Am J Physiol Heart Circ Physiol 293: H2565–H2572, 2007;
doi:10.1152/ajpheart.00759.2007.
Hemoglobin oxygen fractional saturation regulates nitrite-dependent
vasodilation of aortic ring bioassays
T. Scott Isbell,1 Mark T. Gladwin,2 and Rakesh P. Patel1
1
Department of Pathology, and Center for Free Radical Biology, University of Alabama at Birmingham, Alabama; and
Vascular Medicine Branch, Critical Care Medicine Department, National Heart, Lung, and Blood Institute, National
Institutes of Health, Bethesda, Maryland
2
Submitted 2 July 2007; accepted in final form 25 August 2007
THE ROLE OF NITRITE in vascular biology has recently received
much attention with the demonstration in numerous experimental and clinical models that this inorganic anion may
represent an endogenous and/or therapeutic reservoir for nitric
oxide (NO) bioactivity during physiological and pathological
hypoxia (2, 5, 6, 13, 15, 17, 21, 23, 34, 43, 45). In the context
of blood flow, a seminal study by Furchgott et al. (14) showed
that nitrite alone, at high micromolar concentrations, stimulates
vasodilatation. Although it has been appreciated that nitrite is
produced endogenously via NO metabolism [oxidation of NO
produced from NO synthase by the multicopper oxidase ceruloplasmin (42)] and is a ubiquitous element in many diets, its
potential biological role has until recently received little attention, largely due to the relatively high doses of nitrite thought
to be required to elicit a biological effect (29). Acidic conditions (pH 6.6) potentiate nitrite-dependent relaxation, and acidosis can convert nitrite into NO and a variety of other
NO-containing intermediates (4, 30). Importantly, however, is
the observation that the infusion of close to physiological
concentrations of nitrite into the human forearm increases
blood flow by ⬃22% under conditions that are not associated
with significant acidosis (8). Moreover, significant arterialvenous gradients in plasma nitrite are observed basally and
during inhalation of therapeutic NO gas and are associated with
increased NO bioavailability in the microcirculation (7, 25). A
key concept developed from these in vivo and parallel experimental studies is that nitrite biology is regulated by hypoxia such
that at lower O2 tensions, nitrite can stimulate NO-dependent
effects at significantly lower (nanomolar to low micromolar)
concentrations (6, 8, 9, 13, 45). This potency of nitrite in vivo
is particularly surprising when one considers the fact that blood
contains ⬃5 mM of erythrocytic hemoglobin, which would be
expected to potently scavenge intravascularly generated nitritederived NO.
We and others (8, 9, 31, 38) have shown that one mechanism
that links hypoxic sensing with nitrite reduction to NO is a
reaction with deoxyhemoglobin (reaction 1). This reaction is in
contrast to nitrite oxidation to nitrate, which occurs upon
reaction with oxyhemoglobin (reaction 2). The nitrite reductase activity of deoxyhemoglobin was shown to be regulated
allosterically, exhibiting maximal kinetics around the hemoglobin O2 tension at which hemoglobin is 50% deoxygenated
(P50) (9, 12, 20). Evidence that NO produced from this reaction
was competent to stimulate signaling was indicated by stimulation of vasodilation, increased vascular cGMP levels [the
product of soluble guanylate cyclase (sGC) activation], and
inhibition of mitochondrial respiration, which were all reversed
by the NO scavenger 2-carboxyphenyl-4,4 –5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO) (9).
While the detailed mechanisms and biochemical intermediates that lead to NO generation from the reaction of deoxyhemoglobin and nitrite remain under investigation (24), an
interesting paradox arises given that both oxyhemoglobin and
deoxyhemoglobin potently scavenge NO (reactions 3– 4), raising the possibility that deoxyhemoglobin-dependent modula-
Address for reprint requests and other correspondence: R. P. Patel, Dept. of
Pathology, Univ. of Alabama at Birmingham, 901 19th St. S, BMR-2, Rm.
