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`
`Nitrite reduction to nitric oxide by deoxyhemoglobin vasodilates the human
`circulation
`
`Article  in  Nature Medicine · January 2004
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`DOI: 10.1038/nm954 · Source: PubMed
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`A R T I C L E S
`
`Nitrite reduction to nitric oxide by deoxyhemoglobin
`vasodilates the human circulation
`Kenyatta Cosby1, Kristine S Partovi2, Jack H Crawford3, Rakesh P Patel3, Christopher D Reiter2,4,
`Sabrina Martyr2,4, Benjamin K Yang2, Myron A Waclawiw5, Gloria Zalos1, Xiuli Xu6, Kris T Huang7,
`Howard Shields6, Daniel B Kim-Shapiro6,7, Alan N Schechter4, Richard O Cannon III1,8, and Mark T Gladwin2,4,8
`
`Nitrite anions comprise the largest vascular storage pool of nitric oxide (NO), provided that physiological mechanisms exist to
`reduce nitrite to NO. We evaluated the vasodilator properties and mechanisms for bioactivation of nitrite in the human forearm.
`Nitrite infusions of 36 and 0.36 µmol/min into the forearm brachial artery resulted in supra- and near-physiologic intravascular
`nitrite concentrations, respectively, and increased forearm blood flow before and during exercise, with or without NO synthase
`inhibition. Nitrite infusions were associated with rapid formation of erythrocyte iron-nitrosylated hemoglobin and, to a lesser
`extent, S-nitroso-hemoglobin. NO-modified hemoglobin formation was inversely proportional to oxyhemoglobin saturation.
`Vasodilation of rat aortic rings and formation of both NO gas and NO-modified hemoglobin resulted from the nitrite reductase
`activity of deoxyhemoglobin and deoxygenated erythrocytes. This finding links tissue hypoxia, hemoglobin allostery and nitrite
`bioactivation. These results suggest that nitrite represents a major bioavailable pool of NO, and describe a new physiological
`function for hemoglobin as a nitrite reductase, potentially contributing to hypoxic vasodilation.
`
`Nitrite is a vasodilator at high concentrations in vitro1–6. In vivo plasma
`levels of nitrite are in the range of 150–1,000 nM, and the concentration
`in aortic ring tissue is >10 µM (refs. 7–9). This potential storage pool
`for NO is in vast excess of plasma S-nitrosothiols, reported to be
`<10 nM in human plasma9–12. Mechanisms for the in vivo conversion
`of nitrite to NO have been proposed to involve either enzymatic reduc-
`tion with xanthine oxidoreductase, or nonenzymatic disproportiona-
`tion or acidic reduction13–21. Each mechanism would occur
`preferentially in vascular regions with low pH and low partial pressure
`of oxygen (pO2). Indeed, consistent with oxygen- and pH-sensitive
`chemistry, hypoxia and acidosis potentiate NO generation and vasodi-
`lation from both nitrite and NO donors in aortic ring and lung perfu-
`sion bioassay systems22–24. However, the extremely low oxygen tension
`and pH necessary for nitrite reduction by xanthine oxidoreductase and
`disproportionation, as well as the high nitrite concentrations required
`for vasodilation in previous in vitro studies, have cast doubt on the role
`of this anion as a vasodilator. Indeed, no vasodilatory effects were
`reported when nitrite was infused into the forearm circulation of three
`human subjects for 1 min (ref. 25). That study suggested that under
`physiological conditions, nitrite would not function as an intravascular
`storage pool for NO and, thus, was not an intrinsic vasodilator.
