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J Biol Chem, Vol. 273, Issue 48, 32009-32015, November 27, 1998
From the Departments of Pharmacology and Physiology, New York
Medical College, Valhalla, New York 10595
Peroxynitrite (ONOO Exposure of vascular tissue to peroxynitrite
(ONOO Peroxynitrite is also known to undergo additional reactions in the
presence of physiological buffered systems and GSH. One of the first
observed actions of ONOO Nitric Oxide Measurements--
Head space NO measurements were
determined employing a NO chemiluminescence analyzer (model 20B,
Sievers Instruments, Boulder, CO), using a slightly modified version of
the method of Brien et al. (18) that was adapted for our
previous studies on ONOO Chemistry--
Peroxynitrite was prepared from sodium nitrite
and H2O2 in a quenched-flow reactor with minor
modifications (19). H2O2 and oxygen were
removed from the peroxynitrite solution by adding catalytic amounts of
MnO2 followed by bubbling with a gentle stream of dry
nitrogen for 1 h. ONOO Preparation of Standards--
GSNO and GSNO2 were
prepared as described previously (6). Briefly, GSNO2 was
prepared by addition of thiol nitrating reagent, nitronium
tetrafluoroborate (NO2BF4, 0.1 M)
to 25 mM GSH dissolved in 0.5 M
phosphate-buffered saline (pH 7.4), and the mixture was the subjected
to purification by HPLC. The NO2BF4 was
prepared immediately before use as a 1 M solution in 0.1 N HCl. GSNO was obtained via analogous reaction with
nitrosonium tetrafluoroborate (NOBF4). Overnight storage of
GSNO and GSNO2 at ESI/MS/MS--
Mass spectrometry was performed on a triple
quadrupole tandem mass spectrometer (TSQ 700, Finnigan-MAT, San Jose,
CA) equipped with an ESI interface. Both the electrospray needle and
the skimmer were operated at ground potential, whereas the electrospray
chamber and metalized entrance of the glass capillary were operated at HPLC Analyses--
Samples were injected into a 1050 HPLC system
(Hewlett-Packard, Palo Alto CA) equipped with a quaternary pump and a
variable wavelength UV detector and operated by a ChemStation data
system. The compounds were analyzed on a C18 Hypersil column (250 × 4.6 mm, Beckman Instruments) that was eluted isocratically with
acetonitrile (5%, v/v) in water (containing acetic acid, 0.01% v/v,
pH 4) at a flow of 1 ml/min. The effluent from the column was collected in 1-ml fractions using a Gilson FC203B fraction collector. The chromatograms were obtained by monitoring UV absorbance at 215 and 344 nm. Fractions containing UV light-absorbing substances were subjected
to further analyses.
UV Spectroscopy--
Samples (1 ml) containing either
GSNO2 or GSNO, freshly purified by HPLC, were placed in a
semimicro-UV 1-ml quartz cuvette (QS 1.000, Fisher Scientific), and the
spectra were recorded using a diode array spectrophotometer
(Hewlett-Packard HP 8452A). The spectra were taken from 200 to 450 nm
at a scan rate of 250 nm/s. Specific absorbance at 334 nm was 980 M Statistical Analysis--
The data were analyzed by one- and
two-way analysis of variance for multiple comparisons employing a
Bonferroni's modified t test to determine statistical
significance for all studies. Results are reported as means ± S.E., with n equal to the number of separate determinations
in chemical reactions. p < 0.05 was used to determine
statistical significance.
Materials--
Reduced glutathione was from Calbiochem.
NO2BF4 and NOBF4 were purchased
from Aldrich. All solvents were of highest chromatographic grade.
Properties of NO Generation from Reactions of ONOO
The observation of a prolonged release of NO from the interaction of
ONOO
The maximal yield of NO from GSNO2 in the absence of added
GSH at the 30 min time point was approximately 30-40%, and this was
observed to decrease on addition of increasing concentrations of GSH.
