OXIDATION AND NITROSATION OF THIOLS AT LOW MICROMOLAR EXPOSURE TO NITRIC OXIDE EVIDENCE FOR A FREE RADICAL MECHANISM*

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1 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 and the Department of Molecular and Cellular Physiology, Louisiana State University Health Science Center, Shreveport, Louisiana Running title: Thiol Oxidation and Nitrosation by Nitric Oxide and Oxygen Key words: nitric oxide, oxidation, nitrosylation, nitrosation, nitrogen dioxide, glutathione, glutathione disulfide, peroxynitrite, S-nitrosoglutathione, S-nitrosothiol, superoxide, DMPO. *This work was supported by grants CA89366 (to DJ) and HL69029 (to MF) from the National Institute of Health. To whom correspondence should be addressed: Albany Medical College, Center for Cardiovascular Sciences, 47 New Scotland Avenue (MC8), Albany, NY 12208; Tel: (518) ; Fax: (518) ; jourdhd@mail.amc.edu

2 Abstract While the nitric oxide ( NO)-mediated nitrosation of thiol-containing molecules is increasingly recognized as an important posttranslational 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 (N 2 O 3 ), RSNOs may be formed via intermediate oneelectron oxidation of thiols, possibly mediated by nitrogen dioxide ( NO 2 ), and 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%, while 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 does not only depend on the availability of NO and O 2, but also on the degree of oxidative stress by affecting the steady-state concentration of thiyl radicals. 2

3 Introduction S-nitrosothiols (RSNOs) are biological products formed from the interaction of nitric oxide ( NO) with thiol-containing molecules (RSH). Over the 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 pk a 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 3

4 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 (O 2 ) (12). Reactive nitrogen oxide species derived from the reaction of O 2 with NO nitrosate thiols (13-16). The reaction is second order with respect to NO and first order in O 2, consistent with the formation of dinitrogen trioxide (N 2 O 3 ) as the nitrosating agent: 2 NO + O 2 2 NO 2 (1) 2 NO + 2 NO 2 2 N 2 O 3 (2) N 2 O 3 + RSH RSNO + NO H + (3) The oxygen-dependence of this process suggests that low O 2 concentrations, which are in the range of 1-50 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 N 2 O 3 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 O 2 is accelerated several hundred fold 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/O 2 -mediated nitrosation reactions have been delineated in earlier kinetic studies, the importance of nitrogen dioxide ( NO 2 ) as an intermediate in the reaction of thiols with NO has been ignored. Nitrogen dioxide oxidizes 4

5 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 NO 2 with NO (2 x 10 7 M -1.s -1 compared to 1.1 x 10 9 M -1.s -1 (20;21)). RSH + NO 2 RS + NO H + (4) Accordingly, previous studies found negligible thiol oxidation by NO and O 2 since upon bolus addition of authentic NO the initial concentrations of NO and NO 2 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 NO 2 (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 ): RS + NO RSNO (5) Under physiologically relevant conditions, the removal of thiyl radicals occurs either through geminate recombination or reaction with O 2 and thiolates (12). Consequently, reaction 5 directly competes with reactions (6) to (9): RS + RS RSSR (6) RS + O 2 RSOO (7) RS + RS RSSR (8) RSSR +O 2 RSSR + O 2 (9) In the presence of ambient O 2 and excess thiol, reaction 8 dominates due to the rapid removal of the disulfide radical anion (RSSR ) via reaction 9 (12). The effect of different O 2 concentrations on RSNO yields at low NO fluxes is unknown. It may be expected that 5

6 lower, physiologically relevant O 2 tensions will affect the relative amounts of oxidized and nitrosated products not only by reducing the amount of NO 2 formed, but also by limiting the impact of O 2 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 NO 2 and subsequent reaction of the thiyl radical with NO. 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 NO 2. Experimental procedures Spermine NONOate (Sper/NO) was obtained from Cayman Chemicals (Ann Arbor, MI). All other chemicals were purchased from Sigma Chemical Co. (St Louis, MO). Reaction of Glutathione with Nitric Oxide- In a typical experiment, a one 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 K 2 HPO 4, 10 mm tetrabutylammonium hydrogen sulfate (TBAHS) 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 previously described (22;23). Briefly, the solutions were degassed with argon scrubbed from traces of O 2. They were then bubbled with NO gas that was purified by passage through a 5 M NaOH solution. The resulting 6

