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
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ABSTRACT |
We and others recently
demonstrated increased glutathione levels, stimulated cystine uptake,
and induced
-glutamylcysteinyl synthase (
-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
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INTRODUCTION |
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
-glutamylcysteinyl synthase (
-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
-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 |
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.
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RESULTS |
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).
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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).
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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).
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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).
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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).
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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).
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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.
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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.
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DISCUSSION |
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
-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
-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
-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
-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
-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
-GCS activity, such as substrate
availability, may play a role in elevating GSH levels. Furthermore,
induction of
-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.
 |
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