Oxidation and Nitrosation of Thiols at Low Micromolar Exposure to
Nitric Oxide
EVIDENCE FOR A FREE RADICAL MECHANISM*
David
Jourd'heuil
§,
Frances L.
Jourd'heuil
, and
Martin
Feelisch¶
From the Center for
Cardiovascular Sciences, Albany
Medical College, Albany, New York 12208 and the ¶ Department of
Molecular and Cellular Physiology, Louisiana State University Health
Science Center, Shreveport, Louisiana 71130
Received for publication, January 8, 2003, and in revised form, February 17, 2003
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ABSTRACT |
Although the nitric oxide (·NO)-mediated
nitrosation of thiol-containing molecules is increasingly recognized as
an important post-translational modification in cell signaling and
pathology, little is known about the factors that govern this process
in vivo. In the present study, we examined the chemical
pathways of nitrosothiol (RSNO) production at low micromolar
concentrations of ·NO. Our results indicate that, in addition to
nitrosation by the ·NO derivative dinitrogen trioxide
(N2O3), RSNOs may be formed via intermediate
one-electron oxidation of thiols, possibly mediated by nitrogen dioxide
(·NO2), and the subsequent reaction of thiyl
radicals with ·NO. In vitro, the formation of
S-nitrosoglutathione (GSNO) from ·NO and excess
glutathione (GSH) was accompanied by the formation of glutathione
disulfide, which could not be ascribed to the secondary reaction of GSH
with GSNO. Superoxide dismutase significantly increased GSNO yields and
the thiyl radical trap, 5,5-dimethyl-1-pyrroline N-oxide
(DMPO), inhibited by 45 and 98% the formation of GSNO and GSSG,
respectively. Maximum nitrosation yields were obtained at an oxygen
concentration of 3%, whereas higher oxygen tensions decreased GSNO and
increased GSSG formation. When murine fibroblasts were exposed to
exogenous ·NO, RSNO formation was sensitive to DMPO and oxygen
tension in a manner similar to that observed with GSH alone. Our data
indicate that RSNO formation is favored at oxygen concentrations that
typically occur in tissues. Nitrosothiol formation in
vivo depends not only on the availability of ·NO and
O2 but also on the degree of oxidative stress by affecting the steady-state concentration of thiyl radicals.
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INTRODUCTION |
S-Nitrosothiols
(RSNOs)1 are biological
products formed from the interaction of nitric oxide (·NO) with
thiol-containing molecules. In recent years, RSNOs have received
increasing attention as intermediates in the transport, storage, and
delivery of ·NO, as post-translational protein modifications in
cell signaling and inflammatory processes, and as biochemical markers
of reactive nitrogen oxide species (RNOS) (1, 2). Although critical for
understanding their biological roles, the mechanisms by which these
·NO derivatives are formed in vivo are not clearly
understood. Proposed pathways include the reaction of thiols with
dinitrosyl-iron or nitrosylheme complexes (3), direct reaction of
thiols with ·NO in the presence of an electron acceptor such as
NAD+ (4), copper-catalyzed nitrosation (5), and reaction of thiols with peroxynitrite (6-8).
Prevalence of certain reaction pathways is likely to be attained within
the context of specific proteins where the immediate environment may
impact on the reactivity of thiols to RNOS. An increase in
hydrophobicity within the core region of a protein, for example, may
result in the accumulation of ·NO and RNOS with a consequential
acceleration of nitrosation reactions within this region (9). A
decrease in thiol pKa secondary to a change in
the acid/base equilibrium of the surrounding amino acid residues
renders thiols more susceptible to nucleophilic attack by RNOS (10). In
cells, the spatial confinement of protein targets, the presence of
hydrophobic domains, and the occurrence of multiple competing reactions
that limit the availability of ·NO make it difficult to
establish the quantitative contribution of the proposed chemical
pathways (11). Regardless of their contribution to overall RSNO
formation, it is evident that a better understanding of the chemical
principles that dictate the outcome of nitrosation reactions within
cells is required to interpret the possible significance of differences
in RSNO content between different organs and tissues, in particular in
view of a highly reducing environment containing millimolar
concentrations of glutathione (GSH) and limiting micromolar
concentrations of molecular oxygen (O2) (12).