7 NO solutions were maintained in septum sealed glass containers, and the NO concentration was typically 1.5 ± 0.3 mm as determined electrochemicaly using a NOspecific 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 directly determined 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 O 2, 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 %. A 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 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. High Performance Liquid Chromatography (HPLC) analysis of GSH reaction products- The products obtained from the reaction with NO in oxygenated solutions were analyzed by ion-pairing HPLC as previously described (25). Samples were injected onto a 250 X 4.6 mm 5-µm octadecyl silane C 18 ultrasphere column (Beckman Coulter, Inc. Fullerton, CA) isocratically running at a flow rate of 1 ml/min with 10 mm K 2 HPO 4, 10 mm TBAHS 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. 7

8 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 (Rockville, MD). Cells were maintained in 75 cm 2 culture flasks in DMEM 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 cm 2 flasks and grown to 80 % confluence in DMEM 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 DMEM containing 10 % serum, and the flasks were pre-incubated for two hours in humidified atmosphere equilibrated with different O 2 /nitrogen/co 2 gas mixtures, resulting in final O 2 tensions of 1, 3, 21, and 50%. The cells were then incubated with argonpurged Sper/NO for 1 hour at 37 C. Cell viability was not affected by any of the treatment as tested by trypan blue exclusion (data not shown). Chemiluminescence detection of cellular RSNOs- Treated cells were washed once with cold phosphate-buffered saline (PBS) 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 NEM. The samples were kept on ice and in the dark for 15 min before addition of 100 µl of 100 mm sulfanilamide and incubation 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 previously described (26). 8

9 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 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 to represent a statistically significant difference. Results The reaction of 1 mm GSH with authentic NO ( 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.1A 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 9

10 in 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 indicated that the reaction of NO with O 2 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. To further evaluate the nitrosative and oxidative chemistry associated with the reaction of NO with O 2, 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 while GSSG concentration continued to increase over the 3 hr incubation period, at which time the concentration of GSSG exceeded that of GSNO by approximately 400% (Fig. 2A). Again, incubation of pre-formed 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). 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 approximately 20 % the amount of GSNO formed and decreased nitrate formation by about 30 % (Fig. 4A). The formation of GSSG, the increased O 2 consumption, and the effect of SOD were all consistent with a redox 10

11 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 O 2 to produce the superoxide anion (O 2, reactions 6 to 9). Under our experimental conditions, O 2 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 one hour 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. The availability of GS for reaction with NO might be modulated by O 2 not only because the formation of NO 2 is an O 2 -dependent process, but also because O 2 drives the consumption of GS through its reaction with the glutathione disulfide radical with concurrent formation of O 2 (reaction 9, (12)). Thus, we tested the effect of O 2 tension on the reaction of NO and O 2 with GSH. We hypothesized that at high O 2 tension, the reduction of O 2 by the disulfide radical diminishes the concentration of thiyl radicals available for reaction with NO to form GSNO. Alternatively, an increased O 2 formation 11

12 at high O 2 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 % O 2 saturation and that increasing O 2 tension to 50 % inhibited GSNO formation by approximately 30 % (compared to 3% O 2 ; Fig. 5A). Concurrently, GSSG formation increased in a concentration-dependent manner with increasing O 2 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). 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 Materials and Methods. To ascertain that NO reaction sites were thiols, samples were examined for their sensitivity to HgCl 2 and light (26). 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 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 ( µm) to provide initial 12

13 rates of NO release ranging from ~ 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, pre-incubation of the cells with DMPO (10 mm) decreased by approximately 45 % cellular RSNO content (Fig. 7 A). Maximum nitrosation occurred upon incubation of the cells with 3% O 2 and was decreased by 45 % (compared to 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. Discussion The reaction of NO and O 2 with thiols is thought to result in thiol nitrosation through the exclusive intermediacy of dinitrogen trioxide (N 2 O 3 ) formed from the combination of NO with NO 2 (reaction 2; (15;28)). In the context of the cell, however, thiols are in excess relative to NO, and a large fraction of NO 2 should react with thiols rather than NO itself (reaction 4; (21)). An important corollary of the trapping of NO 2 by thiols is the formation of oxidized products including disulfides through the intermediate formation of thiyl radicals (reactions 6 to 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 O 2 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 NO 2. The formation of GSSG, the increased O 2 consumption upon increased GSH concentration, and the effect of SOD were evidence for the occurrence of a free radical mechanism in which O 2 is 13

