Adaptive responses to peroxynitrite: increased glutathione levels and cystine uptake in vascular cells

Barbara J. Buckley and A. Richard Whorton

Departments of Medicine and Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We and others recently demonstrated increased glutathione levels, stimulated cystine uptake, and induced gamma -glutamylcysteinyl synthase (gamma -GCS) in vascular cells exposed to nitric oxide donors. Here we report the effects of peroxynitrite on glutathione levels and cystine uptake. Treatment of bovine aortic endothelial and smooth muscle cells with 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, resulted in transient depletion of glutathione followed by a prolonged increase beginning at 8-9 h. Concentration-dependent increases in glutathione of up to sixfold occurred 16-18 h after 0.05-2.5 mM SIN-1. Responses to SIN-1 were inhibited by copper-zinc superoxide dismutases and manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride, providing evidence for peroxynitrite involvement. Because glutathione synthesis is regulated by amino acid availability, we also studied cystine uptake. SIN-1 treatment resulted in a prolonged increase in cystine uptake beginning at 6-9 h. Increases in cystine uptake after SIN-1 were blocked by inhibitors of protein and RNA synthesis, by extracellular glutamate but not by extracellular sodium. These studies suggest induction of the xc- pathway of amino acid uptake. A close correlation over time was observed for increases in cystine uptake and glutathione levels. In summary, vascular cells respond to chronic peroxynitrite exposure with adaptive increases in cellular glutathione and cystine transport.

endothelial cells; smooth muscle cells


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ACUTE EXPOSURE TO HIGH LEVELS of nitric oxide (·NO) and peroxynitrite (ONOO-) may lead to alterations in cellular signaling and function through the modification of lipids and protein thiols (20, 34, 35). Chronic exposures to lower levels may have different effects. For example, work from our laboratory and others demonstrated increased cellular glutathione (GSH) levels in endothelial and smooth muscle cells treated with S-nitrosothiols and other ·NO donors (21, 27). Elevation of GSH levels is a common response to oxidant stress and can modulate cell injury by enhancing the capacity to scavenge radical species and repair oxidized protein thiols and lipid peroxides (7, 23, 31). Recently, cytokine-stimulated ·NO production was found to be essential for the upregulation of GSH synthesis and the prevention of oxidant injury in hepatocytes (18, 19). These results suggest that the proposed antioxidant role of ·NO may involve more than the direct scavenging of radical species and may also be related to increases in cellular GSH. This adaptive response may result from induction of lambda -glutamylcysteinyl synthase (lambda -GCS), the rate-limiting enzyme in GSH biosynthesis, induction of cystine uptake resulting in greater substrate availability, or a combination of both (7, 23). In our previous study of endothelial cells treated with S-nitrosothiols, elevated GSH levels appeared to result from the increased uptake of cystine by the xc- pathway of amino acid transport (21). By contrast, in vascular smooth muscle cells treated with S-nitrosothiols, elevated GSH was reported to be due to induction of gamma -GCS, the rate-limiting enzyme for GSH synthesis (27).

In the case of ONOO-, many deleterious effects have been reported in vascular cells exposed acutely to high levels, including altered calcium signaling (11), collapse of mitochondrial membrane permeability (14), and decreased cell viability (14). Adaptive responses to ONOO- have not been reported. However, ONOO- is not always injurious. For example, ONOO- is capable of increasing cGMP levels in endothelial cells (25), stimulating relaxation of vascular smooth muscle (42), and inhibiting platelet aggregation (28). Because ONOO- is a stronger oxidant than ·NO, we reasoned that vascular cells exposed to this mediator would respond in a similar manner by elevating GSH levels. In this study, we investigated GSH synthesis and cystine uptake in vascular cells using 3-morpholinosydnonimine (SIN-1), which generates ONOO- from the superoxide (O2-·) and ·NO formed during decomposition.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). All other cell culture reagents were from GIBCO BRL (Grand Island, NY). Tissue culture plasticware was obtained from Nunclon (Fisher Scientific, Raleigh, NC). S-nitrosopenicillamine (SNAP), SIN-1, and manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) were from Alexis (San Diego, CA). Cu,Zn SOD (copper-zinc superoxide dismutases) was from Roche Molecular Biochemicals (Indianapolis, IN). L-[35S]cystine was obtained from Amersham (Piscataway, NJ). All other chemicals were from Sigma (St. Louis, MO).

Cell isolation and culture. Bovine aortic endothelial cells and smooth muscle cells were isolated and cultured by established methods as previously described (4, 21). Briefly, bovine aortic segments were cleaned, opened, and the endothelium was removed by collagenase treatment. Endothelial cell isolates were plated on collagen-coated tissue culture plasticware. Smooth muscle cells were grown from explants of freshly collected aortas. Cells were incubated at 37°C in an atmosphere of 7.5% CO2 in air in Dulbecco's modified Eagle's medium (DMEM) that contained 10% (vol/vol) FBS and antibiotic/antimycotic (100 U/ml penicillin G sodium, 100 ug/ml streptomycin sulfate, and 250 ng/ml amphotericin B). Cells were passaged by treatment with trypsin-EDTA.

