Formation and stability of S-nitrosothiols in RAW 264.7 cells

Yanhong Zhang and Neil Hogg

Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

Submitted 1 October 2003 ; accepted in final form 7 December 2003


    ABSTRACT
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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S-Nitrosothiols have been suggested to be mediators of many nitric oxide-dependent processes, including apoptosis and vascular relaxation. Thiol nitrosation is a poorly understood process in vivo, and the mechanisms by which nitric oxide can be converted into a nitrosating agent have not been established. There is a discrepancy between the suggested biological roles of nitric oxide and its known chemical and physical properties. In this study, we have examined the formation of S-nitrosothiols in lipopolysaccharide-treated RAW 264.7 cells. This treatment generated 17.4 ± 1.0 pmol/mg of protein (means ± SE, n =27) of intracellular S-nitrosothiol that slowly decayed over several hours. S-Nitrosothiol formation depended on the formation of nitric oxide and not on the presence of nitrite. Extracellular thiols were nitrosated by cell-generated nitric oxide. Oxygenated ferrous hemoglobin inhibited the formation of S-nitrosothiol, indicating the nitrosation occurred more slowly than diffusion. We discuss several mechanisms for S-nitrosothiol formation and conclude that the nitrosation propensity of nitric oxide is a freely diffusible element that is not constrained within an individual cell and that both nitric oxide per se and nitric oxide-derived nitrosating agents are able to diffuse across cell membranes. To achieve intracellular localization of the nitrosation reaction, mechanisms must be invoked that do not involve the formation of nitric oxide as an intermediate.

nitric oxide; nitrosation; nitric oxide synthase; lipopolysaccharide; hemoglobin


MANY BIOLOGICAL ACTIONS of nitric oxide have been suggested to occur through the formation of S-nitrosothiols (28, 30, 36, 41, 43). Modification of protein cysteinyl residues, by nitric oxide, to form S-nitrosothiols has been implicated as a mechanism to control enzyme activity and intracellular signaling. For example, the nitrosation of caspase-3 has been implicated in the control of apoptosis (43) and has been shown to be regulated by Fas ligand (34). Strong analogies have been drawn between nitrosation pathways and phosphorylation pathways in cells (49). Phosphorylation is controlled by the activities of kinases and phosphatases and associated proteins, giving a multiplicity of signaling pathways for the control of cellular function. The role of nitrosation is much more controversial since no controlled enzymatic mechanism of S-nitrosothiol formation has been discovered. Although S-nitrosothiol degradation pathways exist (31, 39), it is not clear how they are specifically related to nitric oxide signaling processes. The S-nitrosation signaling paradigm is based on the observation that S-nitrosothiols can be detected in cells (11, 18) exposed to nitric oxide, the ability of S-nitrosothiols to undergo transnitrosation reactions with protein thiols (19, 40, 46), and that, in many cases, nitric oxide appears to exhibit bioactivity through mechanisms that depend on thiol residues (e.g., Refs. 7 and 28). The mechanisms by which nitric oxide can be converted into a nitrosating agent have not yet been established in biological systems.

The known chemical biology (24, 26) and physical properties of nitric oxide (51) would suggest that the nitrosation reaction could not be constrained by the presence of biological membranes or compartmentalized at the cellular or subcellular level. However, it has been suggested that nitrosation can be directed to a particular target by colocalizing nitric oxide synthase (NOS) with a protein thiol at the subcellular level (5, 12). Clearly, there is a discrepancy between the known chemical biology of nitric oxide and its proposed role in nitrosation-mediated signaling processes.

Previous studies that have examined the intracellular levels of S-nitrosothiols, in response to either exogenous or endogenous nitric oxide, have given values that vary by >1,000-fold from 100 nmol/mg of protein (43) to ~100 pmol/mg of protein (11). Although some of the variation is no doubt due to differences in cell type and method of nitric oxide exposure, the major difference can be largely attributable to methodological issues. For example, Clancy et al. (8) reported 25% of cellular glutathione (GSH) was converted to S-nitrosoglutathione (GSNO) upon exposure to 100 µM nitric oxide; however, the assay used could not distinguish between GSNO and oxidized forms of GSH such as glutathione disulfide (GSSG). In addition, the stability of intracellular S-nitrosothiols has not been previously reported. It is imperative, therefore, that a more complete understanding of the formation and behavior of S-nitrosothiols in cellular systems is procured. The recent development of highly sensitive methods for the analytical detection of S-nitrosothiols (35) allows such studies to be performed in the absence of the artifacts (42, 53) that have plagued other such studies.

