SNO-hemoglobin, other putative candidates for endocrine NO species include iron-nitrosylated hemoglobin,^64,65 N-nitrosated proteins,^66,67 nitrated lipids,^68–71 and the anion nitrite.^{15–17,19,72–76} In this article, we focus on 2 of these models, the hemoglobin nitrite reductase hypothesis and the SNO-Hb hypothesis.

	Endocrine NO Transport as Nitrite and the Nitrite Reductase Activity of Hemoglobin

Top Abstract Introduction Destruction of NO by... Preservation of NO Activity... Endocrine NO Transport as... The SNO-Hb Hypothesis Pathology and Therapeutics References

 
Nitrite is produced or accumulates in the blood and tissues by reaction of NO with oxygen and with other yet-to-be identified NO oxidases, and by dietary intake of nitrite and nitrate, with the latter being concentrated in the saliva and then reduced to nitrite by commensal bacteria resident in the posterior crypts of the tongue.^75,77–80 It has long been known that nitrite is vasoactive at high pharmacological concentrations,⁸¹ probably because of its ability to activate sGC.^82,83 Under physiological conditions, nitrite was thought to be biologically inert and incapable of vasodilation.^84–88 However, recent evidence suggests that nitrite is vasoactive under physiological conditions by reduction to NO.^{15–18,25,75,76,89–92} Reduction of nitrite to NO via acidic reduction and the enzymatic activity of xanthine oxidoreductase has been recently reviewed.⁷⁵ Here, we focus on Hb-mediated nitrite reduction and vasodilation, a mechanism that has recently been supported by in vitro biochemical and aortic ring bioassay studies and in vivo nitrite infusions in human volunteers.^15,93

In 2000, Gladwin et al noted gradients in plasma nitrite concentrations during basal conditions and during NO breathing by human subjects.⁹⁴ The authors proposed that circulating nitrite is bioactive and provides a delivery gradient of intravascular NO.⁹⁴ In 2003, this proposal was confirmed by studies showing increased blood flow during infusions of nitrite.¹⁵ Nitrite was infused into the forearm at pharmacological (200 µmol/L) and near physiological levels (0.9 to 2.5 µmol/L, basal plasma levels are &0.3 µmol/L⁶⁷) and an increase in blood flow was measured by strain gauge plethysmography.¹⁵ In the presence of an NO synthase inhibitor, infusion of near physiological levels of nitrite also increased forearm blood flow during exercise, indicating a potential role of the observed nitrite chemistry in hypoxic vasodilation.¹⁵

HbNO formation was detected across the forearm circulation during nitrite infusions, suggesting that nitrite was being reduced to NO during one artery-to-vein transit.¹⁵ Reduction of nitrite to form HbNO has also recently been shown after ingestion of nitrite and nitrate.⁹⁵ The formation of HbNO does not necessarily mean that the reaction between nitrite and Hb is responsible for vasodilation. However, data supporting this notion have been presented using an aortic ring bioassay.^15,93 These vessels spontaneously relax at very low oxygen tensions (10 to 15 mm Hg) in the absence of added red cells or nitrite. However, the oxygen tension at which the vessels relax was shifted to higher pressures in the presence of nitrite (0.5 to 2 µmol/L) and red blood cells (40 mm Hg).¹⁵ In addition, it was shown that deoxygenated Hb in the presence of 100 nM nitrite effected vasodilation at a constant oxygen pressure, whereas oxygenated Hb had no effect even with >50 µmol/L nitrite present.¹⁵ These results indicate that Hb acts as a nitrite reductase under hypoxic conditions and likely contributes to nitrite-dependent vasodilation.