302, Birmingham, AL 35296 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
nitric oxide; hypoxia; blood flow
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Isbell TS, Gladwin MT, Patel RP. Hemoglobin oxygen fractional
saturation regulates nitrite-dependent vasodilation of aortic ring bioassays. Am J Physiol Heart Circ Physiol 293: H2565–H2572, 2007;
doi:10.1152/ajpheart.00759.2007.—Nitrite reacts with deoxyhemoglobin to generate nitric oxide (NO). This reaction has been proposed
to contribute to nitrite-dependent vasodilation in vivo and potentially
regulate physiological hypoxic vasodilation. Paradoxically, while deoxyhemoglobin can generate NO via nitrite reduction, both oxyhemoglobin and deoxyhemoglobin potently scavenge NO. Furthermore, at
the very low O2 tensions required to deoxygenate cell-free hemoglobin solutions in aortic ring bioassays, surprisingly low doses of nitrite
can be reduced to NO directly by the blood vessel, independent of the
presence of hemoglobin; this makes assessments of the role of
hemoglobin in the bioactivation of nitrite difficult to characterize in
these systems. Therefore, to study the O2 dependence and ability of
deoxhemoglobin to generate vasodilatory NO from nitrite, we performed full factorial experiments of oxyhemoglobin, deoxyhemoglobin, and nitrite and found a highly significant interaction between
hemoglobin deoxygenation and nitrite-dependent vasodilation (P ⱕ
0.0002). Furthermore, we compared the effect of hemoglobin oxygenation on authentic NO-dependent vasodilation using a NONOate
NO donor and found that there was no such interaction, i.e., both
oxyhemoglobin and deoxyhemoglobin inhibited NO-mediated vasodilation. Finally, we showed that another NO scavenger, 2-carboxyphenyl-4,4 –5,5-tetramethylimidazoline-1-oxyl-3-oxide, inhibits nitritedependent vasodilation under normoxia and hypoxia, illustrating the
uniqueness of the interaction of nitrite with deoxyhemoglobin. While
both oxyhemoglobin and deoxyhemoglobin potently inhibit NO, deoxyhemoglobin exhibits unique functional duality as an NO scavenger
and nitrite-dependent NO generator, suggesting a model in which
intravascular NO homeostasis is regulated by a balance between NO
scavenging and NO generation that is dynamically regulated by
hemoglobin’s O2 fractional saturation and allosteric nitrite reductase
activity.
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HEMOGLOBIN FRACTIONAL SATURATION REGULATES NITRITE-DEPENDENT VASODILATION
tion of NO homeostasis will be determined by the balance
between NO scavenging and NO generation from nitrite.
Reaction 1: Hb[Fe2⫹] ⫹ NO2⫺ ⫹ H⫹ 3 NO ⫹ Hb[Fe3⫹]
⫹ OH⫺
Reaction 2: 4Hb关Fe2⫹兴O2 ⫹ 4NO2⫺ 3 4NO3⫺ ⫹ 4Hb[Fe3⫹]
⫹ O2 ⫹ 2H2O
Reaction 3: Hb关Fe2⫹兴 ⫹ NO 3 Hb关Fe2⫹兴NO
Reaction 4: Hb关Fe2⫹兴O2 ⫹ NO 3 Hb关Fe3⫹兴 ⫹ NO3⫺
MATERIALS AND METHODS
Materials. All reagents were purchased from Sigma (St. Louis,
MO) except for methylamine hexamethylene methylamine NONOate
(MNO), which was purchased from Axxora Platform (San Diego,
CA). Sodium nitrite was dissolved in PBS (pH 7.4). Indomethacin and
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Recently, Dalsgaard et al. (11) showed, under a constant
contractile stimulus, that hypoxia alone sensitized aortic vascular preparations to nitrite such that the concentration of
nitrite required to vasodilate was significantly lowered to close
to the physiological range. Dalsgaard and colleagues also
showed that nitrite-dependent dilation was cGMP dependent,
indicating metabolism to NO. They concluded from these
experiments that nitrite potently vasodilates aortic rings and
that hemoglobin does not regulate this process. However, their
experimental data clearly show that while oxyhemoglobin
inhibited nitrite-dependent vasodilation, deoxyhemoglobin had
no apparent inhibitory effect. Put more simply, they clearly
observed a potent nitrite vasodilation in aortic rings alone at
low O2, but in the presence of oxyhemoglobin this dilation was
inhibited and in the presence of deoxyhemoglobin this dilation
was preserved. This suggests a unique interaction between
nitrite and deoxyhemoglobin in modulating NO-dependent
vasodilation. Because nitrite-dependent dilation is proceeding
via NO formation, both oxyhemoglobin and deoxyhemoglobin
should inhibit the dilation equally, given the similar and rapid
rates (⬃107 M⫺1 䡠 s⫺1) of NO scavenging (reactions 3– 4),
which are orders of magnitude higher than rates of reactions
with nitrite (24). Therefore, differences in NO scavenging or
nitrite scavenging are unlikely to underlie the differential
effects of oxyhemoglobin versus deoxyhemoglobin on nitritedependent vasodilation. We therefore hypothesized that oxyhemoglobin inhibits nitrite-dependent dilation due to NO scavenging, whereas deoxyhemoglobin does not produce apparent
inhibition because its reactions with nitrite represent a balance
of vasodilator (nitrite reductase) and vasoconstrictor (NO scavenging) activities. We tested this hypothesis by performing full
factorial experiments measuring nitrite- and hemoglobin-dependent dilation of isolated aortic rings at different O2 tensions. Importantly, we also compared these interactions with a
direct NO donor and the NO-scavenger C-PTIO, which does
not react with nitrite. These experiments confirm that the
reactions between nitrite and hemoglobin in aortic ring bioassays at different hemoglobin fractional O2 saturations exhibit a
unique interaction: nitrite-dependent vasodilation is inhibited
at high hemoglobin O2 fractional saturation, whereas vasodilation is promoted when hemoglobin deoxygenates, supporting
the hemoglobin nitrite reductase hypothesis of hypoxic vasodilation.