In separate experiments, which determined the amount of NO released
from GSNO2 in the absence and presence of 1 mM
GSH, it was confirmed that NO generation at 30 min was reduced by 43% in the presence of GSH relative to the control. A reaction of GSH with
GSNO2 resulted most likely in the formation of nitrite and
disulfide (GSSG). This explanation is based on a previous observation
that was suggested as an explanation for the observed decreased
formation of NO from tert-butyl thionitrate via a
competitive reaction with cysteine (20). Whereas
CO2/bicarbonate buffer did not affect the production of NO
from GSNO2 in the presence of GSH and glucose, NO formation
in the absence of glucose was further decreased by an additional 46%
in CO2/bicarbonate buffer (Fig. 2). A strong additional
inhibitory effect of GSH on NO release from GSNO2 in the
presence of CO2/bicarbonate and in the absence of glucose
(Fig. 2, p < 0.05) may explain the minimal release of
NO from ONOO Characterization of GSNO2--
Treatment of GSH with
peroxynitrite at pH 7.4 followed by RP-HPLC analysis using a mobile
phase containing water and 0.1% acetonitrile revealed the presence of
several substances (Fig. 3). Compound
a was confirmed to be unreacted glutathione because it
coeluted with GSH, and its ESI/mass spectrum (Fig.
4) was identical with that of intact GSH.
A novel compound b, eluting at 7.1 min., was detected as a
product of ONOO
Ultraviolet spectra of GSNO2 and GSNO showed minimal
differences (Fig. 5). Both compounds
displayed a relatively strong UV absorbance at 334 nm. The spectrum of
GSNO showed a stronger deflection than the spectrum of
GSNO2 at 218 nm. Because it was difficult to establish the
structure of ONOO
ESI mass spectrum of the ONOO
The abundance of the HPLC peak corresponding to GSNO2
relative to GSH was 6.3% following a mixing of 1 mM GSH
with 400 µM ONOO The results of the present study provide evidence for our recent
suggestion (6) that GSNO2 is a somewhat stable product of
the reaction of ONOO Mass spectra of a key product that results from the reaction of GSH
with ONOO It has been previously reported that RSNO2
(R = tert-butyl) species release detectable
amounts of NO (20). The rate of this decomposition is relatively low
(about 105 s The observation that glucose- and
CO2/bicarbonate-containing buffers reduced the amount of NO
generation from ONOO Observations made in the present study on the reaction of
ONOO We thank Dr. Jing Song Ni from the American
Health Foundation (Valhalla, NY) for performing analyses of samples by
mass spectrometry.
*
This research was supported by Grants HL31069, HL43023, and
HL34300 from the National Institutes of Health and Grant 950325 from
the American Heart Association NY State Affiliate.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.
The abbreviations used are:
ONOO
S-Nitroglutathione, a Product of the Reaction between
Peroxynitrite and Glutathione That Generates Nitric Oxide*
,
ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References
) has been
shown in studies on vascular relaxation and guanylate cyclase
activation to react with glutathione (GSH), generating an intermediate
product that promotes a time-dependent production of nitric
oxide (NO). In this study, reactions of ONOO with GSH
produced a new substance, which was characterized by liquid
chromatography, ultraviolet spectroscopy, and electrospray tandem mass
spectrometry. The mass spectrometric data provided evidence that the
product of this reaction was S-nitroglutathione (GSNO2) and that S-nitrosoglutathione (GSNO)
was not a detectable product of this reaction. Further evidence was
obtained by comparison of the spectral and chromatographic properties
with synthetic standards prepared by reaction of GSH with nitrosonium
or nitronium borofluorates. Both the synthetic and
ONOO/GSH-derived GSNO2 generated a protonated
ion, GSNO2H+, at m/z 353, which was
unusually resistant to decomposition under collision activation, and no
fragmentation was observed at collision energy of 25 eV. In contrast,
an ion at m/z 337 (GSNOH+), generated from the
synthetic GSNO, readily fragmented with the abundant loss of NO at 9 eV. Reactions of ONOO with GSH resulted in the generation
of NO, which was detected by the head space/NO-chemiluminescence
analyzer method. The generation of NO was inhibited by the presence of
glucose and/or CO2 in the buffers employed. Synthetic
GSNO2 spontaneously generated NO in a manner that was not
significantly altered by glucose or CO2. Thus,
ONOO reacts with GSH to form GSNO2, and
GSNO2 decomposes in a manner that generates NO.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
)1 results
in a prolonged relaxation (1) that appears to be mediated through a
glutathione (GSH)-dependent regeneration of NO (2).