14 reduced to O 2 by the glutathione disulfide radical (reaction 9). If true, a fraction of generated NO should then react with O 2 in a diffusion-limited manner to form peroxynitrite (ONOO /ONOOH), 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 2. 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/O 2 and NO/O 2 reactions are accompanied by oxidation reactions. For the most part, the importance of NO 2 in the NO/O 2 reaction and the formation of the glutathionyl radical, GS has been ignored in previous works. Although Pou and Rosen 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 (27). The present work indicates that a large fraction of RSNO formed under these conditions is derived from the combination of GS with NO since we showed that the thiyl radical trap DMPO reduces GSNO yields by approximately 45 %. We also showed that RSNO yields did not increase linearly with O 2 concentration but were maximal at low O 2 tension. Because O 2 drives the removal of thiyl radicals and the consecutive formation of the NO scavenger O 2 (reactions 8 and 9), a decrease in O 2 tension results in a diminished efficacy of these reactions and a consequential increase in the availability of NO and thiyl radicals for combination. Of course, the formation of NO 2 is an oxygendependent process, but the concentrations attained at 3 % O 2 tension (corresponding to ~30 µm) is not limiting compared to the low fluxes of NO generated by Sper/NO. 14

15 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 O 2, spatial and temporal variations in O 2 availability and O 2 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 O 2 concentrations do not limit cellular RSNO formation. Rather, RSNO formation appears to be favored at oxygen concentrations that typically occur in tissues. However, since the rate of NO 2 formation is second order with regard to NO, NO/O 2 -mediated oxidation and nitrosation reactions are hampered by the availability of NO 2 in a fashion similar to the reactions mediated by N 2 O 3. Independent of the role of NO 2 as a key intermediate, the recognition that RSNO formation occurs, at least in part, through a free radical mechanism offers new avenues for investigations on the nature of the pathways that can lead to RSNO formation in vivo, and 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 15

16 with a powerful antioxidative network where intracellular GSH is complemented by e.g. ascorbate and α-tocopherol 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 to modulate the sensitivity of cells and tissues to reactive nitrogen oxide species. Acknowledgement We wish to thank Nadia Azzam-Thorn for her technical assistance. 16

17 References 1. Scharfstein, J. S., Keaney, J., Slivka, A., Welch, G. N., Vita, J. A., and Stamler, J. S. (1994) J.Clin.Invest. 94, Stamler, J. S., Lamas, S., and Fang, F. C. (2001) Cell. 106, Boese, M., Mordvintcev, P. I., Vanin, A. F., Busse, R., and Mülsch, A. (1995) J.Biol.Chem. 270, Gow, A. J., Buerk, D. G., and Ischiropoulos, H. (1997) J.Biol.Chem. 272, Stubauer, G., Giuffrè, A., and Sarti, P. (1999) J.Biol.Chem. 274, Moro, M. A., Darley-Usmar, V. M., Goodwin, D. A., Read, N. G., Zamora-Pino, R., Feelisch, M., Radomski, M. W., and Moncada, S. (1994) Proc.Natl.Acad.Sci. USA 91, Van der Vliet, A., Chr.'t Hoen, P. A., Wong, P. S. Y., Bast, A., and Cross, C. E. (1998) J.Biol.Chem. 273, Balazy, M., Kaminski, P. M., mao, K., Tan, J., and Wolin, M. S. (1998) J.Biol.Chem. 273, Rafikova, O., Rafikov, R., and Nudler, E. (2002) Proc Natl Acad Sci USA 99, Mannick, J. B. and Schonhoff, C. M. (2002) Arch.Biochem.Biophys. 408, Espey, M. G., Miranda, K. M., Thomas, D. D., and Wink, D. A. (2001) J.Biol.Chem. 276, Wardman, P. and von Sonntag, C. (1995) Methods Enzymol. 251, Wink, D. A., Darbyshire, J. F., Nims, R. W., Saavedra, J. E., and Ford, P. C. (1993) Chem.Res.Toxicol. 6,

