ATVB In Focus
Nitric Oxide Redux
Series Editor:
Joseph Loscalzo
Unraveling the Reactions of Nitric Oxide, Nitrite, and
Hemoglobin in Physiology and Therapeutics
Daniel B. Kim-Shapiro, Alan N. Schechter, Mark T. Gladwin
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Abstract—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 ␤-93 cysteine to form S-nitrosohemoglobin (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. (Arterioscler Thromb Vasc Biol. 2006;26:697-705.)
Key Words: nitric oxide 䡲 nitrite 䡲 hemoglobin 䡲 vasodilation 䡲 red blood cell
N
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
itric 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
Original received November 25, 2005; final version accepted January 4, 2006.
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.
The authors are coauthors of a filed patent entitled “Use of nitrite salts for the treatment of cardiovascular conditions.”
Correspondence to Daniel Kim-Shapiro, Department of Physics, Wake Forest University, Winston-Salem, NC 27109. E-mail [email protected]
© 2006 American Heart Association, Inc.
Arterioscler Thromb Vasc Biol. is available at http://www.atvbaha.org
697
DOI: 10.1161/01.ATV.0000204350.44226.9a
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April 2006
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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
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.
(1)
HbO2⫹NO3MetHb⫹NO3⫺.
This reaction occurs at a rate of 6 to 8⫻107 M⫺1s⫺1 26 –28 so
that the half-life of NO in an oxygenated red blood cell (RBC)
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.
is ⬇0.5 ␮s. During this time, intraerythrocytic NO could only
diffuse ⬇0.02 ␮m (assuming an intraerythrocytic diffusion
constant of 1000 ␮m2 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 ␮m2 s⫺1
in the plasma, NO would only have a half life of ⬇1 ␮s and
could only diffuse ⬇0.1 ␮m. 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
Kim-Shapiro et al
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.
External Diffusion Limitations: The Cell-Free
Zone Along Endothelium and the Unstirred Layer
Around the Erythrocyte
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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 ␮m (a reasonable conservative
estimate for many arterioles32,41), 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
Nitric Oxide and Hemoglobin
699
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
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):
(2)
Hb⫹NO3HbNO.
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 6⫻107 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
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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
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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 ␮mol/L) and near physiological levels (0.9 to
2.5 ␮mol/L, basal plasma levels are ⬇0.3 ␮mol/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 ␮mol/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 ␮mol/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:
(3)
K
¢
¡
H⫹⫹NO2⫺ O
HONO
(4)
ko
Hb⫹HONO O
¡MetHb⫹NO⫹OH⫺
(5)
ka
Hb⫹NO O
¡HbNO,
where K is an equilibrium constant, ko is ⬇12.3⫻103 M⫺1s⫺1,
and ka 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–firstorder 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
Kim-Shapiro et al
Nitric Oxide and Hemoglobin
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nitrite reductase enzyme that is likely to play a significant
role in hypoxic vasodilation and hypoxic NO homeostasis
(Figure 2).22,93
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.
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.
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 nitritedependent vasodilation, implicating Hb as a mammalian
The SNO-Hb Hypothesis
A prominent, yet controversial, hypothesis for transducing
NO activity from HbNO involves the formation of Hb that is
S-nitrosated at the ␤-93 cysteine, referred to as SNOHb.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 ␤-93 cysteine when Hb undergoes conformational
changes associated with the T3 R 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 ␤-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 group64,65,115–117; (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 ␤-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
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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 NOdependent vasodilation mediated by the heme-based nitrite
reductase activity of hemoglobin.
Pathology and Therapeutics
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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 ischemicreperfusion 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 synthaseindependent 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.
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Unraveling the Reactions of Nitric Oxide, Nitrite, and Hemoglobin in Physiology and
Therapeutics
Daniel B. Kim-Shapiro, Alan N. Schechter and Mark T. Gladwin
Arterioscler Thromb Vasc Biol. 2006;26:697-705; originally published online January 19, 2006;
doi: 10.1161/01.ATV.0000204350.44226.9a
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