Peroxynitrite has also been observed to stimulate guanylate cyclase
activity in a thiol-dependent manner in vascular
endothelial and smooth muscle preparations (3, 4). Whereas the reaction
of ONOO with GSH has been reported to form small amounts
of S-nitroso-GSH (GSNO) (3, 5), our previous studies
detected a different product of this reaction, which was isolated and
demonstrated to possess potent vascular relaxant activity (2).
Examination of the reaction of nitrogen dioxide (NO2) with
GSH detected the formation of what appears to be the same product as
that observed in the reaction with ONOO (6). Because the
biologically active metabolite of these reactions co-migrated on HPLC
with a the product of a reaction between nitrosonium borofluorate
(NO2BF4) and GSH, the vascular relaxant
detected was suggested to be a nitrated product of GSH
(GSNO2) (6). Thus, additional studies are needed to
identify the biologically active substances derived from the reaction
of GSH with ONOO.
on thiols was that it caused
oxidation reactions, and an analysis of products of these reactions
resulted in a hypothesis that nitrated thiols were a key unstable
intermediate formed during these reactions (7, 8). Recent studies have
also provided evidence that thiol radicals seem to be one of the major
initial products of the reaction of ONOO with thiols (9,
10). Peroxynitrite appears to react with glucose and other hydroxylated
compounds to produce relatively stable products that cause tissue- or
thiol-dependent generation of NO and a prolonged relaxation
of vascular tissue (11, 12). However, the vasoactive products formed
from reactions with glucose seem to be significant only at very
elevated levels of ONOO (13). Peroxynitrite also reacts
with CO2/bicarbonate to generate an intermediate that is a
potent nitrating agent (14-17). Thus, ONOO may interact
with additional components of tissues and buffers to form biologically
active metabolites. The purpose of this study was to determine whether
the reaction of ONOO with GSH generates GSNO2
and to examine aspects of how the formation GSNO2
potentially participates in the generation of NO from
ONOO.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
and NO2 (2, 6). To
quantify the amount of NO produced in the head space gas derived from
0.4 mM ONOO or 0.1 mM
GSNO2, the following protocols were applied: Fernbach flasks (6 ml) containing a 2-ml final volume of 25 mM
potassium phosphate or 25 mM bicarbonate buffer (pH 7.4) in
the presence or absence of combinations of 1 mM GSH and 5.6 mM glucose were incubated at 37 °C equilibrated under an
atmosphere of argon or 95% N2-5% CO2,
respectively. To the sealed Fernbach flasks, aliquots of deoxygenated
ONOO or GSNO2 were injected, and the flasks
containing the various experimental conditions were allowed to
accumulate NO in the head space for 30 min. After 30 min, a single
0.5-ml aliquot was taken of the head space gas from each sealed
Fernbach flask to quantitate the amount of NO produced from each
experimental condition employing the Sievers NO analyzer. Under these
severely hypoxic conditions, NO is stable over the 30-min accumulation
period. The amount of NO formed was quantified, after subtraction of an
injection artifact blank, based on NO standards and recovery of
authentic NO from the Fernbach flask containing 2 ml of buffer.
was concentrated by freezing
the ONOO solution for overnight at 
20 °C, and the
concentrated top yellow layer was carefully thawed and transferred to a
clean glass tube. The concentration of peroxynitrite was determined
spectrophotometrically by measurement of absorbance at 302 nm in 0.1 N NaOH (A302 = 1670 M1·cm1). The stock solution
contained 170-190 mM ONOO, and dillutions
were made in 0.1 N NaOH. The reaction of GSH (1 mM) with ONOO (1 mM) was carried
in 0.5 ml of phosphate buffer (pH 7.4) in a stopper-capped glass tube
with constant stirring at room temperature. The aliquots (20 µl) of
the reaction mixture were taken at various time points and analyzed by
injection into a reverse-phase HPLC column (RP-HPLC). In control
experiments, the GSH was reacted with decomposed ONOO (30 min at 37 °C). The products of the reaction were analyzed by RP-HPLC
and electrospray ionization tandem mass spectrometry (ESI/MS/MS).
20 °C allowed us to obtain enriched
preparations of these compounds as concentrated upper pink-colored
layers, which were collected, purified by RP-HPLC, and analyzed by
ESI/MS/MS.