In this investigation, we have examined the mechanism of formation and stability of intracellular S-nitrosothiols in activated RAW 264.7 macrophages. We conclude that low levels of stable S-nitrosothiols are formed upon exposure of cells to nitric oxide and that the nitrosating propensity of nitric oxide is freely diffusible and not constrained within an individual cell.


    MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
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Materials. Lipopolysaccharide (LPS; from Escherichia coli serotype 026:B6), NG-nitro-L-arginine methyl ester (L-NAME) hydrochloride, GSH, BSA, sodium nitrite, N-ethylmaleimide (NEM), diethylenetriamine pentaacetic acid (DTPA), mercury chloride, and iodine were purchased from Sigma. Dithiothreitol (DTT), potassium iodide, potassium phosphate, and sodium hydroxide were obtained from Fisher. Sulfanilamide and N-(1-naphthyl)ethylenediamine dihydrochloride (NED) were supplied by Aldrich. 2-(4-Carboxyphenyl)-4,5-dihydro-4,4,5,5-tetramethyl-1H-imidazolyl-1-oxy-3-oxide (CPTIO) was purchased from Cayman Chemical. RAW 264.7 cells were purchased from American Type Culture Collection. DMEM, streptomycin/penicillin, PBS, and Hanks’ balanced salt solution (HBSS) were obtained from GIBCO, and FBS was obtained from Hyclone. Oxygenated ferrous hemoglobin (oxyHb) was purified from fresh human blood as previously described (3).

Preparation of S-nitrosothiols. GSNO (17) and S-nitroso-N-acetylpenicillamine (SNAP) (14) were synthesized as previously described. For the synthesis of S-nitroso-BSA (BSA-SNO), BSA (10 mg/ml) was reduced by DTT (1 mM). DTT was then removed by thorough dialysis. S-nitrosation of BSA was carried out via transnitrosation between BSA and GSNO (2 mM). BSA-SNO was purified on a G-25 Sephadex column. The extent of nitrosation was determined by the Saville assay (45).

Cell culture. RAW 264.7 cells were cultured in DMEM supplemented with streptomycin (200 µg/ml), penicillin (200 U/ml), and 10% FBS and incubated at 37°C with 5% CO2 and 95% air. For each experiment, cells were seeded into six-well plates and grown to 80–90% confluence.

Sample preparation. Cells were washed twice with PBS, and 250 µl of lysis buffer (50 mM phosphate, 1 mM DTPA, and 50 mM NEM, pH 7.4) were added. Cells were scraped and sonicated on ice (550 Sonic Dismembrator; Fisher Scientific; 10% power for 15 s) before centrifugation (16,000 g, 5 min). The supernatant was used for measurements of S-nitrosothiols and nitrite. For the detection of extracellular S-nitrosothiols, NEM (10 mM) was added to the medium or HBSS to prevent artifactual S-nitrosothiol formation.

Determination of nitrite levels. The nitrite level in the medium was measured by Griess assay (16). Briefly, 1 ml of sample was mixed sequentially with 50 µl of sulfanilamide (30 mM in 2 N HCl) and 50 µl of NED (30 mM in 0.1 N HCl). The absorbance at 540 nm was measured by UV-visible spectroscopy (HP8453, Hewlett Packard). Nitrite concentration was derived from a standard curve generated using sodium nitrite.

The nitrite level in the lysate was measured by ozone-based chemiluminescence. A solution of potassium iodide (50 mg in 1 ml of double-distilled water) was mixed with 4 ml of glacial acetic acid in the reaction vessel of a Sievers model 280 nitric oxide analyzer and maintained at 30°C. This solution reduces nitrite to nitric oxide, which is carried by argon through an NaOH solution (1 N) and enters the chemiluminescence reaction cell. Nitrite concentration was derived by comparing the integrated peak area to a standard curve generated using sodium nitrite.