Reactive nitrogen oxide species derived from the reaction of
O2 with ·NO nitrosate thiols (13-16). The reaction
is second order with respect to ·NO and first order in
O2, consistent with the formation of dinitrogen trioxide
(N2O3) as the nitrosating agent.
The oxygen dependence of this process suggests that low
O2 concentrations, which are in the range of 1 to 50 µM in most tissues (17), will greatly limit the efficacy
of this reaction. The second order of Reaction 1 with regard to
·NO also dictates that the rate of RSNO formation via Reaction 3 is controlled by the local concentration of ·NO and that
N2O3 would form quantitatively only when the
concentration of ·NO rises to the micromolar range (18).
In vivo, conditions associated with the up-regulation of the
inducible form of nitric oxide synthase (iNOS) are accompanied by the
production of such levels. In addition, the reaction of ·NO with
O2 is accelerated several hundredfold in the interior of
lipid bilayers and proteins, suggesting that hydrophobic environments in cells and tissues may favor the formation of RSNOs via this pathway
(9, 19).
Although the principles governing ·NO/O2-mediated
nitrosation reactions have been delineated in earlier kinetic studies,
the importance of nitrogen dioxide (·NO2) as an
intermediate in the reaction of thiols with ·NO has been
ignored. Nitrogen dioxide oxidizes thiols (Reaction 4) such as GSH with
a rate constant that is approximately two orders of magnitude smaller
than the rate constant for the reaction of ·NO2 with
·NO (2 × 107
M
1·s
1 compared with 1.1 × 109 M
1·s
1 (20,
21)).
Accordingly, previous studies found negligible thiol oxidation by
·NO and O2 because upon bolus addition of authentic
·NO the initial concentrations of ·NO and
·NO2 are very high, which favor Reaction 2 and
result in high RSNO yields (13, 15). The reaction of excess ·NO
with biological substrates is, however, of limited relevance for the
in vivo situation. We reasoned that the slow in
situ generation of ·NO involves rather low concentrations
instead and that excess thiols can effectively compete with ·NO
for ·NO2 (Reaction 4). As a consequence, thiol
oxidation might be greatly increased and a fraction of RSNOs might be
formed through the radical-radical combination reaction of ·NO
with thiyl radicals (RS·).
Under physiologically relevant conditions, the removal of thiyl
radicals occurs through either geminate recombination or reaction with
O2 and thiolates (12). Consequently, Reaction 5 directly
competes with Reactions 6-9.
In the presence of ambient O2 and excess thiol,
Reaction 8 dominates because of the rapid removal of the disulfide
radical anion (RSSR
) via Reaction 9 (12). The effect of
different O2 concentrations on RSNO yields at low
·NO fluxes is unknown. It may be expected that lower,
physiologically relevant O2 tensions will affect the
relative amounts of oxidized and nitrosated products not only by
reducing the amount of ·NO2 formed but also by
limiting the impact of O2 on thiyl radical consumption.
Here, we present evidence that the nitrosation of thiols by ·NO,
both in vitro and in intact cells, occurs via the intermediate oxidation of thiols by ·NO2 and subsequent reaction
of the thiyl radical with impact of O2NO. Under either
condition, RSNO formation was maximal at low, physiologically relevant
oxygen tension. The apparent discrepancy between the present results
and those obtained in previous studies can be understood by taking into
consideration the competition between ·NO and thiols for
·NO2.
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EXPERIMENTAL PROCEDURES |
Materials--
Spermine NONOate (Sper/NO) was obtained
from Cayman Chemicals (Ann Arbor, MI). All other chemicals were
purchased from Sigma.
