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Am J Physiol Heart Circ Physiol 281: H432-H439, 2001;
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Vol. 281, Issue 1, H432-H439, July 2001

Metabolism of S-nitrosoglutathione by endothelial cells

Hong Zeng, Netanya Y. Spencer, and Neil Hogg

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

S-nitrosoglutathione (GSNO) is an inhibitor of platelet aggregation and has also been shown to protect the ischemic heart from reperfusion-mediated injury. Although GSNO is often used in cell culture as a source of nitric oxide, the mechanisms of GSNO metabolism are not well established. We show here that GSNO decomposition by bovine aortic endothelial cells has an absolute dependence on the presence of cystine in the cell culture medium. In addition, GSNO decay is inhibited by diethyl maleate, an intracellular glutathione scavenger, but not by buthionine sulfoximine, a glutathione synthesis inhibitor. This indicates that thiols in general, rather than specifically glutathione, are the major factors that influence GSNO decay. Only 40% of the nitroso group of GSNO could be recovered as nitrite/nitrate, suggesting that the primary route of GSNO decay is reductive and that nitric oxide is only a minor product of GSNO decay. We conclude that the intracellular thiol pool causes the reduction of extracellular disulfides to thiols, which then directly reduce GSNO.

S-nitrosothiols; cystine; thiols; nitric oxide


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

S-NITROSOGLUTATHIONE (GSNO) is a metabolite of glutathione (GSH) and nitric oxide (15). GSNO is formed during the oxygen-dependent oxidation of nitric oxide in the presence of GSH (18, 22, 38), and also as a minor product from the oxidation of GSH by peroxynitrite (26, 37). GSNO has been shown to have several pharmacological activities. These include the inhibition of platelet aggregation (4) and the activation of soluble guanylyl cyclase. In addition, GSNO has a protective effect in cardiac reperfusion injury and during the exposure of cells to oxidants (23, 24). GSNO has also been demonstrated to have a dual role in apoptosis, being both protective and toxic, depending on concentration and cell type (6, 25, 31).

GSNO is often referred to as a "nitric oxide donor" when used in cell culture, and has been employed in many studies as a source of nitric oxide. In many cases, the effects of GSNO in cell culture are automatically ascribed to nitric oxide release. This usage has become common practice because some S-nitrosothiols, such as S-nitrosocysteine, are extremely susceptible to transition metal ion-catalyzed decomposition to yield nitric oxide. This gives the impression of spontaneous nitric oxide release because all buffers and cell culture media contain transition metal ions through design or contamination. GSNO, however, is somewhat resistant to this mode of decomposition (17). In fact, very little is known about the decomposition and metabolism of GSNO in cell culture, and it has not been demonstrated that nitric oxide is a primary product.

The potential biological reactions of GSNO have been well established. The decomposition of GSNO does not occur spontaneously and requires the presence of an additional agent such as light (33), transition metal ions (particularly copper) (5), or thiols. In the latter case, thiols can decay GSNO by two mechanisms. The first, more rapid, mechanism is reversible transnitrosation, which leads to the formation of GSH and another S-nitrosothiol (2, 16). The second, slower, reaction is the reduction of GSNO by thiol to generate nitroxyl anion and a mixed disulfide (28). This latter reaction, though slower than transnitrosation, is thermodynamically favored due to the rapid consumption of nitroxyl.

Gorge et al. (12, 13) have demonstrated that a copper-dependent pathway of GSNO decomposition is responsible for the ability of GSNO to inhibit platelet aggregation. In addition, they identified a putative activity, termed "GSNO lyase" (11), that is responsible for GSNO decay by vascular cells. Specifically, the GSNO lyase activity followed the conversion of GSNO to nitrite by various cell types. The activity of this enzyme was inhibited by high concentrations of metal ion chelators (0.1-1 mM) and a mixture of diamide and Zn2+. This latter treatment increases the glutathione disulfide (GSSG) to GSH ratio and suggested that the GSNO lyase activity was controlled by the redox state of the system (11).