`
`Consistent with the bioconversion of intravascular nitrite to NO, we
`and others have observed arterial-to-venous gradients of nitrite across
`the human forearm at rest and during regional NO synthase inhibi-
`tion, with increased consumption of nitrite occurring during exer-
`cise8,26. Other research groups have reported large arterial-to-venous
`gradients of nitrite also form across the human forearm during NO
`synthase inhibition25. Unlike the simpler case of oxygen extraction
`across a vascular bed, nitrite is both consumed—as evidenced by arte-
`rial-to-venous gradients during NO synthase inhibition and exer-
`cise—and produced
`in the vascular bed by endothelial NO
`synthase–derived reactions of NO with oxygen. Supporting the exis-
`tence of an intravascular NO species capable of storage and distal
`delivery of NO bioactivity, multiple research groups have observed
`that red blood cells and plasma ‘loaded’ with NO, by exposure to NO
`solutions, NO gas or NO donors, can export an ‘NO-like’ bioactivity
`and induce vasodilation in vitro and in vivo11,27–32. We have previously
`evaluated the reaction products formed in human blood during
`inhalation of NO gas, and found significant increases in plasma nitrite
`and limited formation of plasma and erythrocyte S-nitroso-proteins,
`suggesting a role for nitrite in the transduction of NO bioactivity along
`the vasculature29. We therefore considered the possibility that nitrite,
`
`1Cardiovascular Branch, National Heart, Lung and Blood Institute, National Institutes of Health, 10 Center Drive, Building 10, Room 7B15 Bethesda, Maryland
`20892, USA. 2Critical Care Medicine Department, Warren G. Magnuson Clinical Center, National Institutes of Health, 10 Center Drive, Building 10, Room 7D43
`Bethesda, Maryland 20892, USA. 3Department of Pathology, Center for Free Radical Biology, Biomedical Research Building II, Room 307, 901 19th Street South,
`University of Alabama at Birmingham, Birmingham, Alabama 35294, USA. 4Laboratory of Chemical Biology, National Institute of Diabetes, Digestive and Kidney
`Diseases, National Institutes of Health, 10 Center Drive, Building 10, Room 9N307, Bethesda, Maryland 20892, USA. 5Office of Biostatistics Research, National
`Heart, Lung and Blood Institute, National Institutes of Health, 6701 Rockledge Drive, Bethesda, Maryland 20892, USA. 6Department of Physics, Wake Forest
`University, Winston-Salem, North Carolina 27109-7507, USA. 7Department of Biomedical Engineering, Wake Forest University School of Medicine, Winston-Salem,
`North Carolina 27157-1022, USA. 8These authors contributed equally to this work. Correspondence should be addressed to M.T.G. (mgladwin@nih.gov) or R.O.C.
`(cannonr@nih.gov).
`
`Published online 2 November 2003; doi:10.1038/nm954
`
`1 4 9 8
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`VOLUME 9 | NUMBER 12 | DECEMBER 2003 NATURE MEDICINE
`
`©2003 Nature Publishing Group http://www.nature.com/naturemedicine
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`A R T I C L E S
`
`Figure 1 Hemodynamic and metabolic measurements at baseline and
`during exercise (protocol, part I). Measurements were taken without (a) and
`with (b) inhibition of NO synthesis in 18 subjects. Mean arterial pressure
`(MAP), forearm blood flow (FBF), venous oxyhemoglobin saturation (O2
`saturation), pO2 and pH are shown for all experimental conditions. These
`interventions and measurements (part I of the protocol) served as a control
`for part II of the protocol, in which these interventions were performed
`during nitrite infusion. *, P < 0.05 versus baseline 2; **, P < 0.01 versus
`baselines 1 or 2, respectively; †, P < 0.05 versus baseline 1; +, P < 0.01
`versus exercise. Error bars denote s.e.m.
`
`nitrosylated hemoglobin in the ipsilateral antecubital vein increased
`from 55.7 ± 11.4 to 693.4 ± 216.9 nM during nitrite infusion. During
`forearm exercise (with continued nitrite infusion), blood flow
`increased further. Metabolic stress was present, as evidenced by
`reduced forearm venous hemoglobin oxygen saturation, pO2 and pH
`levels compared with baseline values. Venous nitrite levels declined,
`indicating that increased blood flow to the forearm was diluting the
`concentration of infused nitrite. Despite decreasing forearm nitrite
`concentration during exercise, iron-nitrosylated hemoglobin levels
`increased, indicating an augmented rate of NO generation from nitrite
`during exercise stress (Fig. 2a).
`After cessation of nitrite infusion and substitution of saline as the
`intra-arterial infusate for 30 min, repeat baseline measurements showed
`persistent elevations in systemic levels of nitrite, iron-nitrosylated
`hemoglobin and methemoglobin (Fig. 2b) compared with values
`obtained almost 1 h previously, before the infusion of nitrite. A persist-
`ent vasodilator effect was also apparent, as forearm blood flow was
`significantly higher (4.79 ± 0.37 versus 3.94 ± 0.38 ml per min per
`100 ml tissue; P = 0.003) and systemic blood pressure was significantly
`
`a
`
`b
`
`a
`
`b
`
`rather than S-nitrosothiols, is the largest intravascular storage pool for
`NO, and that nitrite bioactivation to NO could vasodilate regions with
`tissue oxygen debt in the human circulation.