Reaction of Glutathione with Nitric Oxide--
In a typical
experiment, a 1-ml reaction volume containing 1 mM GSH, 100 µM DTPA, and either authentic ·NO or the
·NO donor Sper/NO at the indicated concentration was incubated at 37 °C in 20 mM phosphate buffer (pH 7.4). At the
indicated time, the sample was diluted 1:2 (v/v) with ice-cold buffer
containing 10 mM K2HPO4, and 10 mM tetrabutylammonium hydrogen sulfate in acetonitrile-water (5:95, v/v, pH 7.0) and immediately analyzed by high
performance liquid chromatography (HPLC) as described below. Stock
solutions of authentic ·NO were prepared under an inert argon
atmosphere as described previously (22, 23). Briefly, the solutions
were degassed with argon scrubbed from traces of O2. They
were then bubbled with ·NO gas that was purified by passage
through a 5 M NaOH solution. The resulting ·NO
solutions were maintained in septum-sealed glass containers; the
·NO concentration was typically 1.5 ± 0.3 mM
as determined electrochemically using an ·NO-specific electrode
(World Precision Instruments, Sarasota, FL). The ·NO donor
solutions were prepared each day as 10 mM stock solutions in ice-cold 10 mM NaOH and stored on ice until use. The
initial rate of ·NO generation via decomposition of Sper/NO at
37 °C and pH 7.4 was determined electrochemically. In some
instances, the rate of Sper/NO decomposition was determined directly by
measuring the decrease in absorbance at 250 nm (24). For some
experiments, 10-ml solutions of GSH (1 mM) in 20 mM phosphate buffer (pH 7.4) and 100 µM DTPA
were purged with argon in septum-sealed vials. Saturated solutions of
O2, prepared by equilibration of water with 100% oxygen,
were added to each vial using gas tight syringes to obtain final oxygen
tensions of 1, 3, 21, and 50%. 100 µl of a 10 mM stock
solution of argon purged Sper/NO was then injected into each reaction
solution using a gas-tight syringe. After a 60-min incubation at
37 °C, the solutions were diluted 1:2 (v/v) with ice-cold HPLC
eluent and immediately analyzed by HPLC as described below.
HPLC Analysis of GSH Reaction Products--
The products
obtained from the reaction with ·NO in oxygenated solutions were
analyzed by ion-pairing HPLC as described previously (25). Samples were
injected onto a 250 × 4.6 mm 5-µm octadecyl silane
C18 ultrasphere column (Beckman Coulter, Fullerton, CA) isocratically running at a flow rate of 1 ml/min with 10 mM
K2HPO4, 10 mM tetrabutylammonium
hydrogen sulfate in acetonitrile-water (5:95, v/v, pH 7.0). The
reaction products of GSH were detected at 210 nm, and the identity of
each peak was confirmed by co-elution with authentic standards.
Oxygen Consumption--
Oxygen consumption was measured
polarographically using a Cole-Parmer oximeter fitted with a
water-jacketed Clark-type electrode (YSI model 5300), calibrated with
air-saturated distilled water. All experiments were carried out at
37 °C in 20 mM potassium phosphate buffer containing 0.1 mM DTPA.
Cell Culture and Treatment--
The mouse fibroblast cell line
NIH 3T3 was obtained from the American Type Culture Collection
(Manassas, VA). Cells were maintained in 75-cm2 culture
flasks in Dulbecco's modified Eagle's medium supplemented with 10%
(v/v) calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). For experimentation, NIH 3T3 cells were seeded in 25 cm2 flasks and grown to 80% confluence in Dulbecco's
modified Eagle's medium with 10% (v/v) calf serum, penicillin (100 units/ml), and streptomycin (100 µg/ml). Before incubation with the
·NO donor, cell culture media were replaced with fresh
Dulbecco's modified Eagle's medium containing 10% serum, and the
flasks were preincubated for 2 h in humidified atmosphere
equilibrated with different O2/nitrogen/CO2 gas
mixtures, resulting in final O2 tensions of 1, 3, 21, and
50%. The cells were then incubated with argon-purged Sper/NO for
1 h at 37 °C. Cell viability was not affected by any of the
treatments as tested by trypan blue exclusion (data not shown).