Experimental conditions. Early passage primary cultures of endothelial and smooth muscle cells were used. Confluent monolayers of cells were incubated for 1-24 h in DMEM (phenol red free) that contained 1% FBS, 1% antibiotic/antimycotic, 4 mM glutamine, and the ONOO- donor SIN-1 or the ·NO donor SNAP. Immediately before use, SIN-1 and MnTMPyP were dissolved in sterile distilled H2O, and SNAP was dissolved in media. Decomposed SNAP was prepared by incubating a 5 mM stock solution at 22°C for 5 days under fluorescent lights. The absorbance at 340 nM was monitored to verify its decomposition. Decomposed SIN-1 was similarly prepared.

GSH assay. Incubations were stopped by rinsing cells twice with ice-cold PBS and adding 1 M perchloric acid containing 2 mM EDTA. Cells were removed from the culture dishes by scraping and incubated for 30 min at 4°C. The lysates were subjected to centrifugation for 10 min at 6,000 g, and the acid extracts were neutralized with 4 M KOH that contained 0.6 M MOPS. The potassium perchlorate precipitate was removed by centrifugation. Total GSH levels [GSH and glutathione disulfide (GSSG)] were determined spectrophotometrically using the GSH reductase-linked 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) recycling assay (40). The assay conditions were 100 mM KPO4 (pH 7.0), 1 mM EDTA, 100 µM DTNB, 300 µM NADPH, and 0.2 U/ml GSH reductase. Quantification was based on standard curves that were prepared using known concentrations of GSSG. Results are reported in terms of GSH equivalents. The acid-insoluble pellets were analyzed for protein by using the Lowry method.

Measurement of cystine uptake. Uptake was determined using [35S]cystine as previously reported (6, 21). Cells were rinsed twice with PBS and incubated in room air at 37°C for 10-20 min in Hanks' balanced salt solution (HBSS) that contained 10 mM HEPES, pH 7.4 (HHBSS), followed by incubation with 1 ml of HHBSS that contained [35S]cystine (1 µCi/60 µM). After 10 min, cells were rapidly rinsed with ice-cold HHBSS that contained 600 µM cystine, lysed with 1% Triton X-100/HHBSS, and scraped from the culture dishes. Radioactivity was measured by liquid scintillation counting. In some experiments, sodium-free buffer solutions were prepared by using LiCl and Tris as substitutes for the NaCl and NaPO4 in HBSS and by omitting the HEPES (38).

Statistical analysis. Results are expressed as means ± SE. Statistical comparisons between two groups were made using Student's t-test, and comparisons between multiple groups were made using analysis of variance followed by Tukey's post hoc test. P values < 0.05 were considered to be statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We have previously demonstrated that treatment of endothelial cells with S-nitrosothiols resulted in increased levels of cellular GSH (21). Endothelial cells responded similarly to SIN-1, with 2.4- and 6-fold increases in cellular GSH 17 h after treatment with and 0.1 and 1 mM, respectively (Fig. 1). Increased cellular GSH was also observed using 1 mM SNAP as a positive control (Fig. 1). Decomposed SIN-1 and SNAP (0.5 mM) failed to stimulate an increase in GSH levels (data not shown). Concentrations of SIN-1 >1 mM were associated with a decline in cell protein levels of >20% and were not routinely used in these studies. Elevations in cellular GSH of 1.9-fold were observed with concentrations of SIN-1 as low as 0.05 mM (Figs. 2 and 3). To determine whether this response required de novo protein and RNA synthesis, cells were treated with 0.5 mM SIN-1 for 8 h in the presence of cycloheximide (1 µg/ml) or actinomycin D (2.5 µg/ml). Both agents effectively blocked the response (Fig. 4). Control levels were increased with cycloheximide treatment, an effect attributed to a shift in cysteine utilization from protein synthesis to GSH synthesis (6).


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Fig. 1.   Effect of 3-morpholinosydnonimine (SIN-1) and S-nitrosopenicillamine (SNAP) on glutathione (GSH) levels in endothelial cells. Confluent monolayers of bovine aortic endothelial cells were treated with SIN-1 and SNAP in DMEM (phenol red free) that contained 1% fetal bovine serum and 1% antibiotic/antimycotic. After 17 h, cell extracts were prepared and analyzed for GSH and protein as described in METHODS. Data represent means ± SE, n = 3. *Significantly different from control (P < 0.05).



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Fig. 2.   Effect of coincubation with copper-zinc superoxide dismutases (Cu,Zn SOD) on GSH levels in endothelial cells treated with SIN-1. Confluent monolayers of cells were treated with SIN-1 in the presence or absence of 100 U/ml Cu,Zn SOD. After 17 h, cell extracts were prepared as described for determination of GSH and protein. Data represent means ± SE, n = 3. *Significantly different from control (P < 0.05). Inset: Cu,Zn SOD was boiled for 5 min at 100°C before treatment of cells with 0.5 mM SIN-1. Data represent means ± SE, n = 3. **Significantly different from group treated with SIN-1 alone (P < 0.05).



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Fig. 3.   Effect of coincubation with manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride (MnTMPyP) on GSH levels in endothelial cells treated with SIN-1. Confluent monolayers of cells were treated with SIN-1 in the presence or absence of 0.1 mM MnTMPyP. After 17 h, cell extracts were prepared as described for determination of GSH and protein. Data represent means ± SE, n = 3. *Significantly different from control (P < 0.05).



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Fig. 4.   Effect of coincubation with cycloheximide (CHX) or actinomycin D (ACT D) on GSH levels in endothelial cells treated with SIN-1. Confluent monolayers of cells were treated with 0.5 mM SIN-1 in the presence of 1 µg/ml CHX or 2.5 µg/ml ACT D. After 8 h, cell extracts were prepared and analyzed for GSH and protein as previously described. Data represent means ± SE, n = 3. *Significantly different from control (P < 0.05). **Significantly different from group treated with SIN-1 alone (P < 0.05).