C-PTIO were dissolved in 100% ethanol or methanol, respectively,
which was diluted in vessel relaxation experiments to a final solvent
concentration of 0.1% (vol/vol). No effects of these solvents were
observed on vasoactive responses at these concentrations (not shown).
Human cell-free hemoglobin was used for all experiments and was
purified from healthy volunteers according to Institutional Review Boardapproved protocols as previously described (33). Krebs-Henseleit (KH)
buffer was used for all experiments and consisted of the following
composition (final concentrations): 118.0 mM NaCl, 27.2 mM
NaHCO3, 4.8 mM KCl, 1 mM KH2PO4, 1.2 mM MgSO4, and 11.1
mM D-glucose with a pH of 7.4 at 37°C and 5% CO2 perfusion. Male
Sprague-Dawley rats (200 –250 g) were purchased from Harlan (Indianapolis, IN).
Vessel bioassay experiments. For all vessel experiments, thoracic
aortas from male Sprague-Dawley rats were used as previously
described (10, 22) and according to Institutional Animal Care and Use
Committee approval. Briefly, aortas were isolated and submerged in
KH buffer maintained at room temperature. Vessels were cleansed of
fat and connective tissue and segmented into 2- to 4-mm rings. Vessel
rings were placed in vessel bioassay chambers (Radnoti Glass Technologies, Monrovia, CA) containing 15 ml KH buffer and perfused
with a gas mixture composed of 21% O2-5% CO2-74% N2 at 37°C
(pH 7.45). A basal tension of 2 g was applied, and vessel rings were
allowed to equilibrate for 1 h. Following equilibration, a hyperpolarizing dose of KCl (70 mM) was added to the baths to check for
viability and assess maximum constriction.
To assess the effects of O2 on nitrite- or MNO-mediated vasodilation, vessels were equilibrated to 21%, 2%, 1%, or 0% O2 gas
mixtures (containing 5% CO2 and balanced with N2). Gas was
precisely delivered by mass flow controllers (Sierra Instruments) set
to 0.15 l/min, a requirement found to be necessary to achieve reproducible hemoglobin fractional saturations between experiments. In
all runs, vessels were pretreated with indomethacin (5 ␮M) and
NG-monomethyl-L-arginine (L-NMMA;100 ␮M) to block cyclooxygenase and endothelial NO synthase, respectively. Vessels were
preconstricted to ⬃50 –75% of maximal KCl constriction with
L-phenylephrine (PE; 100 nM). Upon lowering O2 tensions, vessel
tone can fluctuate before reaching a stable tone, as illustrated by
Dalsgaard et al. (11). For all experiments reported here, nitrite or
MNO dose responses were initiated after a stable contractile tone had
been reached. Nitrite- or MNO-dependent vasodilation was assessed
by cumulative administration of increasing doses. Experiments were
performed in the absence or presence of either human hemoglobin (20
␮M heme) or C-PTIO (200 ␮M), which were added before the
initiation of the nitrite or MNO dose response. Vasodilatory effects of
cumulative MNO or nitrite additions were determined by measuring
the change in tension and expressing this as the percent relaxation
with respect to the maximal PE constriction. EC50 values were
obtained by directly reading off the concentration (x-axis coordinate)
at 50% relaxation on the dose-response curves.
The protocol employed above uses the same PE and L-NMMA
concentrations at all O2 tensions resulting in lower stable contractile
tensions (i.e., tension immediately before the initiation of the nitrite
dose response) as the O2 tension is decreased (see RESULTS). To test if
differing contractile tensions affect nitrite-dependent dilation and
subsequent modulation by deoxyhemoglobin, a separate series of
experiments was performed whereby upon deoxygenation with 0%
O2, vessel tensions were increased by the addition of higher PE
concentrations (0.5–1 ␮M) and then nitrite was added as described
above.