Determination of S-nitrosothiol concentration. Triiodide-dependent, ozone-based chemiluminescence was used to measure S-nitrosothiol concentration (44). The reaction solution was made fresh daily by dissolving potassium iodide (200 mg) and I2 (130 mg) in glacial acetic acid (28 ml) and double-distilled H2O (8 ml). The solution (5 ml) was added into the reaction vessel and stabilized at 30°C. Antifoaming agent was used to prevent foaming caused by injection of protein-rich samples. Samples were pretreated with 10% (vol/vol) of sulfanilamide (100 mM in 2 N HCl) for 15 min to remove nitrite (35). HgCl2 (5 mM) was used to verify the presence of S-nitrosothiols. The concentration of S-nitrosothiol was derived from a standard curve generated using GSNO. High-performance liquid chromatography of GSNO was performed according to published procedures (47).

Total nitric oxide metabolite measurement. Vanadium(III) chloride (VCl3)-dependent chemiluminescence was used to measure total nitric oxide metabolites (NOx) in samples, including nitrite, nitrate, and S-nitrosothiols. VCl3 (80 mg) was dissolved in a solution containing 800 µl of concentrated HCl and 9.2 ml of double-distilled water. After filtration, 5 ml of the solution was added into the reaction vessel. Antifoaming agent was also added, and the solution was maintained at 95°C. The concentration of total NOx was derived from a standard curve generated using sodium nitrate.


    RESULTS
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 MATERIALS AND METHODS
 RESULTS
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Chemiluminescence detection of S-nitrosothiols. Figure 1A shows typical chemiluminescence data for duplicate injections of GSNO standard using the triiodide/sulfanilamide method of S-nitrosothiol detection. This method selectively oxidizes S-nitrosothiols to liberate nitric oxide, which is detected by chemiluminescence after reaction with ozone (44, 53). Any potential interference from nitrite was removed by pretreating the samples with sulfanilamide under acidic conditions (35, 53). Sulfanilamide did not affect GSNO concentration as determined by both chemiluminescence and high-performance liquid chromatography (data not shown).



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Fig. 1. Representative chemiluminescence data for S-nitrosothiol measurement. A: S-nitrosoglutathione (GSNO) standards were injected into the reaction vessel of a nitric oxide analyzer for S-nitrosothiol measurement. GSNO was also pretreated with HgCl2 (5 mM) before injection to destroy S-nitrosothiols. Inset shows a linear regression of the peak area of the signal and the amount of GSNO injected. B: S-nitrosothiol content in the lysate of control or LPS-treated (1 µg/ml for 15 h) RAW 264.7 cells was detected by chemiluminescence, after sulfanilamide pretreatment, with (n = 3) and without (n = 2) HgCl2 (5 mM).

 
As can be seen (Fig. 1A), the chemiluminescence signal (as measured by peak area) responded linearly to the amount of GSNO injected (Fig. 1A, inset) and was abolished by addition of HgCl2. Recovery of GSNO was 98 ± 4% when GSNO (1 µM) was spiked into cell lysate, indicating, in agreement with previous studies (13), that cell lysate did not intrinsically interfere with the detection methodology.

Figure 1B shows a typical chemiluminescence trace for the triplicate injection (duplicate for mercury treatments) of sulfanilamide-treated RAW 264.7 cell lysate. The signal from the control cells was largely inhibited by HgCl2, indicating a basal level of S-nitrosothiols. The residual signal after mercury treatment was close to noise levels and is likely due to iron nitrosyls and N-nitrosoamines (42). Pretreatment of the cells with LPS for 15 h gave a robust increase in chemiluminescence that was mercury inhibitable, indicating an increased level of intracellular S-nitrosothiol. Again, a residual level of signal was observed upon treatment with HgCl2 that was close to noise levels. The residual signal was not quantifiable and was ignored for quantitative purposes.