`
`RESULTS
`Vasodilatory properties of nitrite in vivo
`Eighteen healthy subjects (nine males and nine females, aged 21–50
`years) were enrolled in a physiological study to determine whether
`nitrite is a vasodilator and to examine nitrite’s in vivo chemistry. In
`part I of the protocol, the normal hemodynamic and metabolic
`responses to exercise and to inhibition of NO synthesis in the forearm
`were measured as controls for part II, in which these interventions
`were done during nitrite infusion (Fig. 1a,b; see Supplementary Note
`online for detailed description of control observations). Parts I and II
`were conducted in random order.
`To determine whether nitrite has vasoactivity in humans, in part II
`of the protocol we infused sodium nitrite in bicarbonate-buffered
`saline (final concentration of 36 µmol/ml) into the brachial arteries of
`the 18 subjects, to achieve an estimated intravascular nitrite concen-
`tration of ∼200 µM (ref. 25). After infusion of sodium nitrite at
`1 ml/min for 5 min, nitrite levels in the ipsilateral antecubital vein
`increased to 221.82 ± 57.59 µM (Fig. 2a). Forearm blood flow
`increased by 175% over resting values; venous hemoglobin oxygen sat-
`uration, pO2 and pH levels increased significantly (all P < 0.01) over
`preinfusion values, indicating increased perfusion of the forearm.
`The systemic nitrite concentration was 16 µM, as measured in the
`contralateral arm, and was associated with a systemic decrease in mean
`blood pressure of ∼7 mm Hg (P < 0.01). Consistent with immediate
`NO generation from nitrite during arterial-to-venous transit, iron-
`
`Figure 2 Effects of nitrite infusion. NaNO2 was infused into the brachial
`arteries of 18 healthy subjects for 5 min at baseline and continued during
`exercise, without (a) or with (b) inhibition of NO synthesis with L-NMMA
`(protocol 1, part II). Values for mean arterial blood pressure (MAP), forearm
`blood flow (FBF), venous oxyhemoglobin saturation, pO2, pH, venous nitrite,
`venous iron-nitrosylated hemoglobin (NO-heme) and venous methemoglobin
`(Met-Hb) are shown for all experimental interventions. *, P ≤ 0.06 versus
`baselines 1 or 2, respectively; **, P < 0.01 versus baselines 1 or 2,
`respectively; †, P < 0.05 versus baseline 1; ††, P < 0.01 versus baseline 1.
`Error bars represent s.e.m.
`
`©2003 Nature Publishing Group http://www.nature.com/naturemedicine
`
`NATURE MEDICINE VOLUME 9 | NUMBER 12 | DECEMBER 2003
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`A R T I C L E S
`
`a
`
`b
`
`c
`
`d
`
`Figure 3 Effects of low-dose nitrite infusion. NaNO2 was infused into the brachial arteries of ten healthy subjects at baseline and during exercise, without or
`with inhibition of NO synthesis. (a) Forearm blood flow at baseline and after a 5-min infusion of NaNO2. (b) Forearm blood flow with and without low-dose
`nitrite infusion at baseline and during L-NMMA infusion, with and without exercise stress (Ex). (c) Venous levels of nitrite from forearm circulation at the time
`of blood flow measurements. (d) Venous levels of S-nitroso-hemoglobin (S-NO) and iron-nitrosylated hemoglobin (Hb-NO) at baseline and after nitrite
`infusion during exercise stress. *, P < 0.05 versus baseline. Error bars represent s.e.m.
`
`lower (82.1 ± 3.7 versus 89.2 ± 3.5 mm Hg; P = 0.002) than initial
`preinfusion values. We then reinfused the brachial artery with sodium
`nitrite (36 µmol/ml) and NG-monomethyl-L-arginine (L-NMMA;
`8 µmol/min) again to inhibit regional synthesis of NO. We observed
`vasodilator effects of nitrite on resting and exercise forearm blood flow
`similar to those observed during nitrite infusion without L-NMMA
`(Fig. 2b). This is in contrast to the vasoconstrictor effect of NO synthase
`inhibition with L-NMMA observed in part I of the protocol (Fig. 1b).