Reduction of nitrite by deoxygenated Hb was first reported by Brooks⁹⁶ and later studied by Doyle et al.⁹⁷ The primary reaction of nitrite with deoxyhemoglobin produces NO and MetHb. The NO can then bind to vacant deoxygenated hemes, forming HbNO. The pH dependence of the kinetics implicates the involvement of nitrous acid (HONO).^22,97 The reaction can be summarized as follows:

where K is an equilibrium constant, k_o is &12.3x10³ M^–1s^–1, and k_a is the rate that NO binds to deoxygenated Hb (&10⁷ M^–1s^–1) referred to in the second equation in this article.⁹⁷ Brooks found that in the presence of excess nitrite, 2 deoxygenated Hb molecules are converted to 1 MetHb and 1 HbNO (in hemes), as predicted by equations 3 to 5.⁹⁶ However, Doyle et al found a stoichiometry of &7:3 MetHb:HbNO.⁹⁷

The reaction of nitrite with deoxygenated Hb has recently been re-examined.^16,21,22,98 Importantly, when samples are thoroughly deoxygenated without significant formation of MetHb, the stoichiometry of the product follows that first observed by Brooks (1HbNO:1MetHb).^21,22 Rifkind et al proposed that significant Fe(III)NO-Hb (or MetHb-NO) also accumulates in the reaction.¹⁶ In fact, it was suggested that in vivo 75% of NO bound to Hb is ligated to ferric hemes (MetHb-NO) rather than to ferrous hemes (HbNO).¹⁶ Although MetHb-NO may be a transient intermediate in the reaction of nitrite and Hb, it is difficult to see how it could be present in steady-state conditions or even accumulate during the reaction at detectable levels because the affinity of MetHb for NO is one million times lower than that of deoxygenated (ferrous) Hb and the dissociation rate of NO from MetHb is &1 s^–1.⁹⁹ Spectral analysis of absorption spectra collected during the reaction of nitrite with deoxygenated Hb does not support the accumulation of significant amounts of MetHb-NO.^21,98

When nitrite is in excess to Hb, one expects pseudo–first-order kinetics to govern the reaction described by equations 3 to 5. However, it has recently been discovered that the kinetics appear closer to zero order and are sigmoidal.^21,22 This surprising result was explained by the notion that the reduction of nitrite by R-state Hb is faster than that by T-state Hb.^21,22 This allosteric mechanism, together with the fact that a single nitrite molecule produces 1 HbNO and 1 MetHb, leads to the reaction speeding up as Hb is converted from T-state to R-state during the progress of the anaerobic reaction.^21,22 Consistent with an allosteric mechanism, it was found that the Hb-mediated nitrite reduction proceeds most rapidly at approximately the p50, where half the Hb is oxygen-bound.²² At the p50, the reduced rate of the reaction caused by fewer deoxygenated hemes being present than at zero oxygen is compensated for by the presence of more deoxygenated hemes being present in R-state Hb tetramers. The oxygen saturation dependence of the reaction rate has been correlated with the efficacy of Hb-mediated nitrite-dependent vasodilation, implicating Hb as a mammalian nitrite reductase enzyme that is likely to play a significant role in hypoxic vasodilation and hypoxic NO homeostasis (Figure 2).^22,93

View larger version (77K): [in this window] [in a new window]   Figure 2. A model of intravascular metabolism of NO. NO produced by eNOS may diffuse into the vascular lumen as well as the underlying smooth muscle. The majority of this NO enters the erythrocyte and reacts with oxyhemoglobin (Oxy Hb) to form nitrate (NO3–); a minor portion may escape the hemoglobin scavenger and react with plasma constituents to form nitros(yl)ated species (RXNO, nitrosothiols: RSNO), nitrated lipids (NO2 lipids), and nitrite (NO2–). Each of these species is capable of transducing NO bioactivity far from its location of formation. Nitrite may diffuse into the erythrocytes, where it appears in a higher concentration than in plasma. In the erythrocyte, nitrite reacts with deoxyhemoglobin (Deoxy Hb) to form NO and methemoglobin (Met Hb) and other NO adducts. NO can then diffuse either out of the erythrocyte directly or via an intermediate NO metabolite. The question mark circled in white refers to the possibility of an intermediate during nitrite bioactivation. NO Hb indicates iron nitrosylhemoglobin; SNO Hb, nitrosohemoglobin; L-Arg, L-arginine; L-Cit, L-citrulline; NxOy, higher N oxides or NO itself (x=y=1). This figure was originally published in Blood92 and is reproduced with permission. © The American Society of Hematology.