S-nitrosothiol formation in LPS-activated RAW 264.7 macrophages. Extracellular nitrite was measured as an index of NOS induction. When RAW 264.7 cells were incubated with LPS (1 µg/ml) for 15 h, extracellular nitrite reached a level of 48.5 ± 1.6 µM (means ± SE, n = 27) compared with 1.6 ± 0.3 µM (means ± SE, n = 27) in untreated cells. The increase in nitrite was abolished by L-NAME (2 mM), a NOS inhibitor, giving a nitrite level of 0.3 ± 0.04 µM (means ± SE, n = 9). Lysate from control cells, which were not stimulated with LPS, contained 5.6 ± 0.5 pmol/mg of protein of S-nitrosothiol (means ± SE, n = 27). Cells incubated with LPS exhibited an approximately threefold larger chemiluminescence signal corresponding to an intracellular S-nitrosothiol content of 17.4 ± 1.0 pmol/mg of protein (means ± SE, n = 27). The presence of L-NAME (2 mM) did not affect the basal level of S-nitrosothiol but substantially inhibited LPS-dependent formation of S-nitrosothiol to 6.3 ± 0.7 pmol/mg of protein (means ± SE, n = 6). This indicates that the increase in intracellular S-nitrosothiols occurs as a result of NOS activity.

There are several possibilities for the mechanism of S-nitrosothiol formation. These include the formation of a nitrosating agent either directly from nitric oxide (i.e., from reaction with oxygen or from an unknown cell-dependent process) or from nitrite, either through local acidification or enzymatic processes (23). To examine these alternatives, the relationship between intracellular nitrite and intracellular S-nitrosothiol was examined. Figure 2 illustrates the levels of both intracellular nitrite and S-nitrosothiol during treatment of cells with both LPS and nitrite. In the absence of LPS, the levels of intracellular nitrite are ~90-fold greater than S-nitrosothiol. Interestingly, LPS increases intracellular nitrite only slightly, whereas the level of S-nitrosothiol more than triples and extracellular nitrite increases by ~50 times (as mentioned above). This indicates that there is no clear relationship between nitrite and S-nitrosothiol content in the intracellular space. The effect of nitrite on S-nitrosothiol formation was tested by adding nitrite directly to cells. As shown in Fig. 2, addition of a nitrite concentration (100 µM), comparable to that generated by LPS treatment, caused a modest increase in intracellular nitrite but did not increase intracellular S-nitrosothiol content. The fact that intracellular nitrite levels appear to be buffered against major changes in extracellular nitrite levels and that intracellular nitrite does not correlate with intracellular S-nitrosothiol strongly suggests that the observed increase in S-nitrosothiol levels is unrelated to the presence of intracellular nitrite.



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Fig. 2. Effect of LPS and nitrite on cellular nitrite and S-nitrosothiol contents. RAW 264.7 cells were treated with LPS (1 µg/ml) or nitrite (100 µM) for 15 h. The cells were washed and lysed. The S-nitrosothiol and nitrite levels in the cell lysate were measured by chemiluminescence. Data represent means ± SE (n = 3).

 
More than 90% of the S-nitrosothiol signal obtained from LPS-treated macrophages was lost upon filtering the cell lysate with a 3,000-Da cut-off filter (data not shown), indicating that the vast majority of S-nitrosothiols are associated with the high-molecular-weight pool of thiol groups. This is in agreement with previous studies of intracellular S-nitrosothiols (11, 18) and suggests that either high-molecular-weight S-nitrosothiols are the major intracellular species or that low-molecular-weight S-nitrosothiols (e.g., GSNO) are lost during the preparation of the lysate. To examine this, three different S-nitrosothiols (GSNO, SNAP, and BSA-SNO) were dissolved in the lysis buffer and added to the cells so that the S-nitrosothiol was exposed to the cellular contents at the time of lysis. Under these conditions, SNAP and BSA-SNO were recovered to the same extent in each trial (72% for SNAP and 100% for BSA-SNO). GSNO, however, was recovered to a different extent (varying from 30 to 80%) for each trial. This indicates that GSNO is variably unstable in cell lysate and is decomposed by as yet unknown factors. This observation is likely to be related to the "GSNO reductase" or "GSNO terminase" activity of GSH-dependent formaldehyde dehydrogenase (GSH-FDH) (22, 32). Consequently, an intracellular pool of GSNO cannot be ruled out in this system. However, the presence of an efficient GSNO decomposition activity makes it unlikely that cells can sustain a detectable steady-state level of GSNO.