Chemiluminescence Detection of Cellular RSNOs--
Treated cells
were washed once with cold phosphate-buffered saline containing 100 µM DTPA. The cells were detached with trypsin-EDTA, collected by centrifugation, counted, and resuspended in 1 ml of 4 mM phosphate buffer containing 100 µM DTPA.
Cell suspensions were homogenized using a Dounce homogenizer. Eight
hundred microliters of each homogenate were transferred to a glass tube
containing 100 µl of 100 mM
N-ethylmaleimide. The samples were kept on ice and in
the dark for 15 min before the addition of 100 µl of 100 mM sulfanilamide and were then incubated for another 15 min
on ice. RSNO formation was evaluated by measuring the amount of
·NO liberated after reductive decomposition of the
S-nitrosothiols in a purge vessel as described previously
(26). Briefly, the purge vessel contained 4.5 ml of glacial acetic acid
and 500 µl of an aqueous mixture containing 450 mM
potassium iodide and 100 mM iodine. The vessel was kept at
70 °C via a water jacket, and the solution was constantly purged
with nitrogen and changed every four injections. The amounts of NO
evolving from the purge vessel were quantified by gas phase
chemiluminescence (NOA 280, Sievers Instruments, Boulder, CO). Peak
integration was performed, and the results were converted to ·NO
concentrations using authentic ·NO as a standard. Calculated
·NO concentrations were further validated using standards of
authentic GSNO.
Statistics--
For groups of three or more, the data were
analyzed by one-way analysis of variance, and when a significant
difference was suggested, the Tukey test was used as a post
hoc test. Comparisons restricted to two groups were analyzed using
the Student's t test. A probability value of less than 0.05 was considered a statistically significant difference.
 |
RESULTS |
The reaction of 1 mM GSH with authentic ·NO
(12.5-375 µM) in 20 mM potassium phosphate
buffer (pH 7.4, 37 °C) equilibrated with ambient air was studied by
HPLC. Glutathione, nitrite, GSSG, nitrate, and GSNO eluted in this
order as confirmed by comparison with standards (Fig.
1, A and B). Both
GSSG and GSNO were formed upon incubation of 1 mM GSH with
375 µM ·NO (Fig. 1B). Table
I summarizes the concentrations of GSSG, GSNO, nitrite, and nitrate formed under these conditions. For all four
concentrations of ·NO tested, GSNO production was accompanied by
GSSG formation. The latter could not be attributed to the reaction of
GSH with GSNO because the incubation of authentic GSNO (50 µM) with GSH (1 mM) did not result in an
increase in the GSSG concentration within the 30-min incubation period
(data not shown). The amount of GSNO formed was two to three times
higher than the amount of GSSG formed at the highest ·NO
concentration tested. In contrast, the amount of GSNO recovered was two
to five times lower than that of GSSG at the lowest concentration of
·NO. These results indicate that the reaction of ·NO with
O2 resulted in the formation of an intermediate capable of
oxidizing GSH to GSSG and that high concentrations of ·NO could
compete with GSH for this oxidant to limit GSSG formation and increase
GSNO formation.

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Fig. 1.
Reaction of GSH with authentic ·NO in
oxygenated solution. A, representative HPLC
chromatogram of GSH, nitrite, GSSG, nitrate, and GSNO standards with
direct UV detection at 210 nm. B, GSH (1 mM) was
incubated with 375 µM authentic ·NO in 20 mM phosphate buffer and 100 µM DTPA for 30 min at 37 °C. The amounts of nitrite, GSSG, nitrate, and GSNO formed
are reported in Table I.
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Table I
Concentrations of GSNO, GSSG, nitrite, and nitrate formed upon
incubation of GSH with ·NO at ambient oxygen concentration
Reduced glutathione (GSH; 1 mM) was incubated for 30 min
with various concentrations of ·NO in 20 mM
potassium phosphate (pH 7.4), and product formation was determined as
described in the legend for Fig. 1. The concentrations are expressed as
the means ± S.D. of three independent experiments.