Because SIN-1 can act as a ·NO donor rather than a ONOO- donor under some conditions (15, 37), we investigated the response to SIN-1 in the presence of Cu,Zn SOD and the SOD mimetic MnTMPyP. By scavenging O2-·, these agents would be expected to block the formation of ONOO- during SIN-1 decomposition, leaving ·NO as the major decomposition product. Incubation of cells for 17 h with 0-0.5 mM SIN-1 in the presence of 100 U/ml Cu,Zn SOD resulted in a 32-67% inhibition of the GSH response (Fig. 2). No further inhibition was observed using 250 U/ml Cu,Zn SOD (data not shown). However, boiled enzyme was much less effective in blocking the response to 0.5 mM SIN-1, demonstrating that inhibition required active enzyme (Fig. 2, inset). With the use of 0.1 mM MnTMPyP, the increase in cellular GSH observed in response to SIN-1 was diminished by 69-92% (Fig. 3). A concentration of 0.05 mM MnTMPyP was equally effective. These results indicate that at least part of the response to SIN-1 was mediated by ONOO- and that ·NO, which may have been generated during SIN-1 decomposition, could not fully account for the response.

We also tested the effects of Cu,Zn SOD on SNAP-mediated increases in cellular GSH levels because O2-· produced endogenously by endothelial cells might react with SNAP-derived ·NO, forming ONOO-. These experiments demonstrated a small and variable effect of Cu,Zn SOD that was not altered by boiling the enzyme (data not shown). Additionally, we found that Cu,Zn SOD had no effect on increases in cellular GSH levels in endothelial cells treated with another ·NO donor, spermine NONOate (data not shown). These results provided evidence that the effects of the ·NO donors were not secondary to ONOO- production.

Increased cellular GSH in smooth muscle cells after treatment with S-nitrosothiols has also been recently reported (27). To determine whether SIN-1 has similar effects, we incubated smooth muscle cells with 0-2.5 mM SIN-1 or SNAP for 18 h. Increases in cellular GSH of 2.6- to 2.7-fold were observed with these agents (Fig. 5). However, a decline in cellular protein levels of 40% was observed in smooth muscle cells incubated with 1 and 2.5 mM SNAP (data not shown). Cellular protein levels decreased by 5-10% with the same doses of SIN-1 (data not shown). It should be noted that smooth muscle cell GSH levels were variable between experiments, as has been previously reported (27).


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Fig. 5.   Effect of SIN-1 (A) and SNAP (B) on GSH levels in smooth muscle cells. Confluent monolayers of cells were treated with SNAP and SIN-1. After 18 h, cell extracts were prepared and analyzed for GSH and protein, as previously described. Data represent means ± SE, n = 3. *Significantly different from controls (P < 0.05).

The availability of precursor amino acids, particularly cysteine, is an important regulator of GSH synthesis (2, 7), and cells in culture rely on extracellular cystine for their intracellular cysteine pool (2). We have previously reported that ·NO donors increase cystine uptake in endothelial cells, but the effects of ONOO- were not determined. Furthermore, the role of increased cystine uptake in elevating GSH levels in vascular smooth muscle treated with ·NO donors or ONOO- has not been investigated. Thus we next studied the effects of SIN-1 on cystine uptake. Endothelial and smooth muscle cells were incubated with 1 mM SNAP and SIN-1 for 12 h, and the uptake of radiolabeled cystine during 10 min was determined. No decline in cellular protein levels was observed under these experimental conditions. The response to SIN-1 was similar in both cell types (2.3- to 2.4-fold), whereas the response to SNAP was much greater in endothelial cells (3.7-fold) than in smooth muscle cells (52%; Fig. 6). Interestingly, basal cystine uptake was fourfold higher in smooth muscle cells than in endothelial cells.


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Fig. 6.   Effect of SNAP and SIN-1 on cystine uptake in endothelial cells (EC) and smooth muscle cells (SMC). Confluent monolayers of cells were treated with SNAP and SIN-1. After 19 h, cells were rinsed and incubated at 37°C with [35S]cystine [1 µg/60 µmol in 1 ml of Hanks' balanced salt solution that contained 10 mM HEPES (HHBSS)]. After 10 min, cells were rinsed 4 times with ice-cold HHBSS that contained 600 µM cystine and solubilized with 1% Triton X-100 in HHBSS. Radioactivity in cell lysates was determined by liquid scintillation counting. Cellular protein levels were determined in a parallel set of cells. Data represent means ± SE, n = 3. *Significantly different from control (P < 0.05).

To determine whether the increase in cystine uptake was due to induction of amino acid transport, endothelial and smooth muscle cells were incubated with cycloheximide (5 µg/ml) or actinomycin D (1 µg/ml) to inhibit de novo protein and mRNA synthesis, respectively, during treatment with SIN-1. Endothelial cells were incubated with 0.5 mM SIN-1 for 8 h and smooth muscle cells were incubated with 1 mM SIN-1 for 12 h. Different protocols were employed because the two cell types exhibited different sensitivities to cycloheximide and actinomycin D and different lag periods before the increase in cystine uptake occurred. Cycloheximide and actinomycin D fully blocked the increase in cystine uptake in both cell types in response to SIN-1 (Fig. 7).