Hemoglobin fractional saturations. To assess hemoglobin fractional saturation at each O2 tension used, 1-ml aliquots from baths
containing hemoglobin were taken in gas-tight syringes and injected
into argon-purged sealed 1-ml cuvettes under a positive pressure of
argon. Visible spectra (500 –700 nm) of hemoglobin were measured,
and fractional saturation was determined by fitting against previously acquired base spectra (oxyhemoglobin, deoxyhemoglobin,
HEMOGLOBIN FRACTIONAL SATURATION REGULATES NITRITE-DEPENDENT VASODILATION
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and methemoglobin) using a least-squares method as previously
described (20).
Statistical analysis. Evaluation of the effects of either hemoglobin
or C-PTIO on nitrite- or MNO-dependent vasodilation as a function of
O2 tension was assessed by two-way ANOVA with Bonferroni’s post
test. Evaluation of the effects of O2 tension on nitrite- or MNOdependent vasodilation was assessed by one-way ANOVA with Bonferonni’s post test. Significance was set at P ⱕ 0.05, and analysis was
performed using GraphPad Prism software (San Diego, CA).
RESULTS
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Fig. 1. Hemoglobin (Hb) fractional saturations obtained with different O2
tensions. Vessel bioassay baths containing hemoglobin were perfused with gas
mixtures composed of either 21%, 2%, 1%, or 0% O2 (with 5% CO2 and
balanced with N2). A: representative spectra of hemoglobin at each O2 tension.
␭, wavelength. B: calculated fractional saturations after spectral deconvolution.
Data shown means ⫾ SE; n ⫽ 3–5.
system. We note that Dalsgaard et al. (11) observed the same
phenomenon, which they ascribed to a strange artifact of IHP
alone rather than an effect of IHP on promoting hemoglobin
deoxygentation. Importantly, we saw the same effect with
simple hemoglobin deoxygenation in the absence of IHP.
Another possibility is that oxyhemoglobin scavenges nitrite
faster compared with deoxyhemoglobin, a prediction not expected based on reaction kinetics at these nitrite and hemoglobin concentrations (19). To test this, we measured methemoglobin (by spectral deconvolution), a product of both deoxyhemoglobin and oxyhemoglobin reactions with nitrite, before
and after nitrite dose-response curves had been completed.
Methemoglobin was undetectable prior to the addition of nitrite
and did not increase beyond 0.1 ␮M at all O2 tensions (data not
shown). We next tested whether this interaction between nitrite
and deoxyhemoglobin was related to differential NO scavenging effects of deoxyhemoglobin versus oxyhemoglobin (again
not expected based on kinetic considerations).
Hemoglobin-dependent inhibition of NO-mediated vasodilation is not affected by O2 fractional saturation. Figure 2, C and
D, shows representative traces and EC50 values, respectively,
for vasodilation induced by the NO donor MNO at different O2
tensions and in the presence and absence of hemoglobin.
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Hemoglobin fractional saturation in vessel bioassay chambers. To modulate hemoglobin fractional saturation in vessel
bioassay chambers, previous studies have utilized a combination of different O2 contents of the perfused gas together with
coincubation with the negative allosteric effector inositol
hexaphosphate (IHP) (8, 11, 28). Although we have not previously observed basal effects of IHP on vessel tone, other
investigators have suggested such a confounding effect (11,
28). To exclude potential artifacts arising from the use of IHP,
we decided to first determine the conditions required to achieve
hemoglobin fractional saturations spanning 0 –1 (i.e., deoxy ⫺
oxy) without the addition of exogenous allosteric effectors by
testing a variety of gases comprising different O2 tensions
together with different perfusion rates [since most vessel bioassay chambers are open systems, the actual concentration of
dissolved O2 will vary depending on many factors, including
the O2 content of the gas, perfusion pressure, flow rate, or
atmospheric pressure, to name but a few (22)]. Moreover, the
sensitivity of hemoglobin fractional saturation to small changes
in O2 tension around the hemoglobin P50, coupled with the
potential variability in P50 of different cell-free hemoglobin
preparations, necessitate a direct measurement of fractional
saturations. Figure 1A shows representative visible spectra of
hemoglobin; Fig. 1B shows the calculated fractional saturations when baths in our experimental setup were perfused with
a constant flow rate (0.15 l/min) of gases composed of 21%,
2%, 1%, and 0% O2, which resulted in fractional saturations of
0.97, 0.66, 0.26, and 0.04, respectively. We suggest that such
measurements are vitally important in studies that attempt to
evaluate the functional consequences of the allosteric regulation of hemoglobin’s nitrite reductase activity.