Extracellular S-nitrosothiol formation. A significant level of extracellular S-nitrosothiol formation was observed when cells were incubated with nitrite (100 µM) for 15 h. As a consequence, nitric oxide-mediated S-nitrosothiol formation could not be distinguished from nitrite-mediated effects. Whereas a component of the nitrite-mediated S-nitrosothiol formation depended only on the presence of cell culture medium, the detected levels of S-nitrosothiols were also affected by the ability of the cells to modify the medium (unpublished results). Although this nitrite-dependent S-nitrosothiol formation amounted only to a small fraction of the added nitrite (<1%), it was large enough to mask any potential nitric oxide-dependent contribution. To avoid these issues, we conducted experiments in HBSS after LPS pretreatment.

Figure 3 shows the extracellular formation of S-nitrosothiol upon addition of GSH. For these experiments, cells were treated for 15 h with LPS, washed twice with PBS, and incubated for a further 1.5 h in HBSS supplemented with L-arginine (500 µM). During this time, ~8 µM nitrite accumulated in the medium, and little S-nitrosothiol was observed. Upon inclusion of GSH, S-nitrosothiol formation was observed and increased as a function of GSH concentration (Fig. 3). A control incubation of nitrite (15 µM) and GSH (1 mM) in HBSS with LPS-untreated cells did not result in formation of S-nitrosothiols (data not shown). The amount of S-nitrosothiol detected, even at the highest GSH concentration, amounted to only ~3% of the nitrite generated, and no significant decrease in nitrite formation was observed. These data suggest that extracellular thiols are able to intercept the nitrosating propensity of cell-generated nitric oxide. The intracellular level of S-nitrosothiol was not affected by the presence of extracellular GSH (data not shown), suggesting that intracellular S-nitrosothiol content was not dependent on uptake of extracellular S-nitrosothiol. However, because the routes and mechanisms of S-nitrosothiol transport by cells are not firmly established, this conclusion cannot yet be tested pharmacologically.



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Fig. 3. Extracellular S-nitrosothiol and nitrite formation upon incubation with glutathione (GSH). RAW 264.7 cells were treated with LPS (1 µg/ml) for 15 h. The cells were washed with PBS and further incubated in HBSS supplemented with L-arginine (500 µM) in the presence of various concentrations of GSH for 1.5 h. The HBSS was removed and mixed with N-ethylmaleimide (10 mM), and the S-nitrosothiol and nitrite content were measured by chemiluminescence. Data represent means ± SE (n = 3).

 
Intracellular S-nitrosothiol stability. To examine the stability of intracellular S-nitrosothiols, cells were washed after 15 h of treatment with LPS and further incubated with and without L-NAME (4 mM) in fresh serum-free medium. As shown in Fig. 4A, L-NAME treatment prevented accumulation of nitrite in the medium of preactivated cells, indicating that the concentration of inhibitor used was sufficient to greatly suppress NOS activity. As shown in Fig. 4B, the S-nitrosothiol content of the cells slowly decayed over 3 h in the presence of L-NAME, whereas there was a slight increase in intracellular S-nitrosothiols in the absence of L-NAME. This indicates that under these conditions, intracellular S-nitrosothiols are relatively stable and are not subject to rapid metabolism.



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Fig. 4. Decay of S-nitrosothiol in RAW 264.7 cells. RAW 264.7 cells were treated with LPS (1 µg/ml) for 15 h. The cells were washed with PBS and further incubated in fresh serum-free medium with (w) or without (w/o) NG-nitro-L-arginine methyl ester (L-NAME; 4 mM) for 1, 2, or 3 h. A: the nitrite concentration in the medium was measured by Griess assay. B: the intracellular S-nitrosothiol level was detected by chemiluminescence. Data represent means ± SE (n = 3).

 
Effect of nitric oxide scavengers on S-nitrosothiol formation. It is possible that nitric oxide directly nitrosates intracellular thiols without diffusing out of the cell by an as yet unknown rapid and concerted mechanism. This type of process has to be invoked to explain the so-called "intracrine" activities of nitric oxide (5, 12). To test whether the S-nitrosothiol observed here was formed through such a process, we examined the effect of extracellular nitric oxide trapping agents on intracellular S-nitrosothiol stability. Cells were preinduced with LPS for 15 h, washed, and incubated with HBSS (supplemented with 500 µM L-arginine) containing either oxyHb (100 µM) or CPTIO (100 µM) for a further 2 h. In LPS-treated cells, as illustrated in Fig. 5, the vast majority (~80%) of total NOx was nitrite. As expected in oxyHb-treated cells, nitrite did not form (9). Interestingly, total NOx was more than doubled under these conditions. Most importantly, the presence of oxyHb decreases intracellular S-nitrosothiol levels in a similar manner to L-NAME (cf. Fig. 4). This indicates that the presence of an extracellular nitric oxide trap can intercept nitric oxide before it is converted to a nitrosating agent. In addition to oxyHb, the trap CPTIO was also tested. This trap has the property of rapidly converting nitric oxide into nitrogen dioxide (2, 20), potentially diverting the formation of nitrosating agents to the extracellular environment. As shown in Fig. 5, CPTIO increased medium NOx, which (as expected) was almost 100% nitrite, but only slightly reduced intracellular S-nitrosothiol formation. These data suggest that extracellular nitrosating agents can cross cell membranes and nitrosate intracellular thiols.