In this study, we examine the mechanism of decomposition of GSNO by vascular endothelial cells, by directly monitoring the rate of decay of GSNO. We show that GSNO decomposition is slow, not prevented by inhibitors of GSH formation, and absolutely dependent on the presence of either L-cystine or D-cystine in the cell culture medium. Unlike GSNO lyase activity, GSNO decay is not saturable and is unlikely to be due to a direct interaction between GSNO and an enzyme active site. We conclude that the mechanism of GSNO decomposition by endothelial cells involves the reduction of extracellular disulfides to form thiols, which then reduce GSNO. These data suggest that GSNO is not metabolized by a specific protein on the cell surface but is degraded by cellular modification of the extracellular environment. In addition, these data suggest that GSNO cannot be considered a nitric oxide donor molecule because its metabolism is complex and because nitric oxide may not be a primary product.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. GSH, N-ethylmaleimide (NEM), sodium nitrite, ferrozine, buthionine-[SR]-sulfoximine (BSO), glutamic acid, o-phthalaldehyde (OPA), L-cystine, D-cysteine, and acivicin were obtained from Sigma. Diphenyleneiodonium chloride (DPI) was obtained from Calbiochem. Diethyl maleate (DEM) and trichloroacetic acid (TCA) were obtained from Aldrich Chemical. Superoxide dismutase (SOD) was obtained from Boehringer-Mannheim. Diethylenetriaminepentaacetic acid (DTPA) was obtained from Fluka Chemical. GSNO was synthesized and purified according to published procedures (8, 15). D-Cystine was synthesized from D-cysteine by oxidation with hydrogen peroxide.

Cell culture. Bovine aorta endothelial cells (BAEC) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing fetal bovine serum (FBS, 15%), penicillin-streptomycin (2%), and L-glutamine (1%). The cells were placed in a 5% CO2 water-jacketed incubator at 37°C. Under these conditions, confluence was normally achieved 4 days after passage.

GSNO determinations. Cells were washed twice with FBS-free DMEM and incubated with FBS-free DMEM in the presence of the compounds being tested. For GSNO measurements, cells were grown in 6-well plates. After the compounds were added, the medium (1 ml) was removed and immediately treated with NEM (10 mM). Any protein was precipitated by the addition of 2% trichloroacetic acid (TCA), followed by centrifugation (5 min at 2,000 g), before high-performance liquid chromatography (HPLC) analysis.

GSNO concentration was determined by HPLC with the use of an HP1100 series apparatus as previously described (33). Samples (100 µl) were injected onto a Kromasil C-18 column and eluted isocratically with a mobile phase consisting of methanol: 0.05% trifluoroacetic acid containing 10 µM DTPA (94:6). Total run time was 35 min per sample, which included a 5-min prerun and 7-min postrun wash with 100% methanol. The effluent was monitored with a diode array spectrophotometer at both 210 and 336 nm. GSNO concentration was determined using authentic standards. Total S-nitrosothiols were also determined using the Saville assay (32).

Thiol measurements. For thiol measurements, cells were grown in 100-mm dishes. Cells were scraped and lysed by five freeze/thaw cycles. Total thiol content was measured by ultraviolet spectrophotometry using 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB). DTNB (5 mM final) was added to the cell lysate (1:5 vol/vol), and the concentration of the thiols was determined at 412 nm using cysteine as a standard.

GSH concentration was determined by HPLC (1). OPA (36.4 mM) was dissolved in ethanol and then diluted 1:10 (vol/vol) with borate buffer (75 mM, pH 8.0). Cell lysate (1 ml) was mixed with OPA (0.5 ml, final concentration of 1.2 mM), and the solution was filtered under centrifugation by using Centricon filtration devices. Effluent (100 µl) was injected onto a Kromasil C-18 column and was eluted isocratically with a mobile phase consisting of methanol: 150 mM sodium acetate (91.5:8.5). The OPA-GSH adduct was monitored with a diode array spectrophotometer and quantified at 338 nm using authentic standards.