`
`Vasodilatory properties of nitrite at physiological concentrations
`As a test of the physiological relevance of vascular nitrite as a vasodila-
`tor, the concentrations of the nitrite infusions were decreased by 2 logs
`to 400 nmol/ml. An infusion of 400 nmol/ml nitrite at 1 ml/min for
`5 min significantly increased forearm blood flow in all ten subjects
`from 3.49 ± 0.24 to 4.51 ± 0.33 ml per min per 100 ml tissue (Fig. 3a;
`P = 0.0006). Blood flow significantly increased at rest and during NO
`synthase inhibition, with or without exercise (Fig. 3b; P < 0.05 under
`all conditions). Mean venous nitrite levels increased from 176 ± 17 nM
`to 2,564 ± 462 nM after a 5-min infusion, and exercise venous nitrite
`levels decreased to 909 ± 113 nM (secondary to the diluting effects of
`increased blood flow during exercise; Fig. 3c). Again, the vasodilator
`effects of nitrite were paralleled by an observed formation of both
`iron-nitrosylated hemoglobin and S-nitroso-hemoglobin across the
`forearm circulation (Fig. 3d). These data suggest that basal levels of
`nitrite, from 150–1,000 nM in plasma to 10,000 nM in vascular tis-
`sue7–9, are likely to contribute to resting vascular tone and hypoxic
`vasodilation.
`The vasodilatory property of nitrite during basal blood flow condi-
`tions, when tissue pO2 and pH are not exceedingly low, was unex-
`pected. These results suggest that the previously hypothesized
`mechanisms for nitrite reduction, nitrite disproportionation and xan-
`thine oxidoreductase activity, all of which require extremely low pO2
`and pH values not typically encountered in normal physiology, must
`be complemented in vivo by additional factors that catalyze nitrite
`reduction. We now report that deoxyhemoglobin effectively reduces
`nitrite to NO, a mechanism described by Doyle et al. in 1981 (ref. 33),
`within one half-circulatory time from artery to vein. This mechanism
`provides graded production of NO along the physiological oxygen
`gradient, tightly regulated by hemoglobin oxygen desaturation.
`
`Intravascular formation of NO and S-nitrosothiol
`Before and during nitrite infusions, blood was drawn from the
`brachial artery and antecubital vein, and the whole blood was immedi-
`ately (at the bedside to minimize processing time) lysed 1:10 in an
`NO-hemoglobin ‘stabilization solution’. The iron-nitrosylated hemo-
`globin and S-nitroso-hemoglobin content was determined by
`tri-iodide–based reductive chemiluminescence and electron paramag-
`netic resonance (EPR) spectroscopy (described in Methods). As previ-
`ously reported30 and recently confirmed9, the baseline levels of
`S-nitroso-hemoglobin and iron-nitrosyl-hemoglobin were at the lim-
`its of detection (<50 nM or 0.0005% NO per heme), with no artery-
`to-vein gradient. After nitrite infusion as in part II of the protocol,
`venous
`levels of
`iron-nitrosylated hemoglobin and S-nitroso-
`hemoglobin rose substantially (Fig. 4a–c). This formation of iron-
`nitrosylated hemoglobin across the forearm circulation was confirmed
`by EPR spectroscopy (Fig. 4b). The formation of both NO-hemoglo-
`bin adducts occurred across the vascular bed, with a half-circulatory
`time of less than 10 s. The rate of NO formation was measured as iron-
`nitrosylated and S-nitroso-hemoglobin content and quantified by
`subtraction of the arterial from the venous levels with the difference
`being multiplied by blood flow. The NO formation rate increased
`greatly during exercise, despite a significant decrease in the venous
`concentration of nitrite secondary to the dilution of regional nitrite
`concentration by increased blood flow (Fig. 4d; P = 0.006 for iron-
`nitrosylated hemoglobin and P = 0.02 for S-nitroso-hemoglobin, by
`repeated-measures ANOVA).