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Fig. 7.   Effect of cycloheximide and actinomycin D on cystine uptake in endothelial cells (A) and smooth muscle cells (B) treated with SIN-1. Cells were incubated with 5 µg/ml CHX or 1 µg/ml ACT D in the presence and absence of SIN-1. After 8 h with 0.5 mM SIN-1 or 12 h with 1 mM SIN-1, cells were rinsed and incubated with [35S]cystine as described above. Data represent means ± SE, n = 3. *Significantly different from control (P < 0.05). **Significantly different from group treated with SIN-1 alone (P < 0.05).

A common pathway for cystine uptake, which is upregulated by oxidants, thiol reagents, and heavy metals is the xc- pathway (2, 7). This pathway is characterized by a sodium-independent counterexchange of cystine and glutamate. To determine whether the increase in cystine uptake observed in SIN-1-treated cells might be occurring by this mechanism, cystine uptake was measured in the presence or absence of extracellular glutamate after SIN-1 treatment. Glutamate (5 and 10 mM) completely blocked the SIN-1-mediated increase in cystine uptake observed in both cell types (Fig. 8). In addition, 10 mM glutamate blocked basal uptake by 82% in smooth muscle cells and by 38% in endothelial cells. These results indicate that that the exchange of cystine for glutamate accounts for the majority of basal cystine uptake in smooth muscle cells, a minority of basal cystine uptake in endothelial cells, and all of the increase in cystine uptake observed in both cell types.


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Fig. 8.   Effect of extracellular glutamate on cystine uptake in endothelial cells (A) and smooth muscle cells (B) treated with SIN-1. After 19 h with SIN-1, cells were rinsed and cystine uptake was determined as described above in the presence or absence of 5 or 10 mM glutamate. Data represent means ± SE, n = 3. *Significantly different from control incubated without glutamate (P < 0.05). **Significantly different from SIN-1-treated group incubated without glutamate (P < 0.05).

To provide further evidence for the involvement of the xc- pathway in the SIN-1-mediated increase in cystine uptake, we investigated the sodium dependence of the response. Endothelial and smooth muscle cells were incubated with 1 mM SIN-1 for 19 h, and cystine uptake was measured in the presence and absence of extracellular sodium. We found that the increase in cystine uptake after SIN-1 was independent of sodium status in both cell types (Fig. 9).


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Fig. 9.   Effect of extracellular sodium on cystine uptake in endothelial cells and smooth muscle cells treated with SIN-1. After 19 h with SIN-1, cells were rinsed with sodium-free buffer, and cystine uptake was measured in sodium-free buffer by the method described above. Data represent means ± SE, n = 3. *Significantly different from group incubated in the presence of sodium (P < 0.05).

Because both the increase in cystine uptake and the increase in cellular GSH appeared to be induced in SIN-1-treated cells, it seemed likely that increases would occur over similar time frames. The next experiments examined the time course of the two responses in both cell types. When endothelial cells were incubated with 0.5 mM SIN-1 for 1-24 h, GSH levels were decreased by 41% at 2 h and increased 6.9-fold at 16 h (Fig. 10). Cystine uptake was increased 4.4-fold by 9 h and remained elevated at 23 h. The increase in cystine uptake was first observed at 6 h, whereas the increase in GSH was first observed at 9 h. When smooth muscle cells were similarly treated, GSH levels were decreased by 22% at 6 h and increased 2-fold at 9 h and 2.2-fold at 24 h (Fig. 11). Cystine uptake was increased by 49% at 9 h and remained elevated at 23 h. No decrease in cellular viability was observed in endothelial or smooth muscle cells treated with 0.5 mM SIN-1 for 24 h (data not shown). In fact, at the completion of the study, cell protein levels were similar in control and SIN-1-treated endothelial cells and higher in SIN-1-treated smooth muscle cells compared with controls.


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Fig. 10.   Changes in GSH levels (A) and cystine uptake (B) over time in SIN-1-treated endothelial cells. Cells were treated with 0.5 mM SIN-1 for 1-23 h. At the appropriate times, cell extracts were prepared for the determination of GSH, and protein and cystine uptake was measured in intact cells as previously described. Data represent means ± SE, n = 3.



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Fig. 11.   Changes in GSH levels (A) and cystine uptake (B) over time in SIN-1-treated smooth muscle cells. Cells were treated with 0.5 mM SIN-1 for 3-23 h. At the appropriate times, cell extracts were prepared for the determination of GSH, and protein and cystine uptake was measured in intact cells as previously described. Data represent means ± SE, n = 3.

It should be noted that the increase in cystine uptake occurred several hours later in smooth muscle cells than in endothelial cells. However, in both cell types, increased cystine uptake preceded by a few hours or occurred concomitantly with increased cellular GSH levels. These results demonstrate a close correlation in time for the two responses and suggest a causal relationship.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our results demonstrate for the first time that vascular cells respond to ONOO- by increasing cellular GSH levels. Treatment of endothelial and smooth muscle cells with 0.05-2.5 mM SIN-1 for 16-18 h resulted in 1.9- to 6-fold increases in cellular GSH. In endothelial cells, the magnitude of the response was similar to that seen with SNAP. In smooth muscle cells, the elevation in GSH was greater in response to SIN-1 than to SNAP. Reasons for the smaller response to SNAP than to SIN-1 in smooth muscle cells are unknown but are possibly related to differences in metabolism of S-nitrosothiols and ONOO-. Elevations in GSH have been reported in endothelial cells 8-24 h after treatment with diethyl maleate, hyperoxia, cadmium, arsenite, N,N'-bis(2-chloroethyl)-N-nitrosourea, disulfiram, and pyrrolidine dithiocarbamate (6, 8, 9, 26, 39). In smooth muscle cells, elevations in GSH were found 24 h after treatment with SNAP, sodium nitroprusside, isosorbide dinitrate, and diethyl maleate (27, 33, 41). In the present investigation, GSH levels began to increase 6-12 h after treatment. This response was blocked by inhibitors of protein and RNA synthesis.