Hemoglobin-dependent inhibition of nitrite-mediated vasodilation is regulated by fractional saturation. Figure 2A shows
nitrite-dependent vasodilation in the absence and presence of
hemoglobin at 21% or 0% O2 (traces for 2% or 1% are not
shown for the sake of clarity), and Fig. 2B shows EC50 values
at each O2 tension in the presence or absence of hemoglobin.
Similar to recent data by Dalsgaard et al. (11), nitrite stimulated dilation of rat thoracic aortas in a process that was more
efficient as the O2 tension was lowered. We recognize that this
represents a surprising potency of nitrite, the mechanism for
which remains unclear and which could include interactions
with smooth muscle deoxyheme proteins including deoxymyoglobin (36, 41). We chose to focus on how hemoglobin, and
specifically how hemoglobin O2 fractional saturation, regulates
nitrite-dependent vasodilation. We found that hemoglobin significantly inhibited nitrite-dependent relaxation (indicated by
an increase in EC50) at 21% and 2% but not at 1% or 0% O2,
underscoring a key difference between oxyhemoglobin and
deoxyhemoglobin interactions with nitrite in the aortic ring
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HEMOGLOBIN FRACTIONAL SATURATION REGULATES NITRITE-DEPENDENT VASODILATION
Unlike nitrite, a trend toward increasing EC50 values for
NO-dependent dilation was observed with lowering O2 tensions, which was significant at 0% O2. Most importantly, in
sharp contrast to nitrite-dependent vasodilation, hemoglobin
inhibited NO-dependent vasodilation to similar extents at all
fractional O2 saturations. This suggests that nitrite, compared
with an authentic NO donor, displays a unique interaction with
deoxyhemoglobin that promotes vasodilation. Finally, and as
an additional control, we evaluated the O2 dependence of
dilation stimulated by forskolin, a cAMP-dependent vasodilator, and found that lowering O2 tensions increased EC50 values
from 306 ⫾ 16.4 to 749.8 ⫾ 134 nM (P ⫽ 0.01, n ⫽ 5) as the
O2 tension was lowered from 21% to 0%. This is opposite to
nitrite-dependent effects, and collectively these data underlie
the sensitivity for enhanced nitrite-dependent dilation during
hypoxia.
Contractile tension does not affect nitrite-dependent dilation
at low O2 tensions. Exposure of vessels to low O2 tensions
decreases stable PE-induced contractile tone. For example, in
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the experiments shown in Fig. 2, using a constant PE (100 nM)
concentration at each O2 tension, stable contractile tensions
were 1.7 ⫾ 0.1, 1.14 ⫾ 0.06, 0.98 ⫾ 0.07, and 0.53 ⫾ 0.04 g
at 21%, 2%, 1%, and 0% O2, respectively (data are means ⫾
SE; n ⫽ 15–25). This may affect the potency of nitritedependent dilation at different O2 tensions and, therefore,
hemoglobin-dependent modulation also. To test this, the contractile tension at 0% O2 was increased by the addition of more
PE. Under these constant contractile tension experiments,
nitrite was still a more potent vasodilator compared with 21%
O2, as indicated by lower EC50 values, with this increased
potency not being significantly different to when a constant PE
concentration was used (Fig. 3A). Figure 3B shows that with
the use of the constant contractile tone protocol, deoxyhemoglobin still did not significantly inhibit nitrite-dependent dilation. We note that the EC50 value for nitrite-dependent dilation
at 21% O2 in Fig. 3 was lower than those reported in Fig. 2 and
attribute this interexperimental variance to intrinsic variables
associated with different batches of animals used to obtain
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Fig. 2. Effects of hemoglobin fractional saturation on nitrite or nitric oxide (NO)-mediated vasodilation. A: nitrite dose-dependent relaxation of aortic rings at
21% and 0% O2 in the absence or presence of hemoglobin (20 ␮M). Data are representative of 4 –7 different experiments with each condition repeated 2–3 times
per experiment. Repeated two-way ANOVA analysis indicated a significant (P ⫽ ⬍ 0.05) right shift of nitrite-dependent dilation at 21% O2 by hemoglobin. *P ⱕ
0.001 relative to corresponding the nitrite dose in the absence of hemoglobin at 21% O2 by Bonferroni’s post test. At 0% O2, repeated two-way ANOVA analysis
showed no significant effect (P ⫽ 0.93) of hemoglobin on nitrite dose-dependent dilation. B: EC50 values for nitrite-dependent vasodilation at 21%, 2%, 1%,