`The amounts of
`iron-nitrosylated and S-nitroso-hemoglobin
`formed in vivo in this study are notable. With a transit time of less
`than 10 s through the forearm circulation during exercise, infused
`nitrite (200 µM regional concentration) produced ∼750 nM iron-
`nitrosylated hemoglobin and 200 nM S-nitroso-hemoglobin
`(Fig. 4b,c). The formation of both NO-hemoglobin adducts was
`inversely correlated with hemoglobin-oxygen saturation, which fell
`during exercise stress, as measured from the antecubital vein by co-
`oximetry (r = –0.7 and P < 0.0001 for iron-nitrosylated hemoglobin;
`r = –0.45 and P = 0.04 for S-nitroso-hemoglobin; Fig. 4e). Addition of
`200 µM nitrite to whole blood at different oxygen tensions (0–100%)
`recapitulated the in vivo data, with increasing concentrations of iron-
`nitrosylated hemoglobin being formed at lower oxygen tensions
`
`1 5 0 0
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`VOLUME 9 | NUMBER 12 | DECEMBER 2003 NATURE MEDICINE
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`©2003 Nature Publishing Group http://www.nature.com/naturemedicine
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`A R T I C L E S
`
`(r = –0.968 and P < 0.0001 for iron-nitrosylated hemoglobin;
`r = –0.45 and P = 0.07 for S-nitroso-hemoglobin; data not shown).
`This strongly suggests that iron-nitrosylated hemoglobin and
`S-nitroso-hemoglobin formation was dependent on the reaction of
`nitrite with deoxyhemoglobin.
`These data are consistent with the previous characterization of the
`reaction of nitrite with deoxyhemoglobin to form NO and iron-
`nitrosylated hemoglobin33. Nitrite is first reduced to form NO and
`methemoglobin, with a rate constant of 2.9 M–1s–1 (measured at 25 °C,
`pH 7.0)33. This reaction is pseudo-first order, governed by the vast
`amounts (20 mM) of intraerythrocytic hemoglobin, and limited by
`the rate of nitrite uptake by the erythrocyte membrane. NO then
`either binds to deoxyhemoglobin to form iron-nitrosylated hemoglo-
`bin, escapes the erythrocyte (discussed below) or reacts with other
`higher oxides (such as NO2, to form N2O3 and S-nitroso-hemoglobin;
`discussed later). These reactions are represented by the following
`equations:
`
`– (nitrite) + HbFe2+ (deoxyhemoglobin) + H+ → HbFe3+
`NO2
`(methemoglobin) + NO + OH–
`NO + HbFe2+ (deoxyhemoglobin) → HbFe2+NO (iron-nitrosylated
`hemoglobin)
`
`We confirmed that the reaction of deoxyhemoglobin and nitrite is
`second-order in nitrite and hemoglobin by conducting kinetic meas-
`urements, first with a molar excess of nitrite to hemoglobin, and then
`with an excess of hemoglobin to nitrite. We found the same bimolecu-
`lar rate constant, 0.47 ± 0.07 M–1s–1, for both conditions at 25 °C and
`pH 7.4. This rate constant is similar to that found by Doyle et al. at this
`pH (1 M–1s–1)33.
`
`To explore the effects of red blood cell membrane nitrite uptake rate
`on the formation of intraerythrocytic iron-nitrosylated hemoglobin,
`we examined the kinetics of the reaction of 200 µM nitrite with deoxy-
`genated whole blood at 37 ºC. Iron-nitrosylated hemoglobin formed
`at an observed rate constant (k) of0.0035 ± 0.006 s–1 (Fig. 4f,g).
`Assuming a concentration of 20 mM for the concentration of hemo-
`globin in the red blood cell, this corresponds to a bimolecular rate of
`0.18 ± 0.03 M–1s–1, which is substantially lower than the rate expected
`by measurements made by Doyle et al., and indicates that the in vivo
`rate is limited by erythrocyte nitrite uptake. Using this rate and a 10-s
`artery-to-vein transit time (with the equation (0.28)(200 µM)
`(1 – e–kt)), we would expect 1.9 µM of iron-nitrosylated hemoglobin
`formation in vivo. This result would be similar in magnitude to the
`observed formation of ∼750 nM iron-nitrosylated hemoglobin across
`the arterial-to-venous gradient (Fig. 4).