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Fig. 5. Intracellular S-nitrosothiol and medium nitric oxide metabolite (NOx) formation in the presence of oxygenated ferrous hemoglobin (oxyHb) and CPTIO. RAW 264.7 cells were treated with LPS (1 µg/ml) for 15 h. The cells were washed with PBS and further incubated in HBSS supplemented with L-arginine (500 µM) in the presence of either oxyHb (100 µM heme) or CPTIO (100 µM) for 2 h. The levels of intracellular S-nitrosothiol and the extracellular levels of nitrite and total NOx were measured by chemiluminescence. Data represent means ± SE (n = 6).

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we have examined intracellular and extracellular S-nitrosothiol formation in a macrophage cell line under conditions of high nitric oxide production. It has been previously demonstrated that the treatment of this cell line with endotoxin can elicit induction of inducible NOS (iNOS or NOS II) after a 4- to 6-h delay (50). Nitrite is the major product of aerobic oxidation of nitric oxide and was used as an index of nitric oxide formation. After 15 h of incubation with LPS, the nitrite levels rose to ~48 µM. Experiments in which nitrite formation was followed kinetically after 15 h indicate that at this time the rate of nitrite formation was ~3–5 µM/h.

The measured concentration of intracellular S-nitrosothiols was 17.4 ± 1.0 pmol/mg of protein and was associated with the high-molecular-weight pool (>3,000 Da). As the protein concentration in each well was ~1 mg, this represents ~0.02% of the generated nitrite. If it is assumed the average molecular weight of cytosolic proteins is 70 kDa, then this corresponds to ~0.001 SNO groups per protein. The levels of S-nitrosothiol detected here represent the levels in the cytosolic fraction as cellular membranes are spun down after sonication.

Previous measurements of cellular S-nitrosothiols have been widely variable. Reports of intracellular S-nitrosothiol include Hoffman et al. (18), who measured 100 nmol/mg of protein S-nitrosothiol in endothelial cells with a much lower flux of nitric oxide. The latter value was measured using the Griess assay and is close to the total thiol concentration within an endothelial cell. This value is likely an overestimation resulting from the use of an insensitive method. In reasonable agreement with our data, Eu et al. (11) measured levels of 80–100 pmol/mg of protein S-nitrosothiols in LPS-treated RAW 264.7 cells using a method that relies on mercury-inhibitable photolytic nitric oxide release. Gow et al. (15) reported levels of 10–150 pmol/mg of protein using this same system and methodology. Feelisch et al. (13) reported levels of 30 pmol/106 cells in LPS-treated J774 cells using very similar methodology to that used here. A consensus from these studies indicates that cellular S-nitrosothiol concentration under conditions of high nitric oxide formation lies in the range of 20–100 pmol/mg of protein.

There are three potential routes for S-nitrosothiol formation: 1) the nitrosating ability of nitric oxide in the presence of oxygen, 2) the nitrosating activity of nitrite at low pH or by an enzymatic process (23), and 3) other cellular processes. This study has attempted to examine intracellular and extracellular S-nitrosothiols formation with respect to these three possible routes.

The first route can occur through several mechanisms, the most well established being the nitrosation of thiols (for example, GSH) via the intermediate formation of N2O3 (Eqs. 13) (26).

(1)

(2)

(3)

(4)
The oxidation of nitric oxide by oxygen forms nitrogen dioxide. This step is rate limiting and is second order in nitric oxide. Nitrogen dioxide can rapidly combine with nitric oxide to form N2O3, which then nitrosates thiols. Hydrolysis of N2O3 (Eq. 4) is likely the major source of nitrite. A second mechanism mediated by thiyl radical may occur by the reactions shown in Eqs. 5 and 6 (24).