3.5 kV in the positive ion mode. The mass spectrometer was operated at unit resolution across its entire mass range. Nitrogen was used for
all inlet gases other than the collision gas, which was argon. The
collision gas thickness for tandem mass spectrometry experiments was
250-300 ×1012 molecules/cm2. All samples
(0.5-1 pmol/µl) were dissolved in deionized water containing 5%
acetonitrile and 0.01% acetic acid and infused into the electrospray
ion source by using a syringe pump (model 22, Harvard Apparatus, South
Natick, MA) at a flow of 1 µl/min for 5 min through a 100 µm-inner
diameter fused silica capillary. The capillary transfer tube
temperature was kept at 150 °C. The curtain gas flow was 1.2 liters/min, and the nebulizer gas flow was held at 0.9 liter/min.
Collision-induced dissociations were induced with 1.4 torr, resulting
in 50-60% decrease in ion transmission. Collision energy ranged from
8 to 25 eV.
1·cm1 for GSNO2
and 800 M1·cm1 for GSNO.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
with GSH and from GSNO2--
Our previous work
demonstrated that the release of NO from the reaction of
ONOO with GSH was approximately linear for the first 30 min, and it decreased substantially after this time point (2). This
observation was used to design the experiments shown in Figs.
1 and 2.
The data in Fig. 1 show that GSH stimulates the release of NO from ONOO as previously reported (2). Generation of NO was
significantly lower in CO2/bicarbonate buffer than in
phosphate buffer. This is consistent with a recent observation that
CO2 catalyzes ONOO decomposition via
formation of an unstable intermediate nitrosoperoxycarbonate (ONOOCO2), thereby accelerating formation of
nitrate (14). Addition of glucose alone had no effect on NO release
from ONOO. However, as compared with the reaction with
GSH alone (in phosphate buffer), about 50% less NO was detected when
ONOO was reacted with GSH in the presence of glucose.
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Fig. 1.
Effects of absence and presence of
combinations of glutathione (1 mM) and glucose (5.6 mM) on the release of NO from 0.4 mM
ONOO dissolved in CO2/bicarbonate-
or phosphate-containing buffers (pH 7.4). Experiments were
conducted as described under "Experimental Procedures"
(n = 11-12). Note that bicarbonate buffer
significantly (p < 0.05) decreased the detection of NO
as compared with phosphate buffer under all conditions, glutathione
significantly (p < 0.05) increased NO release under
all conditions except in glucose-free bicarbonate-containing solution,
and glucose decreased (p < 0.05) the formation of NO
from peroxynitrite + glutathione in phosphate buffer.
View larger version (44K):
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Fig. 2.
Effects of absence and presence of
combinations of glutathione (1 mM) and glucose (5.6 mM) on the release of NO from 0.1 mM
GSNO2 dissolved in CO2/bicarbonate- or
phosphate-containing buffers (pH 7.4). Experiments were conducted
as described under "Experimental Procedures" (n = 11-12). Note the depression (p < 0.05) of NO release
by glutathione and that bicarbonate buffer significantly lowered (*,
p < 0.05) NO formation from GSNO2 + GSH in
the absence of glucose.
with GSH suggests that ONOO reacted
with GSH producing an intermediate compound, possibly GSNO2
(2), which released NO. This hypothesis is supported by experiments
shown in Fig. 2, which demonstrate that synthetic GSNO2
spontaneously releases NO in the buffer systems employed. In
preliminary experiments, it was confirmed that the amount of NO formed
from GSNO2 at 15 min was approximately half that detected at 30 min. Data in Fig. 2 indicate that the presence of glucose and
CO2/bicarbonate had minimal inhibitory effects of NO
release from GSNO2, whereas the data in Fig. 1 demonstrate
that the presence of glucose or CO2/bicarbonate suppresses
the generation of NO from ONOO. Thus, the actions of
glucose and CO2/bicarbonate on NO release seem to originate
primarily from an action of these buffers on the ONOO and
not on the stability of GSNO2.
under the same conditions (Fig. 1).
reaction with GSH, and its
chromatographic mobility was similar to that previously observed (6).
The relative concentration and extent of this product formation was
dependent upon the initial concentration of ONOO and GSH.