Because S-nitrosothiols and SIN-1 both increased cellular GSH levels, we considered the possibility that SIN-1 was acting as a ·NO donor rather than a ONOO- donor under the conditions of our study. It is known that SIN-1 generates ONOO- in aqueous solutions from the ·NO and O2-· produced during decomposition (16) and that in MEM, ·NO is produced from 1 mM SIN-1 in the presence of SOD (15). Recent reports have shown that SIN-1 can also act as a ·NO donor in the presence of one-electron oxidizing agents that can be found in plasma (37). Our incubations with SIN-1 were, in fact, carried out in the presence of 1% FBS. If SIN-1 was acting as a ·NO donor by this mechanism in our studies, one would expect that the addition of O2-· scavengers would have a minimal or enhancing effect on the response. We found that Cu,Zn SOD inhibited a large part of the SIN-1 response. These results indicate that SIN-1 was not primarily acting as a ·NO donor in our studies and that ONOO- mediated at least part of the response to SIN-1. Incomplete inhibition of the response by Cu,Zn SOD was not surprising, given the greater efficiency of ·NO compared with SOD for scavenging O2-· (37).

Results using the other O2-· scavenger, MnTMPyP, are less clear-cut but also provide evidence that ONOO- plays a major role in mediating the response to SIN-1. Recent reports demonstrate that MnTMPyP scavenges ONOO- in addition to O2-· (12). Furthermore, in the presence of a low-molecular-weight reductant, MnTMPyP also scavenges ·NO (12, 32). In our experiment, most of the SIN-1 response was inhibited by MnTMPyP, suggesting that either MnTMPyP efficiently scavenged O2-·, resulting in little or no ONOO- production, or that MnTMPyP efficiently scavenged the ONOO- generated by SIN-1. A role for MnTMPyP in scavenging ·NO generated in the culture media seemed unlikely, given the low concentrations of low-molecular-weight reductants present. Furthermore, a role for MnTMPyP in scavenging intracellular ·NO in the presence of high concentrations of low-molecular-weight reductants seemed unlikely because it is not clear that MnTMPyP enters cells (32). Recent studies have also demonstrated that ONOO- reacts rapidly with CO2 in bicarbonate-CO2-buffered media (10, 13), as was used in our experiments. This results in the production of nitrosoperoxycarbonate anion (ONOOCO2-), which decomposes to form the highly reactive carbonate radical (CO3-) (3). MnTMPyP effectively competes with CO2 for reaction with ONOO- and also scavenges ONOOCO2- (12). In our studies, the nearly complete inhibition of the SIN-1 response by MnTMPyP indicates that ONOO- or products of its reaction with CO2 played a major role in the GSH response.

Although ONOO- can oxidize proteins, carbohydrates, lipids, and DNA (10, 34, 35) and can be scavenged by GSH, ascorbate, and uric acid (12), the biological effects of ONOO- more likely depend on its reaction with CO2 to form ONOOCO2- and CO3- (3). These reactants mediate one-electron oxidations and nitrations (3, 10, 13) and can cause the formation of thiyl radicals, albumin disulfide dimers, and 3-nitrotyrosine-containing proteins in plasma (30, 36). The cellular targets of ONOO- in the present study have not been identified. However, SIN-1 decreased cellular GSH levels in both endothelial and smooth muscle cells 1-4 h after treatment.

Our studies show that ONOO- stimulated an increase in cystine uptake, apparently by inducing the xc- transport pathway. Treatment of endothelial and smooth muscle cells with 1 mM SIN-1 led to a 2.5-fold increase in cystine uptake after 12 h. SNAP treatment also increased cystine uptake in both cell types, although the response was much smaller in smooth muscle cells. The increased uptake in SIN-1-treated endothelial and smooth muscle cells exhibited characteristics of the xc- transport pathway, including sodium independence, inhibition by extracellular glutamate, and a requirement for new protein and RNA synthesis (2, 7). This is the first evidence for an inducible xc- transport pathway in smooth muscle cells. However, increased GSH and glutamate uptake in smooth muscle cells treated with diethyl maleate (33) and increased GSH and gamma -GCS in smooth muscle cells treated with SNAP (27) have previously been reported.

Interestingly, basal cystine uptake in smooth muscle cells was fourfold higher than in endothelial cells. In addition, the majority of basal cystine uptake was inhibited by high extracellular glutamate in smooth muscle cells but not in endothelial cells. These results suggest that the xc- pathway is much more active in smooth muscle cells in culture than in endothelial cells. A recent report demonstrated upregulation of the xc- pathway in hepatocytes during isolation and culture (23). Whether or not culture conditions have a similar effect on smooth muscle cells used in the current study is not known. Others have demonstrated cell density-dependent alterations in sodium-independent glutamate uptake, presumably by the xc- pathway, in endothelial cells (38).