(5)

(6)
By this mechanism, electron transfer between a thiol anion and nitrogen dioxide generates a thiyl radical, which subsequently combines with nitric oxide. It is likely that thiols can compete with nitric oxide for nitrogen dioxide and that nitric oxide can compete with oxygen and thiols for thiyl radical. A final mechanism for the first route of nitrosation involves the one-electron reduction of oxygen to form superoxide, which reacts with nitric oxide to form peroxynitrite (ONOO) (Eqs. 710).

(7)

(8)

(9)

(10)
Reaction of peroxynitrite with GSH forms GSNO (37) or GS(O)NO (4) at low yield.

(11)
The second route of nitrosation occurs through the nitrosating ability of nitrous acid (Eq. 11). Because nitrite has a pKa of 3.5, significant acidification is required to generate substantial nitrosation. Our data suggest that nitrite is not a major source of S-nitrosothiol formation in the intracellular compartment, as increasing intracellular nitrite by the addition of extracellular nitrite does not cause a large increase in intracellular S-nitrosothiol.

There is currently no strong evidence for an enzyme-catalyzed route of S-nitrosothiol formation, although it has been suggested that the hydrophobic interiors of both proteins (38) and membranes (33), and transition metal ions (52), may facilitate the formation of nitrosating agents from nitric oxide. In the extracellular space, both albumin (6) and ceruloplasmin (21) have been shown to catalyze S-nitrosothiol formation.

The formation of extracellular S-nitrosothiols is nitrite independent and dependent on the presence of extracellular thiol in a concentration-dependent manner. This indicates that the nitrosating potential of the cell can be exported to the extracellular environment, and, presumably, between cells. This raises the question of whether the observed S-nitrosothiols are generated through an intracrine process in which the production of nitric oxide and the nitrosation of thiols occur by a concerted mechanism that is confined within a cell, or whether the nitrosating potential is a freely diffusible element. One diagnostic test of an intracrine process involving nitric oxide is that an extracellular nitric oxide trap should not affect the outcome (see Ref. 27 for a theoretical treatment of this issue). For example, rat erythrocytes were used to inhibit the formation of an iron-nitrosyl species in cytokine-stimulated rat hepatocytes, indicating that the reactivity of nitric oxide within the cell was not competitive with its diffusion out of the cell (48).

In the presence of extracellular oxyHb, intracellular S-nitrosothiol formation was inhibited to a similar extent to that observed upon addition of L-NAME. This indicates that trapping nitric oxide in the extracellular environment is functionally equivalent to inhibiting nitric oxide production. Consequently, the S-nitrosothiol detected is not formed through a local process and the nitric oxide that forms the S-nitrosothiol must spend some time in the extracellular environment. Interestingly, CPTIO, which promotes the oxygen-independent conversion of nitric oxide into nitrogen dioxide, only slightly inhibits intracellular nitrosation. This suggests that although nitric oxide is trapped outside the cell, the nitrosating agent formed outside the cell is also freely diffusible and can nitrosate intracellular thiols. The formation of nitric oxide or nitrogen dioxide does not appear to restrict or compartmentalize the S-nitrosation reaction to the cellular or subcellular level. To achieve intracellular localization of the nitrosation reaction via NOS trafficking, mechanisms must be invoked that do not involve the formation of nitric oxide as an intermediate.

A tangential observation made from the above studies was that the presence of an extracellular nitric oxide trap results in an increase in the total measurable NOx. A similar observation was previously reported using cytokine-stimulated rat hepatocytes in the presence and absence of erythrocytes (48). The presence of oxyHb almost doubled the level of NOx in the extracellular medium, and CPTIO had a lesser, although similar, effect. Two possibilities for this are either that the presence of the trap decreases the steady-state concentration of nitric oxide within the cell, thus relieving iNOS inhibition by nitric oxide (1), or that the trap intercepts nitric oxide that is otherwise destined to be released into the headspace above the cell culture medium. Release of up to 20% of generated nitric oxide into the headspace above the cell culture medium has previously been demonstrated (29), although this value will clearly depend on the geometry of the system under study. The relative contribution of these two processes is not clear.