and 0% O2 in the absence and presence of hemoglobin. Hemoglobin inhibited nitrite-dependent relaxation at 21% and 2% O2 but not at 1% or 0% O2. Data are
means ⫾ SE; n ⫽ 4 –7. Data were analyzed by two-way ANOVA with Bonferroni’s post test to evaluate significant differences between control and hemoglobin
at each O2 tension. P values are as shown. One-way ANOVA indicated that decreasing O2 tension increases the potency of nitrite-mediated dilation (in the
absence of hemoglobin). *P ⬍ 0.001 relative to nitrite at 21%O2. C and D: data in a similar format to those in A and B except that methylamine hexamethylene
methylamine NONOate (Mahma/NO)-dependent vasodilation was tested. For C, repeated two-way ANOVA analysis indicated significant right shifts of
nitrite-dependent dilation at 21% and 0% O2 by hemoglobin (P ⫽ 0.0003 and P ⫽ 0.005 for 21% and 0% O2, respectively). *P ⱕ 0.001 and #P ⱕ 0.001 relative
to corresponding the nitrite dose in the absence of hemoglobin at 21% and 0% O2, respectively, by Bonferroni’s post test. For D, P values indicate differences
between EC50 values in the presence and absence of hemoglobin at each respective O2 tension by two-way ANOVA with Bonferroni’s post test. Effects of O2
on NO dilation (in the absence of hemoglobin) were assessed by one-way ANOVA with Bonferroni’s posttest. *P ⱕ 0.001 relative to 21% O2. Data in C and
D were obtained from 2– 6 separate experiments and indicate that hemoglobin inhibits NO-dependent vasodilation at all fractional saturations.
HEMOGLOBIN FRACTIONAL SATURATION REGULATES NITRITE-DEPENDENT VASODILATION
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nitrite ⫹ C-PTIO, and nitrite ⫹ deoxyhemoglobin, respectively]. These data suggest that the unique interaction between
nitrite and deoxyhemoglobin cannot be explained by differential NO scavenging at different O2 partial pressures.
DISCUSSION
aortic segments. Importantly, in both Figs. 2 and 3, hypoxia
significantly decreases the EC50 for nitrite-dependent vasodilation.
Comparison of C-PTIO and hemoglobin on nitrite-dependent vasodilation at low O2 tensions. Finally, we evaluated the
effects of C-PTIO, a cell-impermeable non-heme-based NO
scavenger, on nitrite-dependent dilation. Figure 4 shows the
fold change in the EC50 for nitrite-dependent dilation at 21%
and 0% O2 in the presence of either hemoglobin or C-PTIO.
Both C-PTIO and hemoglobin inhibited nitrite-dependent vasodilation, i.e., increased the EC50, at 21% O2 between threeand fourfold. However, at 0% O2, only C-PTIO inhibited
nitrite-dependent dilation, whereas hemoglobin had no effect
[absolute EC50 values at 0% O2 were 6.4 ⫾ 1.2, 19.1 ⫾ 4.9,
and 6.6 ⫾ 0.7 ␮M (means ⫾ SE; n ⫽ 3–7) for nitrite alone,
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Fig. 4. 2-Carboxyphenyl-4,4–5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO)dependent inhibition of nitrite-mediated vasodilation is independent of O2
tension. Nitrite-dependent vasodilation at 21% and 0% O2 was determined in
the absence of presence of either C-PTIO (200 ␮M) or hemoglobin (20 ␮M).
Shown are fold changes in EC50 values in the presence of either C-PTIO or
hemoglobin relative to EC50 values for nitrite alone at respective O2 tensions.
Data are means ⫾ SE; n ⫽ 3–7. *P ⱕ 0.03 relative to the respective O2 tension
control by unpaired t-test.
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Fig. 3. Variations in vessel tension during hypoxia do not affect nitritemediated vasodilation in the presence or absence of hemoglobin. A: EC50
values of nitrite-dependent dilation at 21% or 0% O2. For the 0% O2 condition,
vessels were treated with either a constant dose of L-phenylephrine (PE) or
higher doses of PE to achieve a vessel tension similar to the contractile tension
at 21% O2. Shown are average data from 3 separate experiments in which for
the constant PE condition, vessel tension ranged from 35% to 49% of tension
at 21% O2, whereas with the higher PE condition, vessel tension ranged from
62% to 98% of tension at 21% O2. No significant differences in EC50 values
were observed using either protocol. Data are means ⫾ SE. P value were
determined by t-test (n ⫽ 3). B: nitrite-dependent dilation at 0% O2 in the
absence or presence of hemoglobin using vessels where the contractile tone
was increased to 98% of the contractile tension at 21% O2. No significant
differences (P ⫽ 0.22) between nitrite dose-dependent dilation in the absence
or presence of hemoglobin were observed by repeated-measures two-way
ANOVA.