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20 Figure Legends 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. 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 shown in Fig. 1. Values represent the means ± standard deviation (n = 3). B, glutathione (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 Materials and Methods. Product formation was determined as described in Fig. 1. The ratios of GSNO to GSSG concentrations are expressed as the means ± SD of three different experiments. 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 followed for 60 min as described under Materials and Methods. Values represent the means ± SD (n = 3; *p<0.05 compared to no GSH). Fig. 4. Effect of superoxide dismutase (SOD) and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) on GSSG, GSNO, nitrite, and nitrate formation. A, GSH (1 mm) was 20

21 incubated for two hours 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 to control (no SOD). The values represent the mean ± standard deviation (n = 3; *p<0.05 compared to control). B, GSH (1 mm) was incubated with 100 µm Sper/NO as described in A, in the presence or the absence of the thiyl radical trap DMPO (100 mm). Changes in metabolite concentrations are expressed as percentage compared to control (no DMPO). The values represent the means ± SD (n = 3; *p<0.05 compared to control). 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 one hour at 37 C with 100 µm Sper/NO and various concentrations of O 2 to obtain final O 2 tensions of 1, 3, 21, and 50 % as described under Materials and Methods. GSNO (A) and GSSG (B) concentrations were determined as described in Fig. 1. Values represent the means ± SD (n = 3; *p<0.05 compared to 3 %; #p<0.05 compared to 1%). 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 one hour 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 panel A. The values represent the means ± SD (n = 3; *p<0.05 compared to control). C, timedependent increase in RSNO content from NIH 3T3 fibroblasts incubated with 100 µm Sp/NO as described under Materials and Methods. The values represent the means ± SD of three independent experiments. D, fibroblasts were exposed for one hour to increasing 21

22 concentrations of Sper/NO as described under Materials and Methods. The values represent the means ± SD of three independent experiments. Fig. 7. Effect of 5,5-dimethyl-1-pyrroline N-oxide (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 one hour with 100 µm Sper/NO. RSNO content was determined as described in Fig. 6. The values represent the means ± SD (n = 3; *p<0.05 compared to control). B, cells were preincubated for two hours in humidified atmosphere with different O 2 /nitrogen/co 2 gas mixtures resulting in a final O 2 tension of 1, 3, 21, and 50%. The cells were then incubated with argon-purged Sper/NO (100 µm) for one hour at 37 C and RSNO content determined. The values represent the means ± SD (n = 3; *p<0.05 compared to control). 22

23 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 Fig. 1. The concentrations are expressed as the means ± SD of three independent experiments. [NO] NO - 2 formed NO - 3 formed GSSG formed GSNO formed GSNO/GSSG (µm) (µm) (µm) (µm) (µm) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

24 Figure 1 A Absorbance Units B Absorbance Units Time (min) = GSH 2 = Nitrite 3 = GSSG 4 = Nitrate 5 = GSNO Time (min) 24

25 Figure 2 A Concentration (µμ) B [GSNO]/[GSSG] GSSG GSNO Nitrite Nitrate Time (min) Nitric Oxide (µm/min) 25

26 Figure 3 40 * O 2 Consumption (%) * GSH Concentration (mm) 26

27 Figure 4 A Concentration (% Control) B Concentration (% Control) SOD * * GSSG GSNO Nitrite Nitrate + DMPO * * * GSSG GSNO Nitrite Nitrate 27

28 Figure 5 A GSNO Concentration (µm) B GSSG Concentration (µm) * * Oxygen Tension (%) # # Oxygen Tension (%) 28

29 Figure 6 A 40 C 50 Photoelectric Signal (mv) B NO (pmoles/10 6 cells) CON NO NO + HgCl 2 * * NO + Light CON NO NO NO + + HgCl 2 Light NO (pmoles/10 6 cells) D NO (pmoles/10 6 cells) Time (min) Sper/NO Concentration (µm) 29

30 Figure 7 A 40 B NO (pmoles/10 6 cells) NO (pmoles/10 6 cells) * * NO * NO + DMPO * * Oxygen Tension (%) 30

31 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 J. Biol. Chem. published online February 20, 2003 Access the most updated version of this article at doi: /jbc.M Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's alerts