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To further evaluate the nitrosative and oxidative chemistry associated
with the reaction of ·NO with O2, the
diazeniumdiolate Sper/NO was used as a source of ·NO because of
its predicable rate of ·NO release at neutral pH (24). In the
presence of 100 µM Sper/NO (1.2 ± 0.2 µM/min ·NO generated, pH 7.4), GSNO accumulation
reached saturation after 90 min, whereas GSSG concentration continued
to increase over the 3-h incubation period, at which time the
concentration of GSSG exceeded that of GSNO by ~400% (Fig.
2A). Again, incubation of
preformed GSNO (0-50 µM) with 1 mM GSH did
not result in a statistically significant increase in GSNO
decomposition within the 3-hour incubation period (data not shown).
Consistent with the results obtained with authentic ·NO, the
amount of GSNO relative to GSSG increased with the rate of ·NO
generation (Fig 2B).

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Fig. 2.
Oxidation and nitrosation of GSH at low
micromolar exposure of ·NO. A, time course of
product formation upon incubation of GSH (1 mM) with
Sper/NO (100 µM) in solutions equilibrated with ambient
air containing 20 mM phosphate (pH 7.4) and 100 µM DTPA. Product formation was examined as
described in the legend for Fig. 1. Values represent the means ± standard deviation (n = 3). B, GSH (1 mM) was incubated for 60 min with increasing concentrations
of Sper/NO (0-500 µM) in 20 mM potassium
phosphate (pH 7.4). The rate of ·NO generation by the ·NO
donor was determined electrochemically using a ·NO-specific
electrode as described under "Experimental Procedures." Product
formation was determined as described in the legend for Fig. 1. The
ratios of GSNO to GSSG concentrations are expressed as the means ± S.D. of three different experiments.
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Oxygen uptake was moderate during the generation of ·NO alone
but increased in the presence of GSH (Fig.
3). The addition of superoxide dismutase
(SOD) to the reaction mixture increased by ~20% the amount of GSNO
formed and decreased nitrate formation by about 30% (Fig.
4A). The formation of GSSG,
the increased O2 consumption, and the effect of SOD were
all consistent with a redox pathway in which the thiyl radical,
GS·, formed from the one-electron oxidation of GSH, played a
central role. The thiyl radical reacts with the thiolate (GS-) of
another GSH molecule to form the glutathione disulfide radical anion
(GSSG
), which in turn reduces O2 to produce
the superoxide anion (O
, Reactions 6 to 9). Under our
experimental conditions, O
scavenged a fraction of ·NO
to form peroxynitrite (ONOO-/ONOOH) with a resultant decrease in the
yield of GSNO and an increase in nitrate, the decomposition product of
peroxynitrite. Transient formation of the glutathionyl radical
GS· has been shown to occur upon incubation of Sper/NO with GSH
(27). To evaluate the role of GS· in the formation of GSSG and
GSNO under our experimental conditions, we tested the effect of the
thiyl radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO)
(Fig. 4B). We found that DMPO (100 mM) inhibited the formation of GSSG, GSNO, and nitrate by 98, 45, and 45%,
respectively, upon incubation of 100 µM Sper/NO with 1 mM GSH for 1 h at 37 °C. The concentration of
nitrite could not be determined because DMPO (100 mM) and
nitrite co-eluted. These results further support the formation of
GS· and the occurrence of a free radical pathway for the
oxidation and nitrosation of thiols at low micromolar exposure to
·NO.

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Fig. 3.
Effect of GSH on ·NO-mediated oxygen
consumption. Sper/NO (100 µM) was incubated in the
absence and presence of GSH in air-equilibrated 20 mM
phosphate buffer (pH 7.4) supplemented with 100 µM DTPA,
and oxygen consumption was followed for 60 min as described
under "Experimental Procedures." Values represent the means ± S.D. (n = 3; *, p < 0.05 compared with
no GSH).
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Fig. 4.