Nitrite is generally considered an inert end product of NO
metabolism. However, the recent appreciation that hypoxia
regulates nitrite biology and the advent of methods sensitive
enough to measure nitrite in biological matrixes have led to
multiple studies strongly supporting a role for circulating
nitrite in mediating NO-dependent signaling in the vasculature
(6, 8, 27, 29, 31, 35, 38, 44). For example, nitrite protects
against tissue injury in a variety of ischemic diseases (liver and
heart ischemia-reperfusion injury, hypoxic pulmonary vasoconstriction, and hemorrhagic stroke) via NO-dependent mechanisms (13, 21, 23, 34, 43, 45). Moreover, nitrite also has
effects under conditions not characterized by ischemia per se
but by the loss of endogenous NO bioactivity. Examples
include nitrite transducing extrapulmonary effects of inhaled
NO therapy and nitrite-dependent effects on blood pressure (3,
7, 26). In addition, recent insights have indicated that circulating nitrite is a predictor of exercise capacity, and arterialvenous gradients in nitrite have suggested a consumptive
pathway across the microcirculation that is associated with
increased blood flow (18, 37). Potential mechanisms for nitrite
bioconversion into NO and other NO-containing species include reactions with deoxyheme proteins or xanthine oxidase
under ischemic conditions (17).
Although mechanisms that convert nitrite to NO in the
vascular compartment remain under investigation, a viable
mechanism that couples O2 sensing (and hence hypoxia) with
nitrite reduction at physiological pH is reactions with hemoglobin (8, 9, 31). Key data that support this model were
obtained using relaxation of isolated aortic vessels as a functional readout for NO signaling (8, 9). We observed previously
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HEMOGLOBIN FRACTIONAL SATURATION REGULATES NITRITE-DEPENDENT VASODILATION
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that the latter was not observed (Fig. 2B) suggests the third
possibility: in the presence of deoxyhemoglobin, nitrite-dependent bioactivity is a consequence of a net effect of vasoconstriction (NO scavenging) and vasodilation (NO formation).
This is best experimentally defined by a highly significant
interaction between hemoglobin deoxygenation (by low O2 in
our study or the use of IHP in the study of Dalsgaard et al.) and
nitrite. Two-way ANOVA evaluating hemoglobin and O2 fractional saturation on nitrite-dependent vasodilation can be used
to test the significance of this interaction. We note that we fully
reproduce the experiments of Dalsgaard et al., but we can now
clarify that there was a significant nitrite reductase activity of
deoxyhemoglobin in their experiments that was not appreciated.
It remains unclear how these dual reactivities of deoxyhemoglobin are regulated, although recent suggestions have included a possible role for intermediate reactive nitrogen species including N2O3 (15, 20, 39). We also cannot test the more
relevant question of high hematocrits present in vivo. In this
circumstance, it is possible that the nitrite reduction to NO
occurs in specific compartments within the erythrocyte submembrane that promote these reactions. Such physiological
considerations have been recently reviewed (16).
In previous studies, we and others (9, 20) have shown that
the initial rate of nitrite reduction by hemoglobin follows a
bell-shaped dependency with respect to hemoglobin fractional
saturation that reaches a maximum around the hemoglobin P50.
At the P50, an optimal balance of deoxyheme substrate availability and rate constant result in maximal nitrite reduction
kinetics (20, 40). Based on these kinetic observations, one
would predict to see a maximal dilation by nitrite at hemoglobin fractional saturations close to the P50. However, vasodilation experiments assess the magnitude of relaxation and not the
kinetics. In other words, the vasodilation experiments performed here reflect the capacity for a given dose of nitrite to
stimulate vasodilation but not the rate at which this occurs.
Our study was focused on elucidating interactions between
O2, hemoglobin, and nitrite bioactivity. Similar to Dalsgaard
et al. (11), we noted a significant potentiation of nitrite dilation
of aortic vessels at low O2 tensions. The mechanisms intrinsic
to the vessel wall that regulated this hypoxic reactivity are not
known but ultimately feed into NO-dependent activation of
sGC pathways. Whether hypoxia will potentiate nitrite reactivity in resistance vessels remains to be studied, which is an
important consideration since large vessels like the aorta are
not typically exposed to low O2 tensions.