Nitrate/nitrite determination. Nitrate was determined using the Griess assay as previously described (35). Nitrate concentrations were measured using a chemiluminescence analyzer (Sievers Instruments) according to the manufacturer's directions. To measure the combination of nitrate, nitrite, and S-nitrosothiol, vanadium(III) chloride and glacial acetic acid were added to the purge vessel, and the solution was heated to 98°C. The concentration of nitrate was determined by subtraction of the previously determined concentrations of nitrite and S-nitrosothiol.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Decomposition of GSNO by endothelial cells: the effect of serum. To examine the decomposition of GSNO by endothelial cells, it was first necessary to define conditions in which GSNO decay was minimized when incubated in cell culture medium in the absence of cells. As shown in Fig. 1A, incubation of GSNO in DMEM containing FBS (10%) resulted in a loss of GSNO over a period of 8-10 h in both the absence and presence of cells. The rate of decay was marginally faster in the presence of cells. The presence of the metal ion chelator DTPA did not affect the rate of decay (data not shown). In the presence of 0.5% FBS, the difference between GSNO decay in the presence and absence of cells was larger than in 10% FBS. However, GSNO was completely destroyed within 10 h in the absence of cells (data not shown). In the absence of serum, however, cell-dependent GSNO decay occurred over a similar time range, whereas cell-independent GSNO decay was considerably slower, with only ~20-40% loss of GSNO after 10-h incubation. GSNO (100 µM) incubated in the absence of cells in DTPA-containing phosphate buffer did not decay significantly over 8 h (data not shown). The amount of GSNO decay in the absence of cells was inconsistent between experiments and appeared to be related to the age of the medium. The kinetics of cell-dependent GSNO decay were substantially different in the presence and absence of serum. In the presence of serum, GSNO decay was essentially exponential, whereas in the absence of serum, a more linear decay was observed. In addition, a short lag period was also seen before GSNO decay commenced. To examine specifically the mechanisms of GSNO decay, rather than the products formed from the interaction between GSNO and serum, we performed all of the following experiments under serum-free conditions. It should be noted that the absence of serum for a prolonged period of time may place these cells under significant stress. GSNO did not cause any appreciable cell death, as detected using trypan blue exclusion, during 10 h of exposure (data not shown).


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  		Fig. 1.   Decomposition of S-nitroglutathione (GSNO) by bovine aortic endothelial cells (BAEC) in the presence and absence of serum. GSNO (100 µM) was incubated in the presence (A) and absence (B) of serum and in the presence () and absence (open circle ) of cells. Samples were taken at the indicated time points and immediately treated with N-ethylmaleimide (NEM; 10 mM). After protein precipitation with trichloroacetic acid (TCA), GSNO concentration was determined by HPLC. Data are means ± SE, n = 3.

Effect of metal ion chelators, DPI, and SOD on GSNO decay. It has been previously demonstrated that transition metal ions (particularly iron and copper) can catalytically decompose S-nitrosothiols (5, 33). In addition, Gorge et al. (12) proposed a copper protein-mediated mechanism of S-nitrosothiol decay. The effect of three metal ion chelators, ferrozine, an iron(II) chelator, DTPA, an iron(III) and copper(II) chelator, and bathocuproine sulfonate, a copper(I) chelator, were each tested for their ability to inhibit cell-dependent GSNO decay (Fig. 2). Little, if any, effect was observed for each chelator, indicating that transition-metal ion-dependent decomposition is not a significant mechanism of GSNO decay in this system.


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  		Fig. 2.   Effect of metal ion chelators in the decomposition of GSNO by BAEC. GSNO (100 µM) was incubated either in the absence of cells (open circle ) or in the presence of cells with the addition of ferrozine (, 100 µM), bathocuproine sulfonate (, 100 µM), or diethylenetriaminepentaacetic acid (triangle , 100 µM). Samples were taken at the indicated time points and immediately treated with NEM (10 mM). After protein precipitation with TCA, GSNO concentration was determined by HPLC.