This product also showed a strong chromatographic peak when the HPLC
analysis was performed with monitoring of UV absorbance at 334 nm,
typical for nitro- and nitroso-thiols (2). Product b was not
formed when GSH was treated with ONOO at pH greater than
8 or with decomposed ONOO, indicating that peroxynitrous
acid was essential for formation of this product. A peak at 2.5 min and
a broad peak at 5 min in the chromatogram on Fig. 3 (top)
originated from the decomposition of ONOO. Freezing of
the ONOO/GSH reaction mixture at 20 °C for 2-3 h
markedly increased the abundance of product b near the upper
frozen layer, via a freeze distillation, which allowed us to collect
sufficient amounts of this material for mass spectrometric analyses.
Product b, as well as synthetic GSNO and GSNO2,
displayed sharp and reproducible chromatographic peaks on an RP-HPLC
column; however, these three compounds had almost the same retention
time and could not be separated on several HPLC systems. Thus, the
identification of b based on retention time comparison with
synthetic standards was not conclusive.
View larger version (19K):
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Fig. 3.
Chromatograms obtained by monitoring UV
absorbance at 215 nm of the following samples analyzed by RP-HPLC.
Top, reaction mixture obtained by incubation of GSH with
peroxynitrite for 5 min at room temp; middle, GSH standard;
bottom, GSNO2 standard.
View larger version (17K):
[in a new window]
Fig. 4.
Electrospray tandem mass spectrometry of
products a and b obtained by reaction of
glutathione with peroxynitrite and purified by RP-HPLC (as shown in
Fig. 3). Top: mass spectrum of product
b, identified as GSNO2 (collision energy, 25 eV); Bottom: mass spectrum of product a, which
corresponds with GSH (collision energy, 10 eV).
/GSH product based on comparison with
synthetic standards using UV and HPLC, we employed electrospray tandem
mass spectrometry to elucidate the structure of this product.
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Fig. 5.
UV spectra of synthetic GSNO2
(top) and GSNO (bottom).
/GSH-derived product
b contained a prominent ion at m/z 353 (Fig. 4),
which presumably corresponds to a protonated form of the molecule.
Thus, the molecular mass of this product was 352 atomic mass units,
suggesting that product b had a structure consistent with
S-nitroglutathione. This product displayed unusual stability
under collision-induced activation tandem mass spectrometry, where ions
were dissociated as a result of interaction with a neutral gas, argon.
Our attempts to decompose ion m/z 353 at an energy of 25 eV
caused a decrease in the detection of this ion by more than 90%,
without producing any detectable ion fragments. In particular, ions
that might be expected to originate from the loss of NO2,
NO from the GSNO2H+, or decomposition of a GSH
backbone could not be detected. Treatment of GSH with
NO2BF4 yielded a compound that also produced a
protonated GSNO2 ion at m/z 353, and it was also
resistant to collisional activation (Fig.
6). Although the spectrum of
ONOO/GSH product was identical to that of synthetic
GSNO2, the spectrum of synthetic GSNO was very different
(Fig. 6). Collision-induced dissociation of the protonated GSNO ion
(m/z 337) resulted in a loss of nitric oxide, producing an
abundant fragment ion at m/z 307 (GSH+). The
GSNOH+ 
GSH+ fragmentation occurred readily,
and therefore, the cleavage of the S-NO bond required
relatively mild conditions. In the absence of additional ion
activation, the mass spectrum of GSNO showed an ~8% relative
abundance of the m/z 307 ion, suggesting that GSNOH+
ion was releasing NO (not shown). Additional loss of NO radical was
observed under condition of a collision-induced dissociation of
GSNOH+ ion at 9 eV, where approximately 80% of the
S-NO bonds appear to have been broken (Fig. 6). Several
minor ions were also observed in the mass spectrum of GSNO that
originated from dissociation of the GSH backbone. Our mass
spectrometric data provided evidence that the reaction of GSH with
ONOO produces a S-nitro derivative of GSH, and
the protonated GSNO2 ion is considerably more stable to
collision-activated dissociation than the protonated GSNO ion.
View larger version (16K):
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Fig. 6.