Our data also suggest that nitrite may be an adjunct therapy
for attenuating the vasoconstrictive effects associated with the
use of cell-free hemoglobin-based blood substitutes. The latter
constitutes a variety of hemoglobins whereby the oxygen
binding and structural properties have been modified to improve circulatory lifetimes and O2 delivery and limit nephrotoxicity (1, 32). In addition to these structural modifications
and in the context of this report, we propose that the coadministration of nitrite with hemoglobin-based substitutes may
promote or at the very least preserve NO signaling in the
vasculature. Moreover, our present and recent data underscore
the novel concept that designing cell-free hemoglobins that
possess more efficient nitrite reductase activity may provide
more efficacious resuscitation formulations that improve blood
flow by coupling O2 sensing to local NO production.
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that deoxygenated, but not oxygenated, red blood cells potentiate nitrite-dependent vasodilation in a NO-dependent manner.
More recently, Dalsgaard et al. (11) reported that using cellfree hemoglobin, oxyhemoglobin inhibits, whereas deoxyhemoglobin potentiated, nitrite-dependent vasodilation, but the
effect of the latter was attributed to the presence of the
allosteric effector IHP. In addition, the authors concluded that
the lack of an inhibition on nitrite-dependent vasodilation by
deoxyhemoglobin may be due to higher reaction rates between
nitrite and oxyhemoglobin versus nitrite and deoxyhemoglobin. However, considering that nitrite-dependent dilation proceeded via NO (indicated by a role for sGC) and that hemoglobin reactions with NO are significantly (⬎6 orders of
magnitude) faster compared with those with nitrite, we reasoned that the major effect of oxyhemoglobin and deoxyhemoglobin with respect to inhibition of vasodilation in this
reaction system would be NO and not nitrite scavenging.
To test these concepts, we first standardized our experimental system. Perfusion of bioassay chambers with gases composed of different O2 tensions at a fixed flow rate resulted in
systematic alteration of the hemoglobin fractional saturation
that encompassed the oxyhemoglobin to deoxygenated conformations without the use of exogenous allosteric mediators. The
effects of hemoglobin fractional saturation on both nitrite- and
NO-dependent (using MNO) vasodilation were then tested.
Figure 2, C and D, shows that at all fractional saturations,
hemoglobin inhibited NO-dependent vasodilation, consistent
with the rapid reaction of either oxyhemoglobin or deoxyhemoglobin with NO (reactions 3– 4). This confirmed that
under these experimental conditions, both oxyhemoglobin and
deoxyhemoglobin were competent to react and inhibit NO
signaling. Similar to NO, oxyhemoglobin inhibited nitritedependent vasodilation (Fig. 2B). Unlike NO, however, as
hemoglobin was deoxygenated, the potency of inhibition was
decreased with no significant inhibition being observed at
hemoglobin fractional saturations of 0.26 or 0.04. These data
may be explained by either 1) slower rates of nitrite scavenging
by deoxyhemoglobin over oxyhemoglobin, 2) transition of
nitrite-dependent dilation from an NO-dependent to NO-independent mechanism as O2 tensions are decreased, or 3) the
presence of both vasoconstrictive (NO scavenging) and vasodilatory (NO formation) activities for deoxyhemoglobin.
The first is unlikely to play a significant role on the basis that
both oxyhemoglobin and deoxyhemoglobin react with nitrite
slowly such that over the time scale of vasodilation responses
(⬍5 min/dose) relatively little nitrite will be scavenged. Note
that the nitrite reaction with oxyhemoglobin under these conditions proceeds in the lag or slow phase and is therefore
slower than the reaction of nitrite with deoxyhemoglobin (19).
Indeed, assessment of hemoglobin oxidation and ligation state
indicated that throughout the course of our experiments, ⬎95%
of the hemoglobin remained in the oxy or deoxy conformation,
suggesting little direct scavenging of nitrite (not shown). These
results also indicate that the hemoglobin was in a form competent to rapidly scavenge NO. To probe the second option, the
effects of C-PTIO, an NO scavenger that unlike hemoglobin
does not react with nitrite, was tested. C-PTIO inhibited nitritedependent vasodilation at both 21% and 0% O2 (Fig. 4). These
data indicate that nitrite dilates vessels via NO at all O2
tensions and predicts, therefore, that deoxyhemoglobin should
also scavenge the formed NO and inhibit vasodilation. The fact
HEMOGLOBIN FRACTIONAL SATURATION REGULATES NITRITE-DEPENDENT VASODILATION
In summary, this study sheds insights into how hemoglobin
fractional saturation critically regulates nitrite-dependent NO
formation and subsequent vasodilation and further support the
concept that vascular functions of nitrite may be controlled by
hemoglobin fractional saturation both endogenously and during therapeutic applications.
18.
19.
GRANTS
We acknowledge funds from American Heart Association Southeast Affiliate Grant 0655312B (to R. P. Patel) and a Cardiovascular Pathophysiology
Training Fellowship (to T. S. Isbell).
20.
21.
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