Neither the molecular components of the xc- transport pathway nor the mechanisms by which it is upregulated have been defined. However, agents that induce the xc- pathway, such as pyrrolidine dithicarbamate, disulfiram, arsenite, cadmium, hyperoxia, N,N'-bis(2-chloroethyl)-N-nitrosourea, and SNAP, share common features as oxidants and/or thiol reagents (6, 8, 9, 26, 29, 39). These agents stimulate induction of the xc- pathway in endothelial cells, fibroblasts, and Chinese hamster ovary cells (6, 8, 21, 26) but not in alveolar epithelial cells (17). The significance of the xc- pathway of cystine uptake is that it provides a mechanism whereby cells can increase their supply of intracellular cysteine. Although the ASC pathway of cysteine uptake in cells is generally more active than the xc- pathway, it is not inducible (2, 7). Furthermore, cystine is more readily available in cell culture medium than cysteine, which is rapidly oxidized (2). After uptake of cystine, it is reduced to cysteine and made available for protein or GSH synthesis (2). Cellular GSH synthesis is regulated by negative feedback by GSH, the activity of the rate-limiting enzyme gamma -GCS, and the availability of precursor amino acids (7, 23). Because cysteine concentrations are often limiting, the xc- pathway can play an important role in enhancing cellular GSH levels (7, 23).

Our results also demonstrate that increases in GSH and cystine uptake follow a similar time course, suggesting that the availability of cysteine is rate limiting for GSH synthesis after SIN-1 treatment. Results of the current study do not rule out the possibility that gamma -GCS was induced in response to SIN-1 treatment and contributed to the elevations in GSH observed in endothelial and smooth muscle cells. Induction of gamma -GCS has been demonstrated in cytokine and hormone-treated hepatocytes (18, 23), tert-butylhydroquinone-treated lung epithelial cells (22), oxidized low-density lipoprotein-treated endothelial cells (5), and SNAP-treated smooth muscle cells (27). However, different patterns of induction of the heavy (catalytic) and light (regulatory) subunits of gamma -GCS were observed in both tert-butylhydroquinone-treated epithelial cells and SNAP-treated smooth muscle cells (22, 27). In addition, the increase in heavy subunit mRNA and immunoreactive protein preceded by several hours the increase in cellular GSH levels in the smooth muscle cell study (27). These results suggest that other factors regulating gamma -GCS activity, such as substrate availability, may play a role in elevating GSH levels. Furthermore, induction of gamma -GCS is not always required for increases in GSH levels to occur, as was demonstrated in arsenite-treated fibroblasts (29).

Results of the present study demonstrate a close correlation over time between the increases in cystine uptake and the increases in cellular GSH in endothelial and smooth muscle cells treated with SIN-1. We found a similar correlation in SNAP-treated endothelial cells (21). These results suggest that intracellular cysteine levels may be rate limiting for GSH synthesis and that increased cystine uptake by the xc- pathway may play a key role in elevating cellular GSH levels.

Our studies demonstrate that ONOO- stimulates adaptive responses in vascular cells. These results suggest that ONOO-, like ·NO, may protect cells against oxidative/nitrosative stress by increasing cellular GSH levels. This may be considered an adaptive response due to the many cytoprotective functions of this low-molecular-weight antioxidant. In fact, a GSH-dependent detoxification mechanism has recently been reported in vascular tissue exposed to ONOO- (24).

In the present study, it should be noted that GSH levels were decreased over the first few hours of exposure to ONOO- in both endothelial and smooth muscle cells. This suggests that cellular GSH may have reacted directly with ONOO- or ONOO--derived species such as ONOOCO2- and CO3- and may have been consumed in the process (36). The possibility that significant amounts of S-nitrosoglutathione were formed by the direct reaction of ONOO- with GSH seems unlikely, given the extremely low yield of this product in other models (1). Because the concentration of low-molecular-weight thiols in the media was very low, extracellular reactions of ONOO- or its byproducts with GSH was most likely to be negligible. However, one-electron oxidation reactions of ONOOCO2- or CO3- with protein thiols in the media or cells may have occurred and resulted in the formation of thiyl radicals, protein disulfides/mixed disulfides, and nitrothiols, which can release ·NO under certain circumstances (36). In addition, cellular GSH may have been consumed by glutaredoxin-catalyzed reduction of protein mixed disulfides formed subsequent to the reaction of protein thiols with ONOO- or its byproducts (31). Alternatively, GSH may have participated as a cofactor in GSH peroxidase-catalyzed repair of lipid peroxidation that resulted from ONOO- exposure (7, 23). In these ways, cellular GSH may have played a key role in maintaining cellular integrity and function after exposure to ONOO-. Consequently, the elevation in cellular GSH observed may represent an important adaptive response to chronic oxidative and/or nitrosative stress.


    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-61377 and HL-42444.


    FOOTNOTES

Address for reprint requests and other correspondence: B. J. Buckley, Box 3813, Duke Univ. Medical Center, Durham, NC 27710 (E-mail: barbara.buckley{at}duke.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 7 January 2000; accepted in final form 2 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Balazy, M, Kaminski PM, Mao K, Tan J, and Wolin MS. S-nitroglutathione, a product of the reaction between peroxynitrite and glutathione that generates nitric oxide. J Biol Chem 273: 32009-32015, 1998[Abstract/Free Full Text].

2.   Bannai, S. Exchange of cystine and glutamate across plasma membrane of human fibroblasts. J Biol Chem 261: 2256-2263, 1986[Abstract/Free Full Text].