DPI, a nonspecific flavoprotein reductase inhibitor, had no effect on GSNO decay (data not shown), indicating that GSNO reduction by plasma membrane flavoprotein reductases, such as the reduced nicotinamide adenine dinucleotide (phosphate) oxidase family of enzymes, is not responsible for GSNO decay. Because superoxide has been shown to reduce GSNO (21, 36), and because the combination of SOD and GSH has been shown to decompose GSNO (20), we tested the effect of SOD on GSNO decay. SOD (50 and 100 U/ml) had no effect on cell-dependent or cell-free GSNO decomposition (data not shown). This indicates that neither superoxide, nor a combination of SOD and thiol, affect GSNO decomposition in this system.

Effect of BAEC on GSNO decay. It is possible that cells enhance the decay of GSNO by conditioning the cell culture medium. To determine if the presence of BAEC was required throughout the entire time-course of GSNO decay, GSNO was incubated with cells and the medium was removed after 2, 4, or 8 h. After the medium was removed, it was transferred to a new, cell-free plate and replaced in the tissue culture incubator. As shown in Fig. 3A, GSNO decay occurs faster in the presence of cells than in their absence. If GSNO is removed from the cells and placed in cell-free conditions, it continues to decay, but at the rate associated with cell-free conditions. This indicates that viable cells are required throughout the entire time course of GSNO decay.


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  		Fig. 3.   Effect of cells and [GSNO] on the rate of decomposition of GSNO. A: GSNO (100 µM) was incubated in the presence () or absence (open circle ) of BAEC. In addition, GSNO was incubated in the presence of BAEC and subsequently transferred to a cell-free plate after 2 (black-triangle), 4 (down-triangle), or 6 (black-lozenge ) h. B: GSNO (100 µM, ; 250 µM, ; 500 µM, black-triangle; 750 µM, black-down-triangle ; 1,000 µM, open circle ; and 2,000 µM, ) was incubated in the presence of cells. Samples were taken at the indicated time points and immediately treated with NEM (10 mM). After protein precipitation with TCA, GSNO concentration was determined by HPLC. Inset: the rate of decay of GSNO between 2 and 8 h was calculated from the data in B and is plotted as a function of [GSNO] (). The cell-dependent component () was calculated by subtracting data from an identical experiment performed in the absence of cells.

Effect of GSNO concentration on GSNO decay. To determine if the rate of GSNO decay was saturable as a function of GSNO concentration, the rate of GSNO decay was monitored at initial GSNO concentrations ranging from 100 to 2,500 µM. GSNO decayed at all concentrations (Fig. 3B) after a short lag period, in an approximately linear manner. The rate of decay of GSNO was determined between 2 and 8 h and plotted as a function of initial GSNO concentration (Fig. 3B, inset). An identical experiment was performed in the absence of cells (data not shown), and the calculated rates of decay were subtracted to give the cell-dependent component of GSNO decay (Fig. 3B, inset). The concentration dependences of both total GSNO decay and cell-dependent GSNO decay were linear. This suggests that a specific, saturable, GSNO binding site on the cell surface is not involved in BAEC-dependent GSNO decomposition.

Effect of intracellular GSH on GSNO decay. The effect of intracellular GSH on GSNO decay was examined using the specific GSH scavenger DEM and the inhibitor of GSH synthesis, BSO. DEM is an alpha ,beta -unsaturated carbonyl that reacts with GSH through a reaction catalyzed by the glutathione S-transferases (30). The effect of DEM on GSNO decay is shown in Fig. 4A. It should be noted that to measure total intracellular thiols, these experiments were performed in 100-mm dishes, rather than in 6-well plates. In this circumstance, the rate of GSNO decay was greatly enhanced, with total GSNO decomposition occurring over 4-6 h. DEM decreased intracellular GSH levels from 4.2 ± 0.5 to 0.96 ± 0.10 nmol/mg of protein and decreased total thiol content by 25 ± 5%. This reduction in thiol content was accompanied by a significant decrease in the rate of decomposition of GSNO with ~40% of GSNO remaining after 4 h. DEM had no effect on cell-free GSNO decay (Fig. 4A).