If the red cell is an NO black hole, how could NO formed by reduction of nitrite get out and effect vasodilation? Recently, calculations and experimental data were presented that show that it is extremely unlikely that NO itself, formed from the nitrite reaction in the red cell, could be exported in sufficient amounts as to effect vasodilation.¹⁰⁰ It is possible that special circumstances exist in the red cell so that NO formed by reduction of nitrite is formed in a compartmentalized way near the surface of the red cell so that export can be facilitated. Analogous to endothelial nitric oxide synthase (eNOS) compartmentalization within the caveolae, a nitrite reductase metabolin may form in the lipid rafts. Band 3/AE1 transports anions (possibly nitrite) and binds carbonic anhydrase (which produces proton), deoxyhemoglobin (which will reduce nitrite), and aquaphorin and Rh proteins (which may transport NO or other gas molecules). Another possibility is that there is an intermediate in the reaction of nitrite and Hb such as a nitrosothiol, peroxynitrite, nitrogen dioxide, hydrated NO, or a nitrated lipid, which can diffuse out of the RBC and transduce NO activity. Whether additional chemistry to that described by equations 3 to 5, wherein an NO activity exporting intermediate is involved, remains to be elucidated and is the subject of intense current research.

	The SNO-Hb Hypothesis

Top Abstract Introduction Destruction of NO by... Preservation of NO Activity... Endocrine NO Transport as... The SNO-Hb Hypothesis Pathology and Therapeutics References

 
A prominent, yet controversial, hypothesis for transducing NO activity from HbNO involves the formation of Hb that is S-nitrosated at the ß-93 cysteine, referred to as SNO-Hb.^14,101–107 The SNO-Hb hypothesis holds that NO is captured by a deoxyheme on oxygenated hemoglobin via allosterically controlled association kinetics and is then transferred to the ß-93 cysteine when Hb undergoes conformational changes associated with the TR state transition on re-oxygenation. This allosterically controlled transition is thought to reverse on deoxygenation along with some of the NO (actually NO⁺) on the ß-93 cysteine being transferred to thiols on the anion exchange protein within the red blood cell membrane and then exported out of the cell as an "X-NO." The identity of X-NO and mechanism of export has not been determined. Thus, Hb is envisioned as a transporter rather than a destroyer of NO activity.

Many aspects supporting and refuting aspects of the SNO-Hb hypothesis have been reviewed previously.^{59,106,108–114} The challenges include: (1) an inability to measure the micromolar concentrations and artery-to-vein gradients of SNO-Hb or HbNO reported by the Stamler group^{64,65,115–117}; (2) an inability to reproduce the observation of preferential binding of NO on the deoxyhemes of R-state (oxygenated) hemoglobin (ie, allosterically controlled association kinetics)^65,118–122; (3) an inability to observe oxygenation dependent transfer of the NO from the ß-chain heme to the cysteine 93 and the deoxygenation-dependent transfer of NO from the cysteine 93 back to the heme (ie, cycling)^123,124; and (4) an inability to detect the oxygen dependency of SNO-Hb instability in the presence of erythrocytic concentrations of glutathione (ie, the S-NO linkage decays independent of oxygen tension in the presence of mM concentrations of glutathione).^115,125

We support the general principle that Hb can transduce NO-dependent vasoactivity as originally proposed by Stamler et al in 1996.¹⁴ However, we believe that current data better-support the mechanism based on nitrite ions as the primary storage molecule, with the transduction of NO-dependent vasodilation mediated by the heme-based nitrite reductase activity of hemoglobin.

	Pathology and Therapeutics

Top Abstract Introduction Destruction of NO by... Preservation of NO Activity... Endocrine NO Transport as... The SNO-Hb Hypothesis Pathology and Therapeutics References