Effect of SOD and DMPO on GSSG, GSNO,
nitrite, and nitrate formation. A, GSH (1 mM) was incubated for 2 h with 100 µM
Sper/NO in solutions equilibrated with ambient air containing 20 mM phosphate (pH 7.4), 100 µM DTPA, and 0.5 mg/ml SOD. Changes in metabolites are expressed as percentage compared
with control (no SOD). The values represent the mean ± standard
deviation (n = 3; *, p < 0.05 compared
with control). B, GSH (1 mM) was incubated with
100 µM Sper/NO as described in A, in the
presence or absence of the thiyl radical trap DMPO (100 mM). Changes in metabolite concentrations are expressed as
percentage compared with control (no DMPO). The values represent the
means ± S.D. (n = 3; *, p < 0.05 compared with control).
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The availability of GS· for reaction with ·NO might be
modulated by O2 not only because the formation of
·NO2 is an O2-dependent
process but also because O2 drives the consumption of
GS· through its reaction with the glutathione disulfide radical
with concurrent formation of O
(Reaction 9 and Ref. 12).
Thus, we tested the effect of O2 tension on the reaction of
·NO and O2 with GSH. We hypothesized that at high
O2 tension, the reduction of O2 by the
disulfide radical diminishes the concentration of thiyl radicals
available for reaction with ·NO to form GSNO. Alternatively, an
increased O
formation at high O2 tension might
lead to a decrease in the yield of GSNO through peroxynitrite
formation. We found that the amount of GSNO formed upon incubation of
GSH (1 mM) with Sper/NO (100 µM) reached a
maximum at 3% O2 saturation and that increasing O2 tension to 50% inhibited GSNO formation by ~30%
(compared with 3% O2; Fig.
5A). Concurrently, GSSG
formation increased in a concentration-dependent manner
with increasing O2 tension (Fig. 5B). In these
experiments, we verified that the rate of Sper/NO decomposition was not
altered by changes in oxygen tension and that the pH of the solutions did not change upon incubation with the different gas mixtures (data
not shown).

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Fig. 5.
Effect of oxygen tension on the reaction of
·NO with GSH. GSH (1 mM) in 20 mM
phosphate buffer and 100 µM DTPA was incubated for 1 h at 37 °C with 100 µM Sper/NO and various
concentrations of O2 to obtain final O2
tensions of 1, 3, 21, and 50% as described under "Experimental
Procedures." GSNO (A) and GSSG (B)
concentrations were determined as described in the legend for Fig. 1.
Values represent the means ± S.D. (n = 3; *,
p < 0.05 compared with 3%; #, p < 0.05 compared with 1%).
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To explore the relevance of these new pathways for cellular nitrosation
reactions, we examined the sensitivity of RSNO formation to the free
radical scavenger DMPO and to changes in oxygen tension. Mouse
fibroblasts (NIH 3T3 cell line) were exposed to exogenous ·NO
by incubation with Sper/NO at 37 °C. The cells were then processed for RSNO determination by gas phase chemiluminescence as described under "Experimental Procedures." To ascertain that ·NO
reaction sites were thiols, samples were examined for their sensitivity
to HgCl2 and light (26). The results, illustrated in Fig.
6, A and B,
demonstrate that more than 75% of the signal obtained from cells
exposed to ·NO represented RSNOs, but we also identified a
mercury- and light-insensitive component of unknown identity. A
concentration of 100 µM Sper/NO was utilized to establish
the time course of RSNO formation from the NIH 3T3 fibroblasts.
Increased RSNO content was detected as early as 15 min and maximized at
60 min of exposure (Fig. 6C). No RSNO was found when the
cells were incubated with decomposed Sper/NO (data not shown). The
fibroblasts were exposed for 60 min to different concentrations of
Sper/NO (10-100 µM) to provide initial rates of
·NO release ranging from ~0.1 to 1.0 µM/min.
RSNO formation was apparent upon exposure to 10 µM
Sper/NO and increased with increasing ·NO fluxes (Fig
6D). In the presence of 100 µM Sper/NO,
preincubation of the cells with DMPO (10 mM)
decreased cellular RSNO content by ~45% (Fig.
7A). Maximum nitrosation
occurred upon incubation of the cells with 3% O2 and was
decreased by 45% (compared with the 3%) with 50% oxygen (Fig.