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  		Fig. 4.   Effect of diethyl maleate (DEM) and buthionine-[SR]-sulfoximine (BSO) on GSNO decay. A: GSNO (100 µM) was incubated in either the presence (closed symbols) or absence (open symbols) of BAEC, in the presence of (circles) or absence (squares) of DEM (200 µM). B: GSNO (100 µM) was incubated in the presence () or absence (open circle ) of BSO (250 µM). Samples were taken at the indicated time points and immediately treated with NEM (10 mM). After protein precipitation with TCA, GSNO concentration was determined by HPLC. Data are means ± SE, n = 3.

The gamma -glutamylcysteine synthase inhibitor BSO prevents GSH formation by inhibiting the initial step in GSH synthesis (14). In contrast to DEM, BSO did not inhibit, but slightly enhanced, endothelial cell-dependent GSNO decay (Fig. 4B). Although BSO treatment depleted cellular GSH levels from 4.5 ± 0.6 to 0.54 ± 0.04 nmol/mg protein, it caused only a 7 ± 3% drop in total cellular thiols, as measured by DTNB. This suggests that cellular thiol content, rather than GSH per se, is an important factor in the mechanism of GSNO metabolism.

Effect of cystine on GSNO decay. Cystine, the oxidized disulfide form of cysteine, is present in the cell culture medium used in these studies. The effect of extracellular cystine on GSNO decay was examined by incubating GSNO with BAEC in cystine-free DMEM. As shown in Fig. 5A, the use of cystine-free medium completely inhibited the cell-dependent decomposition of GSNO over 6 h. These data indicate that the presence of cystine is essential for cell-dependent GSNO decomposition.


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  		Fig. 5.   Effect of cystine on the rate of decomposition of GSNO by BAEC. A: GSNO (100 µM) was incubated in the presence () or absence (open circle ) of BAEC in cystine-free medium. B: GSNO (100 µM) was incubated in the presence (closed symbols) or absence (open symbols) of BAEC in cystine-free medium, supplemented with either L-cystine (25 µM, , ) or D-cystine (25 µM, , open circle ). Samples were taken at the indicated time points and immediately treated with NEM (10 mM). After the protein was precipitated with TCA, GSNO concentration was determined by HPLC. Data are means ± SD, n = 3.

To examine if uptake and metabolism of extracellular cystine are responsible for GSNO consumption, cystine-free medium was supplemented with either L-cystine or D-cystine. Figure 5B demonstrates that both L- and D-cystine supported cell-dependent GSNO decay. In addition, neither sodium glutamate, an inhibitor of cystine uptake, nor acivicin, a transpeptidase inhibitor, inhibited GSNO decay (data not shown). This suggests that intracellular cystine metabolism is not required for the observed effects and that stereospecific recognition of cystine, by the cell, is not required for the decomposition of GSNO.

In contrast to cystine, GSSG (250 µM) had little effect on the rate of GSNO decay in either cystine-free or cystine-replete media (data not shown). This indicates that the ability of cystine to promote GSNO decay is not shared by all disulfides.

Fate of the nitroso group of GSNO during metabolism by BAEC. It is often assumed that the nitroso group of GSNO decomposes to form nitric oxide, which then oxidizes, by reaction with oxygen and water, ultimately to form nitrite and nitrate. The kinetics of nitrite and nitrate formation were analyzed by a combination of the Griess assay (to measure nitrite), the Saville assay (to measure nitrite and GSNO), and chemiluminescence after reduction with vanadium(III) chloride (to measure nitrate, nitrite, and GSNO) (Fig. 6A). As shown in Fig. 6B, after appropriate subtraction, very little nitrate was formed during cell-dependent decomposition of GSNO until 3-4 h. This is in good agreement with Gordge et al. (11), who did not observe nitrite formation during a 30-min incubation. Fig. 6B also shows that only ~40% of the decomposed GSNO could be accounted for by nitrite/nitrate formation after 4 h. Nitrite (100 µM) could not be metabolized to other products by BAEC during 24 h of incubation (data not shown). Intracellular levels of nitrite and RSNO accounted for only 1% and 0.2% of the initial GSNO, respectively. From an initial amount of 1 µmol of GSNO (10 ml of 100 µM), only ~10 nmol of nitrite and 2 nmol of RSNO could be detected in cell lysate after 1 h of incubation. These intracellular concentrations decreased over 10 h (data not shown). Almost 60% of the nitroso group did not form nitrite or nitrate and remained unaccounted for.