`We also observed the formation of significant amounts of S-nitroso-
`hemoglobin in vivo during nitrite infusion. It was recently proposed
`that nitrite reacts with deoxyhemoglobin to make iron-nitrosylated
`hemoglobin, and that the subsequent ‘transfer’ of the NO to the cys-
`teine 93 of the β-chain of hemoglobin to form S-nitroso-hemoglobin
`is mediated by reoxygenation and the quaternary T-to-R structural
`transition state of hemoglobin34. However, a direct transfer of NO
`from the heme to the thiol would require NO oxidation to NO+, and
`such ‘cycling’ has not been reproduced by other research groups35. It
`has recently been suggested that nitrite catalyzes the reductive nitrosy-
`lation of methemoglobin by NO, a process that generates the interme-
`diate nitrosating species dinitrogen trioxide (N2O3)36. Additional
`reactions of nitrite with hemoglobin produce reactive oxygen metabo-
`lites (such as superoxide and hydrogen peroxide37–39). Such reactions
`of NO radicals with oxygen radicals will provide competitive pathways
`
`a
`
`b
`
`c
`
`d
`
`©2003 Nature Publishing Group http://www.nature.com/naturemedicine
`
`f
`
`g
`
`e
`
`Figure 4 Formation of iron-nitrosylated
`hemoglobin and S-nitroso-hemoglobin after
`nitrite infusion in vivo and in vitro. (a) NO
`levels in arterial and venous blood
`hemoglobin, as measured by ozone-based
`chemiluminescence. Samples incubated
`without mercury (–HgCl2) represent total
`iron-nitrosylated and S-nitroso-hemoglobin,
`whereas samples incubated with mercury
`(+HgCl2) represent only iron-nitrosylated
`hemoglobin. The difference in peak area
`represents S-nitroso-hemoglobin. (b,c)
`Levels of iron-nitrosylated hemoglobin (b) and S-nitroso-hemoglobin (c) increased from artery to vein, indicating formation across the vascular bed after
`nitrite infusion. Inset in b shows arterial blood EPR spectra subtracted from venous blood EPR spectra, showing an increase in iron-nitrosylated hemoglobin
`from artery to vein. Difference spectra from three patients during exercise with nitrite infusion are shown. (d) Formation of iron-nitrosylated hemoglobin (NO-
`heme) and S-nitroso-hemoglobin (S-NO) at baseline, during nitrite infusion and during nitrite infusion with exercise, quantified by subtracting arterial from
`venous levels and multiplying the result by blood flow. (e) Formation of both NO-hemoglobin adducts was inversely correlated with hemoglobin oxygen
`saturation during nitrite infusion. (f,g) Representative EPR spectra (f) and kinetic traces (g) for reaction of nitrite with hemoglobin in venous blood at 37 °C,
`with deoxygenation performed under argon. *, P < 0.05 compared with baseline (b–d) and arterial levels (b,c).
`
`NATURE MEDICINE VOLUME 9 | NUMBER 12 | DECEMBER 2003
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`A R T I C L E S
`
`Oxygen concentration (mmHg)
`
`80
`
`60
`
`40
`
`20
`
`0
`
`0
`
`100
`
`200
`Time (s)
`
`300
`
`1.0
`0.8
`0.6
`0.4
`0.2
`0.0
`
`Fraction of maximal tension
`
`c
`
`10 n M
`
`100 n M
`
`500 n M
`
`100 M
`
`200 M
`
`500 M
`
`}
`
`[Nitrite]
`
` Control
` IHP (100 M)
` Hb (25 M heme)
` Hb + IHP
`
`0
`
`400
`
`800
`Time (s)
`
`1,200
`
`1,600
`
`f
`
`1.2
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Fraction of maximal tension
`
`P ≤ 0.004
`
`P ≤ 0.03
`
`Control Nitrite
`
`RBC RBC + nitrite
`
`b
`
`40
`
`30
`
`20
`
`10
`
`0
`
`pO at which relaxation starts (mmHg)
`
`2
`
`Control
`Nitrite
`
`RBC
`RBC + nitrite
`
`e
`
`1.2
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Fraction of maximum tension
`
`0
`
`50
`
`40
`30
`20
`10
`Oxygen tension (mmHg)
`80
`60
`40
`20
`Oxygen tension (mmHg)
`
`0
`
`a
`
`d
`
`1.2
`
`1.0
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Fraction of maximum tension
`
`Figure 5 Production of NO gas and vasodilation are augmented by nitrite reaction with deoxyhemoglobin. (a) NO release after injection of nitrite into PBS
`and deoxygenated and oxygenated red blood cells. Arrows indicate time of nitrite injection into the system. (b) Rate of NO formation from nitrite mixed with
`PBS and oxygenated and deoxygenated red blood cells. (c) Representative time courses for pO2 (red trace) and vessel tension during deoxygenation of vessels
`treated with control (rat erythrocytes alone; black trace) or rat erythrocytes and nitrite (blue trace). (d) Aortic ring bioassay threshold curves showing the
`relationship between pO2 and vessel tension during different treatments. Inset shows data under same conditions with 500 nM nitrite. (e) Oxygen tensions at
`which relaxation began were determined from individual traces. Data represent mean ± s.e.m. (n = 3). (f) Representative traces (n = 3) showing effects of
`sodium nitrite (added at concentrations indicated) of relaxation of rat thoracic aorta at 15 mm Hg O2 under control conditions (black trace) or in the
`presence of 100 µM IHP (green trace), human hemoglobin (red trace) or IHP and human hemoglobin together (blue trace).