3.   Bonini, MG, Radi R, Ferrer-Sueta G, Ferreira AM, and Augusto O. Direct EPR detection of the carbonate radical anion produced from peroxynitrite and carbon dioxide. J Biol Chem 274: 10802-10806, 1999[Abstract/Free Full Text].

4.   Buckley, BJ, and Whorton AR. Arachidonic acid stimulates protein tyrosine phosphorylation in vascular cells. Am J Physiol Cell Physiol 269: C1489-C1495, 1995[Abstract/Free Full Text].

5.   Cho, S, Hazama M, Urata Y, Goto S, Horiuchi S, Sumikawa K, and Kondo T. Protective role of glutathione synthesis in response to oxidized low density lipoprotein in human vascular endothelial cells. Free Radic Biol Med 26: 589-602, 1999[ISI][Medline].

6.   Deneke, SM, Baxter DF, Phelps DT, and Fanburg BL. Increase in endothelial cell glutathione and precursor amino acid uptake by diethyl maleate and hyperoxia. Am J Physiol Lung Cell Mol Physiol 257: L265-L271, 1989[Abstract/Free Full Text].

7.   Deneke, SM, and Fanburg BL. Regulation of cellular glutathione. Am J Physiol Lung Cell Mol Physiol 257: L163-L173, 1989[Abstract/Free Full Text].

8.   Deneke, SM, Harford PH, Lee KY, Deneke CF, Wright SW, and Jenkinson SG. Induction of cystine transport and other stress proteins by disulfiram: effects on glutathione levels in cultured cells. Am J Respir Cell Mol Biol 17: 227-234, 1997[Abstract/Free Full Text].

9.   Deneke, SM, Lawrence RA, and Jenkinson SG. Endothelial cell cystine uptake and glutathione increase with N,N-bis(2-chlorethyl)-N-nitrosourea exposure. Am J Physiol Lung Cell Mol Physiol 262: L301-L304, 1992[Abstract/Free Full Text].

10.   Denicola, A, Freeman BA, Trujillo M, and Radi R. Peroxynitrite reaction with carbon dioxide/bicarbonate: kinetics and influence on peroxynitrite-mediated oxidations. Arch Biochem Biophys 333: 49-58, 1996[ISI][Medline].

11.   Elliott, SJ. Peroxynitrite modulates receptor-activated Ca2+ signaling in vascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 270: L954-L961, 1996[Abstract/Free Full Text].

12.   Ferrer-Sueta, G, Batinic-Haberle I, Spasojevic I, Fridovich I, and Radi R. Catalytic scavenging of peroxynitrite by isomeric Mn(III) N-methylpyridylporphyrins in the presence of reductants. Chem Res Toxicol 12: 442-449, 1999[ISI][Medline].

13.   Gow, A, Duran D, Thom SR, and Ischiropoulos H. Carbon dioxide enhancement of peroxynitrite-mediated protein tyrosine nitration. Arch Biochem Biophys 333: 42-48, 1996[ISI][Medline].

14.   Gow, AJ, Thom SR, and Ischiropoulos H. Nitric oxide and peroxynitrite-mediated pulmonary cell death. Am J Physiol Lung Cell Mol Physiol 274: L112-L118, 1998[Abstract/Free Full Text].

15.   Haddad, IY, Zhu S, Crow J, Barefield E, Gadilhe T, and Matalan S. Inhibition of alveolar type II ATP and surfactant synthesis by nitric oxide. Am J Physiol Lung Cell Mol Physiol 270: L898-L906, 1996[Abstract/Free Full Text].

16.   Hogg, N, Darley-Usmar VM, Wilson MT, and Moncada S. Production of hydroxyl radicals from the simultaneous generation of superoxide and nitric oxide. Biochem J 281: 419-424, 1992[ISI][Medline].

17.   Jenkinson, SG, Lawrence RA, Zamora CA, and Deneke SM. Induction of intracellular glutathione in alveolar type II pneumocytes following BCNU exposure. Am J Physiol Lung Cell Mol Physiol 266: L125-L130, 1994[Abstract/Free Full Text].

18.   Kuo, PC, Abe KY, and Schroeder RA. Interleukin-1-induced nitric oxide production modulates glutathione synthesis in cultured rat hepatocytes. Am J Physiol Cell Physiol 271: C851-C862, 1996[Abstract/Free Full Text].

19.   Kuo, PC, and Slivka A. Nitric oxide decreases oxidant-mediated hepatocyte injury. J Surg Res 56: 594-600, 1994[ISI][Medline].

20.   Lander, HM, Ogiste JS, Teng KK, and Novogrodsky A. p21ras as a common signaling target of reactive free radicals and cellular redox stress. J Biol Chem 270: 21195-21198, 1995[Abstract/Free Full Text].

21.   Li, H, Marshall ZM, and Whorton AR. Stimulation of cystine uptake by nitric oxide: regulation of endothelial cell glutathione levels. Am J Physiol Cell Physiol 276: C803-C811, 1999[Abstract/Free Full Text].

22.   Liu, R, Hu H, Robison TW, and Forman HJ. Differential enhancement of gamma -glutamyl transpeptidase and gamma -glutamylcysteine synthetase by tert-butylhydroquinone in rat lung epithelial L2 cells. Am J Respir Cell Mol Biol 14: 186-191, 1996[Abstract].

23.   Lu, SC. Regulation of hepatic glutathione synthesis: current concepts and controversies. FASEB J 13: 1169-1183, 1999[Abstract/Free Full Text].