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  		Fig. 6.   Formation of nitric oxide (NO) oxidation products from the decomposition of GSNO by BAEC. A: GSNO (100 µM) was incubated in the presence (closed symbols) or absence (open symbols) of BAEC. Medium was removed at the indicated time points and treated with NEM (10 mM). Samples were analyzed for nitrite using the Griess assay (, open circle ), for GSNO and nitrite using the Saville assay (, ), and for GSNO, nitrite and nitrate using chemiluminescence after reduction by VCl3 (black-triangle, triangle ). Data are means ± SD; n = 3. B: data from A, in the presence of cells, appropriately subtracted to show GSNO consumed (), nitrite formed (open circle ), and nitrate formed () as a function of time.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The mechanism by which cultured cells metabolize S-nitrosothiols is not known. In most of the studies that have examined the effects of S-nitrosothiols in tissue culture conditions, serum has been used. The possible reactions of S-nitrosothiols with the multiple components of serum, especially protein thiol groups, make it impossible in such studies to follow the fate of a particular compound. Such reactions are exemplified by comparison of Fig. 1, A and B, where, in the absence of cells, GSNO decay is dramatically suppressed by removal of serum. In these studies we have monitored the decomposition of GSNO in serum-free conditions.

In the presence of the metal ion chelator DTPA, GSNO spontaneously decays faster in serum-free cell culture medium, than it does in phosphate buffer under the same conditions. This suggests that the cell culture medium contains a component that can accelerate GSNO decay. In the presence of cells, GSNO decay is greatly accelerated, indicating that cells actively contribute to the decomposition of GSNO.

GSNO decay is not characterized by saturable kinetics, strongly suggesting that GSNO decay does not involve the interaction between GSNO and the saturable binding site of an enzymatic catalyst or transporter. Consequently, cellular uptake of GSNO is not required for GSNO metabolism, and an interaction between GSNO and a cell surface enzyme is not required. Gordge et al. (11) reported that nitrite formation from GSNO exhibited saturable decay kinetics, with NRK-49F fibroblasts, and reported a Michaelis-Menten constant of 16.5 µM. However, they also noted that the kinetics of nitrite formation did not follow Michaelis-Menten kinetics. The differences between this result and the data presented here may be attributable to differences in cell type; however, Gordge et al. (11) did not measure GSNO decay but monitored nitrite formation. We show here that nitrite is not formed stoichiometrically from the decay of GSNO in cell culture and appears to be formed as a result of more complex chemistry than the presumed NO release and oxidation. The data presented here do not support the presence of a GSNO lyase enzyme activity in endothelial cells as proposed by Gordge et al. (11) for fibroblasts.

In most experiments, the decomposition of GSNO was preceded by a lag period of ~1 h. The reason for this lag is at present unknown but is likely due to the cells altering the composition of the medium. Such conditioning would have to be transient, however, because if the GSNO is removed from the cells and placed in an identical cell-free culture dish, GSNO decay effectively ceases (Fig. 3A). One possible explanation for this effect is that a component of the medium is constantly being reduced and reoxidized during GSNO decay.

The effects of modulating intracellular thiol/GSH levels on GSNO decay are complex. DEM, which scavenges GSH through the action of glutathione S-transferases, caused a dramatic reduction in cellular thiols and a 25% reduction in total cellular thiols. Such treatment retarded GSNO decay. In contrast, BSO, an inhibitor of GSH synthesis, which reduced GSH levels by almost 90% but decreased total thiols by only ~7%, slightly enhanced GSNO decay. This is in agreement with Gordge et al. (11), who observed an enhanced biotransformation of GSNO to nitrite in the presence of BSO. Intracellular thiols appear to promote the reduction of an extracellular component responsible for GSNO decay. Total thiol content, rather than specifically GSH concentration, is a critical factor for this reduction to occur. The observation that both D- and L-cystine enhance GSNO decay when supplemented to cystine-free medium suggests that cystine uptake is not a requirement for GSNO decay but that cystine is reduced to cysteine in the extracellular space. It is probable that this reduction does not occur at a specific protein locus on the surface of a cell because the effect of cystine is not stereospecific. In addition, the lack of effect of sodium glutamate and acivicin make it unlikely that cystine uptake and metabolism play an important role in this system.