7B). Taken together, these results were consistent with
those obtained with GSH in aqueous buffer systems, indicating that a
free radical pathway may represent a relevant process for RSNO
formation in cells.

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Fig. 6.
Determination of nitrosothiol content of
mouse fibroblasts exposed to exogenous ·NO. A,
typical chemiluminescence detector responses after duplicate injection
of cell lysates obtained from NIH 3T3 fibroblasts incubated for 1 h with or without 100 µM Sper/NO. The presence of RSNOs,
mercury-resistant, and light-resistant species was examined.
B, quantitative determination of the results presented in
A. The values represent the means ± S.D.
(n = 3; *, p < 0.05 compared with
control). C, time-dependent increase in RSNO
content from NIH 3T3 fibroblasts incubated with 100 µM
Sper/NO as described under "Experimental Procedures." The
values represent the means ± S.D. of three independent
experiments. D, fibroblasts were exposed for 1 h to
increasing concentrations of Sper/NO as described under "Experimental
Procedures." The values represent the means ± S.D. of three
independent experiments.
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Fig. 7.
Effect of DMPO and oxygen tension on RSNO
content of fibroblasts exposed to exogenous ·NO.
A, NIH 3T3 fibroblasts were incubated for 30 min with the
thiyl radical trap DMPO (10 mM) before treatment for 1 h with 100 µM Sper/NO. RSNO content was determined as
described in the legend for Fig. 6. The values represent the means ± S.D. (n = 3; *, p < 0.05 compared
with control). B, cells were preincubated for 2 h in
humidified atmosphere with different
O2/nitrogen/CO2 gas mixtures, resulting in a
final O2 tension of 1, 3, 21, and 50%. The cells were then
incubated with argon-purged Sper/NO (100 µM) for 1 h
at 37 °C, and RSNO content was determined. The values
represent the means ± S.D. (n = 3; *,
p < 0.05 compared with control).
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DISCUSSION |
The reaction of ·NO and O2 with thiols is
thought to result in thiol nitrosation through the exclusive
intermediacy of dinitrogen trioxide (N2O3)
formed from the combination of ·NO with ·NO2
(Reaction 2; Refs. 15 and 28). In the context of the cell, however,
thiols are in excess relative to ·NO, and a large fraction of
·NO2 should react with thiols rather than ·NO
itself (Reaction 4; Ref. 21). An important corollary of the trapping of
·NO2 by thiols is the formation of oxidized
products, including disulfides, through the intermediate formation of
thiyl radicals (Reactions 6-8). Previous studies found negligible or
no oxidation products suggesting key differences between the
experimental approach and theoretical principles (13-15). In the
present study, we found that ·NO and O2 both
oxidized and nitrosated GSH. The yields of GSNO and GSSG depended upon
the relative concentrations of ·NO and GSH, consistent with a
competition between ·NO and GSH for ·NO2. The
formation of GSSG, the increased O2 consumption upon increased GSH concentration, and the effect of SOD were evidence for
the occurrence of a free radical mechanism in which O2 is reduced to O
by the glutathione disulfide radical (Reaction
9). If true, a fraction of generated ·NO should then react with
O
in a diffusion-limited manner to form peroxynitrite
(ONOO-/ONOOH), and hence the sensitivity of GSNO and nitrate
formation to superoxide dismutase. Independent of peroxynitrite
formation, the occurrence of GSSG in amounts exceeding those of GSNO
indicates that ·NO autoxidation might initiate oxidative
reactions that were previously only ascribed to the reaction of
·NO with O
. Thus, the dichotomy between the reactivity
of ·NO autoxidation intermediates and peroxynitrite may not be
as clear as previously thought, in as much as both the
·NO/O2 and ·NO/O
reactions are
accompanied by oxidation reactions.
For the most part, the importance of ·NO2 in the
·NO/O2 reaction and the formation of the
glutathionyl radical, GS·, has been ignored in previous works.