`
`for S-nitrosothiol formation in the presence of high-affinity NO sinks
`such as hemoglobin and deserve further study.
`
`Nitrite reductase activity of deoxyhemoglobin
`To determine whether free NO radicals can be formed from the reac-
`tion of nitrite and deoxyhemoglobin, we reacted 100 and 200 µM
`nitrite with deoxygenated erythrocytes (5-ml volume containing a
`total of 660 and 1,000 µM in heme) in a light-protected, helium-
`purged reaction vessel in line with a chemiluminescent NO analyzer.
`The injection of nitrite into a solution of deoxygenated erythrocytes
`resulted in the liberation of NO into the gas phase (Fig. 5a,b). There
`was no release from nitrite in a buffer control under the same condi-
`tions, and significantly (P < 0.05) less NO was released upon addition
`of nitrite to oxygenated erythrocytes (100% oxygen). The observed
`rate (area under the curve of increased steady-state NO generation
`after nitrite injection, calculated over 120 s) of NO production in the
`5-ml reaction volume was consistent with an NO production rate of
`47 pM/s (corresponding to an estimated rate of 300–500 pM/s in
`whole blood). Although NO formation rates in this experimental sys-
`tem cannot be extrapolated to rates of NO formation in vivo, the
`experiments illustrate two important concepts. First, a fraction of the
`free NO can escape autocapture by the remaining heme groups; this is
`likely to be possible only because nitrite is only converted to NO by
`reacting with deoxyhemoglobin, and because its ‘leaving-group’ is the
`met(ferric)heme protein that will limit scavenging and inactivation of
`NO33. Second, the rate of NO production is increased under anaerobic
`conditions, which indicates a nitrite-deoxyhemoglobin reaction.
`
`We next evaluated whether the vasodilator properties of nitrite
`could be reproduced in an aortic ring bioassay system, and whether
`this vasodilation is potentiated by deoxygenated erythrocytes. Rat aor-
`tic rings were suspended in custom-made vessel baths that were sealed
`and fitted with an oxygen electrode, allowing simultaneous measure-
`ment of vessel tension and pO2. Figure 5c shows representative trac-
`ings of vessel tension and pO2, measured during deoxygenation of rat
`aortic rings exposed to red blood cells (0.3% hematocrit, 300 µM
`heme) and nitrite (2 µM). To determine the relationship between oxy-
`gen tension, vessel tone and the impact of erythrocytes and nitrite, ves-
`sel tension was plotted as a function of pO2 (Fig. 5d). Control vessels
`spontaneously relaxed after reaching a pO2 of ∼10 mm Hg (Fig. 5e).
`Addition of 2 µM nitrite did not affect this process. Addition of rat
`erythrocytes alone increased the oxygen tension at which dilation was
`initiated (Fig. 5e), and addition of nitrite and erythrocytes together
`significantly (P < 0.004) left-shifted the vessel tension– pO2 threshold
`curve (Fig. 5c,d) such that vasodilation was observed at oxygen ten-
`sions <40 mm Hg (P < 0.004). Lower concentrations of nitrite
`(500 nM) that fall within the physiological range also significantly pro-
`moted vasodilation in the presence of red blood cells, compared with
`red blood cells or nitrite alone (Fig. 5d) (P < 0.05).
