Oxidation and Nitrosation of Thiols at Low Micromolar Exposure to Nitric Oxide

EVIDENCE FOR A FREE RADICAL MECHANISM*

David Jourd'heuilDagger §, Frances L. Jourd'heuilDagger , and Martin Feelisch

From the Center for Dagger  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

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.
<UP>2<SUP>⋅</SUP>NO + O<SUB>2</SUB> → 2<SUP>⋅</SUP>NO<SUB>2</SUB></UP>

<UP>R<SC>eaction</SC> 1</UP>

<UP>2<SUP>⋅</SUP>NO + 2<SUP>⋅</SUP>NO<SUB>2</SUB> → 2 N<SUB>2</SUB>O<SUB>3</SUB></UP>

<UP>R<SC>eaction</SC> 2</UP>

<UP>N<SUB>2</SUB>O<SUB>3</SUB> + RSH → RSNO + NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB><UP> + H<SUP>+</SUP></UP>

<UP>R<SC>eaction</SC> 3</UP>
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)).
<UP>RSH + <SUP>⋅</SUP>NO<SUB>2</SUB> → RS<SUP>⋅</SUP> + NO</UP><SUP><UP>−</UP></SUP><SUB><UP>2</UP></SUB><UP> + H<SUP>+</SUP></UP>

<UP>R<SC>eaction</SC> 4</UP>
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·).
<UP>RS<SUP>⋅</SUP> + <SUP>⋅</SUP>NO → RSNO</UP>

<UP>R<SC>eaction</SC> 5</UP>
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.
<UP>RS<SUP>⋅</SUP> + RS<SUP>⋅</SUP> → RSSR</UP>

<UP>R<SC>eaction</SC> 6</UP>

<UP>RS<SUP>⋅</SUP> + O<SUB>2</SUB> ↔ RSOO<SUP>⋅</SUP></UP>

<UP>R<SC>eaction</SC> 7</UP>

<UP>RS<SUP>⋅</SUP> + RS<SUP>−</SUP> ↔ RSSR&cjs1138;</UP>

<UP>R<SC>eaction</SC> 8</UP>

<UP>RSSR&cjs1138; + O<SUB>2</SUB> → RSSR + O</UP><SUB><UP>2</UP></SUB><SUP><UP>&cjs1138;</UP></SUP>

<UP>R<SC>eaction</SC> 9</UP>
In the presence of ambient O2 and excess thiol, Reaction 8 dominates because of the rapid removal of the disulfide radical anion (RSSR&cjs1138;) 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.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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.

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.

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&cjs1138;), which in turn reduces O2 to produce the superoxide anion (O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>, Reactions 6 to 9). Under our experimental conditions, O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> 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).

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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> 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%).

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).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> by the glutathione disulfide radical (Reaction 9). If true, a fraction of generated ·NO should then react with O<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> 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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP>. 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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> 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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> (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<UP><SUB>2</SUB><SUP>&cjs1138;</SUP></UP> 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 alpha -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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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