The most startling finding of this study was that the intracellular S-nitrosothiols, formed upon LPS stimulation, are remarkably stable and decay with a half-life of ~3 h. To maintain a steady state, this requires that the rate of formation is also very slow, in the range of 3–4 pmol/mg of protein/h. Given the ability of GSH to "repair" exposed thiols via transnitrosation, and given a putative GSNO reductase activity that will rapidly consume GSNO (22, 31), it is likely that the stable pool of S-nitrosothiols is in a compartment that is not exposed to GSH. Such thiols could be either in nonsolvent exposed protein sites or in specific cellular microenvironments. Whether these are specifically targeted sites, due to heightened propensity of the target thiol to nitrosation, or whether they represent the poorly repairable remnants of a general nitrosation process remains to be determined. It is highly unlikely that the measured S-nitrosothiols are a part of short-term signaling processes due to their stability. However, we cannot rule out the possibility that the level of observed S-nitrosothiol is an aggregate measurement of a dynamic pool, but if this pool is in a GSH-depleted environment, the mechanism by which the S-nitroso group can be transferred is unclear. It should be noted the cells used in this study were LPS treated and may have altered S-nitrosothiol repair mechanisms compared with nonactivated cells. The identification and characterization of S-nitrosated proteins are currently under way.

Our current hypothesis regarding the dynamics of S-nitrosothiol formation is illustrated in Fig. 6. LPS treatment leads to the induction of iNOS in RAW 264.7 cells, which catalyzes rapid production of nitric oxide. Nitric oxide can freely diffuse across cell membranes, and nitrosation is a significantly slower process than diffusion. The nitric oxide/oxygen reaction generates nitrosating agents, either NO2 or N2O3, in both the intracellular and extracellular compartments. In the extracellular space, this reaction is likely controlled by a third-order rate law with rate constant 2 x 106 M–2s–1 (25). In the intracellular space, the situation may be different since nitric oxide consumption by some cell types has been reported to be first order in nitric oxide (10, 51). NO2 and N2O3 are gaseous molecules and are also membrane permeable, although they are likely to be consumed more rapidly than nitric oxide. The majority of the nitrosating agent undergoes hydrolysis to form nitrite, and this is, by far, the major product. The local concentration of thiols will provide a target for nitrosation to form S-nitrosothiols. Because both protein thiols and GSH are present at similar concentrations in the intracellular space, it is likely that they represent approximately equal initial targets. In addition, transnitrosation between S-nitrosothiols and free thiols will tend to equilibrate the nitrosation pattern, favoring thermodynamically more stable S-nitrosothiols (19). However, there exists a strong kinetic pull on these equilibria, in the form of enzymatic GSNO metabolism by GSH-FDH, that will rapidly denitrosate all thiols in kinetic contact with GSH. This strongly suggests that the stable pool of S-nitrosothiols observed in this study is isolated from the GSH pool and is therefore directly nitrosated, and not formed, via transnitrosation. S-nitrosothiols at GSH-inaccessible sites have a relatively long lifetime and are slowly metabolized by unknown cellular mechanisms.



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Fig. 6. A model for S-nitrosothiol formation and metabolism in RAW 264.7 cells. GSSG, glutathione disulfide; GSH-FDH, GSH-dependent formaldehyde hehydrogenase; iNOS, inducible nitric oxide synthase; NO, nitric oxide.

 
This study has focused on thiol nitrosation as a product of intracellular nitric oxide formation. It should be stressed that this modification is extremely inefficient and may represent only a small fraction of the oxidative propensity of the nitric oxide/oxygen reaction. It has been recently demonstrated that the major product of the reaction between nitric oxide, oxygen, and GSH is GSSG and not GSNO, although the precise product ratio depends on thiol and oxygen concentration (24). S-Nitrosation may have specific effects on the phenotype of the cell, yet it is possible that thiol oxidation to disulfides also plays a major role in nitric oxide-mediated biological processes.


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This work was supported by National Institute of General Medical Sciences Grant GM-55792 and American Heart Association Predoctoral Fellowship 0310032Z.


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Address for reprint requests and other correspondence: N. Hogg, Dept. Of Biophysics, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226 (E-mail: nhogg2{at}mcw.edu)

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.


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