24.   Ma, XL, Lopez BL, Liu GL, Christopher TA, Gao F, Guo Y, Feuerstein GZ, Ruffolo RR, Barone FC, and Yue TL. Hypercholesterolemia impairs a detoxification mechanism against peroxynitrite and renders the vascular tissue more susceptible to oxidative injury. Circ Res 80: 894-901, 1997[Abstract/Free Full Text].

25.   Mayer, B, Schrammel A, Klatt P, Koesling D, and Schmidt K. Peroxynitrite-induced accumulation of cyclic GMP in endothelial cells and stimulation of purified soluble guanylyl cyclase. J Biol Chem 270: 17355-17360, 1995[Abstract/Free Full Text].

26.   Moellering, D, McAndrew J, Jo H, and Darley-Usmar VM. Effects of pyrrolidine dithiocarbamate on endothelial cells: protection against oxidative stress. Free Radic Biol Med 26: 1138-1145, 1999[ISI][Medline].

27.   Moellering, D, McAndrew J, Patel RP, Cornwell T, Lincoln T, Cao X, Messina JL, Forman HJ, Jo H, and Darley-Usmar VM. Nitric-oxide dependent induction of glutathione synthesis through increased expression of gamma -glutamylcysteine synthetase. Arch Biochem Biophys 358: 74-82, 1998[ISI][Medline].

28.   Moro, MA, Darley-Usmar VM, Goodwin DA, Read NG, Zamora-Pino R, Feelisch M, Radomski MW, and Moncada S. Paradoxical fate and biological action of peroxynitrite on human platelets. Proc Natl Acad Sci USA 91: 6702-6706, 1994[Abstract].

29.   Ochi, T. Arsenic compound-induced increases in glutathione levels in cultured Chinese hamster V79 cells and mechanisms associated with changes in gamma -glutamylcysteine synthetase activity, cystine uptake and utilization of cysteine. Arch Toxicol 71: 730-740, 1997[ISI][Medline].

30.   Ohshima, H, Celan I, Chazotte L, Pignatelli B, and Mower HF. Analysis of 3-nitrotyrosine in biological fluids and protein hydrolyzates by high-performance liquid chromatography using a postseparation, on-line reduction column and electrochemical detection: results with various nitrating agents. Nitric Oxide 3: 132-141, 1999[ISI][Medline].

31.   Padgett, CM, and Whorton AR. Cellular responses to nitric oxide: role of protein S-thiolation/dethiolation. Arch Biochem Biophys 358: 232-242, 1998[ISI][Medline].

32.   Pfeiffer, S, Schrammel A, Koesling D, Schmidt K, and Mayer B. Molecular actions of a Mn(III)porphyrin superoxide dismutase mimetic and peroxynitrite scavenger: reaction with nitric oxide and direct inhibition of NO synthase and soluble guanylyl cyclase. Mol Pharmacol 53: 795-800, 1998[Abstract/Free Full Text].

33.   Phelps, DT, Deneke SM, Baxter DF, and Fanburg BL. Erythrocytes fail to induce glutathione in response to diethyl maleate or hyperoxia. Am J Physiol Lung Cell Mol Physiol 257: L272-L276, 1989[Abstract/Free Full Text].

34.   Radi, R, Beckman JS, Bush KM, and Freeman BA. Peroxynitrite oxidation of sulfhydryls: the cytotoxic potential of superoxide and nitric oxide. J Biol Chem 266: 4244-4250, 1991[Abstract/Free Full Text].

35.   Radi, R, Beckman JS, Bush KM, and Freeman BA. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 288: 481-487, 1991[ISI][Medline].

36.   Scorza, G, and Minetti M. One-electron oxidation pathway of thiols by peroxynitrite in biological fluids: bicarbonate and ascorbate promote the formation of albumin disulfide dimers in human blood plasma. Biochem J 329: 405-413, 1998[ISI][Medline].

37.   Singh, RJ, Hogg N, Joseph J, Konorev E, and Kalyanaraman B. The peroxynitrite generator SIN-1 becomes a nitric oxide donor in the presence of electron acceptors. Arch Biochem Biophys 361: 331-339, 1999[ISI][Medline].

38.   Steiger, V, Deneke SM, and Fanburg BL. Characterization of glutamic acid uptake by bovine pulmonary arterial endothelial cells. J Appl Physiol 63: 1961-1965, 1987[Abstract/Free Full Text].

39.   Susanto, I, Wright SE, Lawson RS, Williams CE, and Deneke SM. Metallothionein, glutathione and cystine transport in pulmonary artery endothelial cells and NIH/3T3 cells. Am J Physiol Lung Cell Mol Physiol 274: L296-L300, 1998[Abstract/Free Full Text].

40.   Tietze, F. Enzymic method for quantitative determination of nanogram amounts of total and oxidized glutathione. Anal Biochem 27: 502-522, 1969[ISI][Medline].

41.   White, AC, Maloney EK, Boustani MR, Hassoun PM, and Fanburg BL. Nitric oxide increases cellular glutathione levels in rat lung fibroblasts. Am J Respir Cell Mol Biol 13: 442-448, 1995[Abstract].

42.   Wu, M, Pritchard KA, Jr, Kaminski PM, Fayngersh RP, Hintze TH, and Wolin MS. Involvement of nitric oxide and nitrosothiols in relaxation of pulmonary arteries to peroxynitrite. Am J Physiol Heart Circ Physiol 266: H2108-H2113, 1994[Abstract/Free Full Text].


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