These data point to the mechanism outlined in Fig. 7. The intracellular thiol pool reduces extracellular cystine to cysteine by an as yet unidentified mechanism that does not appear to involve uptake of cystine, nor reduction at a chiral protein locus. The cysteine, once formed, reduces GSNO to form a mixed disulfide and nitroxyl anion. If nitric oxide were the primary decomposition product of GSNO decay, it would be expected that the GSNO would be stoichiometrically converted to nitrite because nitrite is by far the major oxidation product of nitric oxide (19). However, our data show that only ~25% of GSNO was converted to nitrite and ~15% converted to nitrate after 4 h. This suggests that nitric oxide is not a major primary product of GSNO decay. The reaction between GSNO and thiols is well established in chemical systems (2, 18, 34, 39) and ultimately leads to the formation of nitrous oxide (under anaerobic conditions), hydroxylamine, ammonia, nitrate, and nitrite. The mechanism of nitroxyl decomposition is complex due to reactions with thiols, oxygen, and itself (3, 7, 10). In addition, cells may have additional targets for nitroxyl that convert nitroxyl to nitric oxide (e.g., SOD and cytochrome c) (27). Such reactions, as well as the reaction of nitroxyl with oxygen (10), may be responsible for the observed nitrite and nitrate formation (see Fig. 7).


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  		Fig. 7.   Proposed mechanism of decomposition of GSNO by BAEC. Intracellular thiols (either cysteine or glutathione) elicit the reduction of extracellular cystine to form cysteine in the cell culture medium. The reaction between cysteine and glutathione ultimately forms mixed disulfides and nitroxyl anion (NO-). The fate of nitroxyl is complex, and, in the presence of reducing agents and oxygen, can include the formation of both reduced (nitrous oxide hydroxylamine and ammonia) and oxidized (nitrate and nitrite) products.

Understanding the mechanism of GSNO decay in cell culture is of prime importance to a full comprehension of how this molecule elicits changes in cell function. In certain cell types, GSNO has been shown to be proapoptotic and it is commercially available as an apoptotic mediator. However, in other cell types, GSNO will inhibit apoptosis caused by other mediators. Most of the effects of GSNO in cell culture are thought to occur through either the release of nitric oxide or through transnitrosation of the nitroso group to a protein thiol. The data presented here suggest that the major mechanism of GSNO in cell culture is reductive denitroxylation generating nitroxyl anion and a thiol disulfide as primary products. Although nitroxyl has been reported to activate guanylyl cyclase (9), its biological chemistry is poorly defined. It is relevant to note that glutathione-protein mixed disulfides have been reported (29) as a major cellular product after nitric oxide exposure.

In conclusion, GSNO decay by BAEC depends absolutely on the cellular reduction of extracellular oxidized thiols using the reducing power of the intracellular thiol pool. Nitrite/nitrate, and consequently nitric oxide, is not the only product of GSNO decomposition by endothelial cells.


    ACKNOWLEDGEMENTS

The authors thank Dr. B. Kalyanaraman for stimulating discussions.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grant GM-55792 and Research Resource Development Program Grant RR-01008.

Address for reprint requests and other correspondence: N. Hogg, Biophysics Research Institute and Free Radical Research Center, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226 (E-mail: nhogg{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.

Received 1 September 2000; accepted in final form 8 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Akerboom, TP, and Sies H. Assay of glutathione, glutathione disulfide, and glutathione mixed disulfides in biological samples. Methods Enzymol 77: 373-382, 1981[Medline].

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3.   Bonner, FT, and Stedman G. The chemistry of nitric oxide and redox-related species. In: Methods in Nitric Oxide Research, edited by Feelisch M, and Stamler JS.. New York: Wiley, 1996, p. 3-18.

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Am J Physiol Heart Circ Physiol 281(1):H432-H439
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