Electrospray tandem mass spectra of synthetic
standards. S-nitroglutathione (GSNO2)
(collision energy, 25 eV); S-nitrosoglutathione (GSNO)
(collision energy, 9 eV).
for 5 min, suggesting that
about 16% of the ONOO appears to have been converted to
GSNO2. Thus, our studies identified the biologically active
substance derived from the reaction of GSH with ONOO as
GSNO2, and GSNO was not a detectable product of
ONOO/GSH reaction.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
with GSH. We have previously
observed that a product of the reaction between ONOO and
GSH has the properties of a substance that slowly releases NO, and a
product of this reaction was isolated and shown to cause what appeared
to be a NO-mediated relaxation (2). In the present study,
GSNO2 was synthesized, and its ability to spontaneously release NO was demonstrated. The data obtained also suggest that nitrated derivatives of glucose or carbon dioxide do not seem to be
primary species contributing to the generation of NO from ONOO under the conditions examined. Thus,
GSNO2 may be a key participant in the generation of NO from
ONOO in the presence of GSH (Fig.
7).
View larger version (10K):
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Fig. 7.
Proposed pathways involving
ONOO , GSH, and GSNO2.
detected an ion with an m/z of 353 that had a mass spectrum comparable with that of synthetic
GSNO2 produced from the reaction of GSH with
NO2BF4. The ONOO/GSH-derived
product was clearly different from synthetic GSNO, which was not a
detectable product of this reaction. Interestingly, collisional
activation of GSNO2 derived from either ONOO
or NO2BF4 with 25 eV did not produce fragment
ions, whereas the ion with an m/z at 337 of GSNO readily
fragmented at 9 eV. Although the actual origin of the gas phase
stability of protonated form of GSNO2 is not yet known,
previously reported (21) molecular orbital calculations on model thiol
nitrates (RSNO2) suggest that they have a very low barrier
for rearrangement to sulfenyl nitrites (RSONO). Thus, it is possible
that rapid equilibration between these species provides a level of
stabilization sufficient to prevent the fragmentation of
GSNO2. The relative stability of GSNO2 in the
electrospray conditions could be also influenced by the attachment of
proton to GSNO2, a process that is less likely to occur in
solution at neutral pH. In a buffered solution, other factors, such as
solvation and ionic strength, may contribute to the release of NO from
GSNO2. Further studies are needed to establish whether the
protonation of GSNO2 enhances its stability in solution.
1) and pH-independent in the
neutral pH region of acetonitrile/phosphate buffer at room temperature
(20). As a result of molecular orbital calculations, it was suggested
that the rearrangement of thiol nitrates (RSNO2) to
sulfenyl nitrites (RSONO) followed by the spontaneous decomposition of
RSONO was a potential mechanism of NO generation (20, 21). It was also
previously observed that tert-butylthionitrate releases NO
through a mechanism that was inhibited by thiols, and this inhibitory
effect of glutathione was seen in the present study with purified
GSNO2. A reaction of thiols with RSNO2
resulting in the formation of nitrite and disulfide was suggested as an
explanation for the observed decreased formation of NO (20). Thus, the
release of NO from GSNO2 has similarities with previous
observations of NO release from tert-butylthionitrate. Although it has been suggested that nitrated thiols are very unstable species (7, 8), the observed release NO form GSNO2 over a
prolonged period suggests that these substances may be more stable than
they were previously considered to be. Because the release of
NO2 via a homolytic cleavage of the S-N bond is
not likely to occur in thionitrates (21), a question that remains to be
answered is whether NO derives from sulfinyl nitrite (GS(O)NO) or
sulfenyl nitrite (GSONO) after rearrangement of the initially formed
thionitrate (GSNO2) (Fig. 7).
in the presence of GSH and the
absence of an identifiable effect of these buffer components on the
release of NO from GSNO2 are consistent with previous
observations on the chemistry of ONOO. Whereas
ONOO reacts with CO2/bicarbonate to form an
intermediate that enhances the efficiency of some of the nitration
reactions caused by ONOO (14-16), data in the present
study suggest that this process does not enhance the formation of the
key NO-generating species, which appears to be GSNO2.