Although Pou and Rosen (27) observed the transient formation of
GS· upon incubation of GSH with Sper/NO in the presence of a
spin trap, no effort was made to further investigate product formation. The present work indicates that a large fraction of RSNO formed under
these conditions is derived from the combination of GS· with
·NO because we showed that the thiyl radical trap DMPO reduces GSNO yields by ~45%. We also showed that RSNO yields did not
increase linearly with O2 concentration but were maximal at
low O2 tension. Because O2 drives the removal
of thiyl radicals and the consecutive formation of the ·NO
scavenger O
(Reactions 8 and 9), a decrease in O2
tension results in a diminished efficacy of these reactions and a
resultant increase in the availability of ·NO and thiyl
radicals for combination. Of course, the formation of
·NO2 is an oxygen-dependent process, but
the concentration attained at 3% O2 tension
(corresponding to ~30 µM) is not limiting compared with
the low fluxes of ·NO generated by Sper/NO.
As outlined in the Introduction, it would be very surprising if
experimental results obtained from studies in aqueous solutions allowed
a comprehensive understanding of the relevant chemical pathways that
dictate cellular nitrosation. Among other considerations, compartmentalization of target molecules, the existence of hydrophobic environments that concentrate NO and O2, spatial and
temporal variations in O2 availability and O
production, as well as changes in enzymatic and non-enzymatic antioxidant levels will all affect cellular nitrosation reactions (11),
making the extrapolation of in vitro results to the in vivo situation a pointless exercise. Nevertheless, the results of
our present investigation provide a strong foundation from which
general principles may be developed that are amenable to further
investigation in relevant animal models. An important implication of
the present study is that low, physiological O2 concentrations do not limit cellular RSNO formation. Rather, RSNO formation appears to be favored at oxygen concentrations that typically
occur in tissues. However, because the rate of ·NO2
formation is second order with regard to ·NO,
·NO/O2-mediated oxidation and nitrosation reactions
are hampered by the availability of ·NO2 in a
fashion similar to the reactions mediated by
N2O3. Independent of the role of
·NO2 as a key intermediate, the recognition that
RSNO formation occurs, at least in part, through a free radical
mechanism offers new avenues for investigations of the nature of the
pathways that lead to RSNO formation in vivo and of
our ability to modulate these processes. Another important implication
of these findings is that the concentration of RSNOs in vivo
may not only be limited by the formation or availability of ·NO
but also by the prevailing steady-state concentration of thiyl radicals, which may be influenced by the local availability of antioxidants. Importantly, RSNO formation was observed not only with
GSH in vitro but also in intact cells endowed with a
powerful antioxidative network in which intracellular GSH is
complemented by ascorbate and
-tocopherol, for example, as well as
superoxide dismutase, catalase, and other enzymes. It is intriguing to
speculate that conditions that are associated with an increased
oxidative stress and a consecutive increase in one-electron oxidation
of thiols may provide an important source of thiyl radicals available for reaction with ·NO. Under such conditions, therapeutic
strategies aimed at limiting the production and availability of thiyl
radicals would be critical in modulating the sensitivity of cells and
tissues to reactive nitrogen oxide species.
 |
ACKNOWLEDGEMENT |
We thank Nadia Azzam-Thorn for technical assistance.
 |
FOOTNOTES |
*
This work was supported by Grants CA89366 (to D. J.) and
HL69029 (to M. F.) from the National Institutes of Health.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.
§
To whom correspondence should be addressed: Albany Medical College,
Center for Cardiovascular Sciences, 47 New Scotland Ave. (MC8), Albany,
NY 12208. Tel.: 518-262-8104; Fax: 518-262-8101; E-mail:
jourdhd@mail.amc.edu.
Published, JBC Papers in Press, February 20, 2003, DOI 10.1074/jbc.M300203200
 |
ABBREVIATIONS |
The abbreviations used are:
RSNO, nitrosothiol;
RNOS, reactive nitrogen oxide species;
HPLC, high performance
liquid chromatography;
DTPA, diethylenetriaminepentaacetic acid;
GSNO, S-nitrosoglutathione;
SOD, superoxide dismutase;
Sper/NO, spermine NONOate.
 |
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