 
NO plays a central role in a multitude of diseases and their treatments.⁷⁸ We focus on those that are related to Hb. Perhaps the most significant pathology is that related to intravascular hemolysis in the acquired and hereditary hemolytic anemias.^126–128 Hemolysis results in a significant accumulation of cell free plasma Hb. As discussed earlier in this review, this cell-free Hb is capable of scavenging endothelial-derived NO much more efficiently than RBC-encapsulated Hb because cell-free Hb (1) can enter the RBC cell-free zone, (2) can extravasate into the endothelium and interstitium, and (3) is not surrounded by RBC-based diffusion barriers. The importance of NO scavenging caused by hemolysis in disease was perhaps first most clearly illustrated for the case of sickle cell anemia.^126,129,130 However, there are additional disease states in which NO scavenging by cell-free Hb is recognized to play a major role, including other hereditary hemolytic anemias, such as paroxysmal nocturnal hemoglobinuria, where NO scavenging has been mechanistically linked to erectile dysfunction, pulmonary hypertension, and gastrointestinal dystonias.¹²⁸

A therapeutic application to counter pathology caused by increased NO scavenging by cell-free Hb is to give inhaled NO (&80 ppm) to oxidize the cell-free Hb, thereby reducing its ability to scavenge NO.^126,127,131 For NO-based therapies to be effective in a wide range of applications, alternative methods of delivery besides NO breathing are desirable. Several factors make nitrite an ideal Hb-mediated source of NO formation in vivo. Under normoxic conditions, plasma nitrite is relatively stable and abundant (several hundred nanomolar in plasma⁶⁷). Under relative hypoxia, Hb reacts with the nitrite (as well as other enzymes) to produce NO.

Nitrite has recently shown promise as a therapeutic agent in several preclinical models.^23–25 Inhaled nebulized nitrite was shown to reduce hypoxia-induced pulmonary hypertension in newborn lambs.²⁵ These effects were coupled to the presence of deoxygenated Hb and formation of HbNO.²⁵ Inhaled nebulized nitrite thus shows promise for the treatment of neonatal pulmonary hypertension.²⁵ In another test of potential therapeutic application, administered nitrite produced significant protective effects in models of ischemic-reperfusion injury in mice.²³ The cytoprotective effects were dependent on nitrite dosage and NO generation.²³ This nitrite-mediated cytoprotection has also been observed in Langendorff rat heart preparations and has been attributed to xanthine oxidoreductase-mediated nitrite reduction to NO.¹³² Finally, infused nitrite prevented delayed cerebral vasospasm in monkeys.²⁴ A blood clot was placed in the monkeys’ cerebral arteries, which led to significant vasospasm in control animals (not administered nitrite) but in none of the monkeys infused with nitrite.²⁴ These studies show that nitrite infusions may (if further tests are successful) prevent cerebral vasospasm, a disabling and sometimes fatal complication in many stroke patients.²⁴

Conclusions
Over the past 10 years, our appreciation of the complexity of NO–hemoglobin biochemistry has deepened and the contribution of hemoglobin and the red blood cell to NO homeostasis is more clear. Several mechanisms have evolved to greatly limit the extent that Hb scavenges NO. The nature and relative importance of these mechanisms remains an active and vital area of study. The notion that NO is stored and allosterically delivered by Hb as SNO-Hb remains controversial, but the principle that Hb participates in hypoxic NO generation by additional mechanisms is being actively explored. We and others have hypothesized that nitrite is the storage molecule for NO activity that is transduced under hypoxia by reactions with Hb. This hypothesis and the more global concept that nitrite is an intrinsic NO synthase-independent source of NO and signaling molecule are currently the subjects of intense investigations in the areas of biochemistry, physiology, pathophysiology, and therapeutics.

	Acknowledgments

 
This work was supported by HL58091 and K02078706 (D.B.K.-S.). We thank Annemarie B. Johnson for her art work.

	Footnotes

 
The authors are coauthors of a filed patent entitled "Use of nitrite salts for the treatment of cardiovascular conditions."

Received November 25, 2005; accepted January 4, 2006.

	References

Top Abstract Introduction Destruction of NO by... Preservation of NO Activity... Endocrine NO Transport as... The SNO-Hb Hypothesis Pathology and Therapeutics References

 
1. Ignarro LJ, Byrns RE, Buga GM, Wood KS. Endothelium-derived relaxing factor from pulmonary-artery and vein possesses pharmacological and chemical-properties identical to those of nitric-oxide radical. Circ Res. 1987; 61: 866–879.[Abstract/Free Full Text]

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