Although ONOO reacts with glucose (and other hydroxylated
substances) to produce nitrovasodilators (11, 12), it appears that the
products that form may have less of a potential to release NO in the
presence of excess GSH than the thiol-derived NO-releasing species that form in the absence of glucose. Thus, the NO-releasing species hypothesized in the present study to be GSNO2 appears to
result from a reaction of GSH with ONOO that is not
catalyzed by glucose or CO2/bicarbonate. Because thiol
radicals and nitrogen dioxide seem to be key initial products of the
aqueous chemistry of ONOO and thiols (8-10), perhaps
thiol nitration is a result of a radical-radical reaction between these
two reactive species.
with GSH are consistent with GSNO2 being
a key intermediate that participates in the generation of NO. Our
recent studies on bovine coronary (22) and pulmonary (23) arteries have
already provided evidence that concentrations of NO as low as 50 nM cause a level of intracellular ONOO
formation that produces a prolonged relaxation of these vascular segments as a result of a thiol-dependent process that
participates in the regeneration of NO. In addition, GSNO2
was hypothesized to be a key metabolite of nitroglycerin over 20 years
ago (24). Therefore, GSNO2 may be an important biologically
active metabolite of NO oxidation and nitrovasodilator drug action.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Dept. of Pharmacology,
New York Medical College, Valhalla, NY 10595. Tel. and Fax:
914-594-3621; E-mail: michael_balazy{at}nymc.edu.
, peroxynitrite; GSH, glutathione; NO, nitric oxide; GSNO2, S-nitroglutathione; GSNO, S-nitrosoglutathione; HPLC, high performance liquid
chromatography; RP, reverse-phase; ESI, electrospray ionization; MS/MS, tandem mass spectrometry.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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 V. Venketaraman, Y. K. Dayaram, A. G. Amin, R. Ngo, R. M. Green, M. T. Talaue, J. Mann, and N. D. Connell Role of Glutathione in Macrophage Control of Mycobacteria Infect. Immun., April 1, 2003; 71(4): 1864 - 1871. [Abstract] [Full Text]
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J. K. Leach, S. M. Black, R. K. Schmidt-Ullrich, and R. B. Mikkelsen Activation of Constitutive Nitric-oxide Synthase Activity Is an Early Signaling Event Induced by Ionizing Radiation J. Biol. Chem., May 3, 2002; 277(18): 15400 - 15406. [Abstract] [Full Text] [PDF]
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 M. Balazy, T. Iesaki, J. L. Park, H. Jiang, P. M. Kaminski, and M. S. Wolin Vicinal Nitrohydroxyeicosatrienoic Acids: Vasodilator Lipids Formed by Reaction of Nitrogen Dioxide with Arachidonic Acid J. Pharmacol. Exp. Ther., November 1, 2001; 299(2): 611 - 619. [Abstract] [Full Text] [PDF]
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 H.-W. Shin and S. C. George Microscopic modeling of NO and S-nitrosoglutathione kinetics and transport in human airways J Appl Physiol, March 1, 2001; 90(3): 777 - 788. [Abstract] [Full Text] [PDF]
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 A. L. Kleschyov, B. Muller, T. Keravis, M.-E. Stoeckel, and J.-C. Stoclet Adventitia-derived nitric oxide in rat aortas exposed to endotoxin: cell origin and functional consequences Am J Physiol Heart Circ Physiol, December 1, 2000; 279(6): H2743 - H2751. [Abstract] [Full Text] [PDF]
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 B. J. Buckley and A. R. Whorton Adaptive responses to peroxynitrite: increased glutathione levels and cystine uptake in vascular cells Am J Physiol Cell Physiol, October 1, 2000; 279(4): C1168 - C1176. [Abstract] [Full Text] [PDF]
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M. S. Wolin Interactions of Oxidants With Vascular Signaling Systems Arterioscler. Thromb. Vasc. Biol., June 1, 2000; 20(6): 1430 - 1442. [Abstract] [Full Text] [PDF]
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C. Boulos, H. Jiang, and M. Balazy Diffusion of Peroxynitrite into the Human Platelet Inhibits Cyclooxygenase via Nitration of Tyrosine Residues J. Pharmacol. Exp. Ther., April 1, 2000; 293(1): 222 - 229. [Abstract] [Full Text]
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X.-L. Ma, F. Gao, B. L. Lopez, T. A. Christopher, and J. Vinten-Johansen Peroxynitrite, a Two-Edged Sword in Post-Ischemic Myocardial Injury---Dichotomy of Action in Crystalloid- Versus Blood-Perfused Hearts J. Pharmacol. Exp. Ther., March 1, 2000; 292(3): 912 - 920. [Abstract] [Full Text]
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