`To test whether the effect of red blood cells on nitrite-dependent
`vasodilation could be mediated by deoxyhemoglobin, vessel dilation
`experiments were conducted at a pO2 of 15 mm Hg, with cell-free
`hemoglobin, in the absence or presence of inositol hexaphosphate
`(IHP; Fig. 5f). IHP was used to modulate the oxygenation state of
`hemoglobin in this system. In the absence of IHP, hemoglobin
`
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`VOLUME 9 | NUMBER 12 | DECEMBER 2003 NATURE MEDICINE
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`©2003 Nature Publishing Group http://www.nature.com/naturemedicine
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`A R T I C L E S
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`remained saturated with oxygen at a pO2 of 15 mm Hg (p50 = 9 mm
`Hg oxygen; p50 is the pO2 at which hemoglobin is 50% saturated with
`oxygen), but was deoxygenated in the presence of IHP (p50 =
`45 mm Hg oxygen). At a pO2 of 15 mm Hg and using a Hill coefficient
`of n = 2.8, the oxygen saturation would be 81% for hemoglobin with
`p50 = 9 mm Hg, and 4% for hemoglobin with p50 = 45 mm Hg. With
`a heme concentration of 25 µM, ∼82% of oxyheme is tetrameric.
`Addition of nitrite alone stimulated vasodilation, with a half-maximal
`effective concentration of 100–200 µM. IHP alone slightly inhibited
`this effect, whereas hemoglobin alone right-shifted the dose-depend-
`ence of nitrite, indicating an oxyhemoglobin-dependent oxidation of
`nitrite to nitrate. However, in the presence of IHP and hemoglobin at a
`pO2 of 15 mm Hg, nitrite-dependent vasodilation was potentiated by
`three orders of magnitude (half-maximal effective concentration of
`100–200 nM). These results support a physiological model for nitrite
`reduction to NO by reaction with erythrocyte deoxyhemoglobin.
`
`DISCUSSION
`We show that nitrite-induced vasodilation in humans is associated
`with reduction of nitrite to NO by deoxyhemoglobin. Systemic levels
`of 16 µM resulted in systemic vasodilation and decreased blood pres-
`sure, and regional forearm levels of only 1–2 µM significantly
`increased blood flow at rest and with exercise-induced stress. In addi-
`tion, conversion of nitrite to NO and S-nitrosothiol was mediated by
`reaction with deoxyhemoglobin, providing a mechanism for hypoxia-
`regulated catalytic NO production by erythrocytes or endothelial or
`tissue heme proteins. A nitrite-hemoglobin chemistry would support a
`role for the red blood cells in oxygen-dependent NO homeostasis, a
`concept first advanced by Stamler et al. but ascribed to S-nitroso-
`hemoglobin40,41. It would also provide a mechanism for the observa-
`tion that red blood cells and plasma ‘loaded’ with NO, by exposure to
`high concentrations in solution or to NO gas or donors (invariably in
`equilibrium with high concentrations of nitrite), can export NO and
`induce vasodilation in vitro and in vivo11,27–32. We realize that the high
`concentrations of hemoglobin in red blood cells, coupled with the
`near-diffusion-limited reaction rates (∼107 M–1s–1) of NO with hemo-
`globin, seem to prohibit NO from being exported from the red blood
`cell. However, our data (Fig. 5) argue to the contrary. Perhaps the
`unique characteristics of the erythrocyte membrane, with its submem-
`brane proteins and methemoglobin-rich microenvironment42, and
`the relatively lipophilic nature of NO, allow compartmentalized NO
`production at the red blood cell membrane. This, coupled with the
`small amounts of NO necessary for vasodilation, could account for the
`export of NO despite these kinetic constraints. Further study will be
`required to determine whether this reaction occurs primarily in the
`erythrocyte, as data in Figure 5 suggest, with subsequent export of NO
`or of S-nitrosothiol, or whether this is a primary reaction of nitrite
`with endothelial or smooth muscle heme proteins, such as myoglobin,
`soluble guanylyl cyclase, cytochrome P450 or mitochondrial
`cytochromes.
`Three factors uniquely position nitrite, rather than S-nitrosothiol,
`as the major vascular storage pool of NO. First, nitrite is present in
`substantial concentrations in plasma, erythrocytes and tissues7.
`Second, nitrite is relatively stable because it is not readily reduced by
`intracellular reductants (as are S-nitrosothiols30), and its reaction rate
`with heme proteins is 10,000 times less than that of authentic NO.
`Third, nitrite is only converted to NO by reacting with deoxyhemoglo-
`bin (or presumably deoxymyoglobin, deoxycytoglobin, deoxyneuro-
`globin or other oxygen-binding heme proteins), and its leaving-group
`is the met(ferric)heme protein that limits scavenging and inactivation
`of NO33. The nitrite pool therefore provides the ideal substrate for NO
`
`generation along the physiological oxygen gradient, potentially pro-
`vi