Stimulation of cystine uptake by nitric oxide: regulation of
endothelial cell glutathione levels
Hongfei
Li,
Zermeena M.
Marshall, and
A. Richard
Whorton
Department of Pharmacology and Cancer Biology, Duke University
Medical Center, Durham, North Carolina 27710
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ABSTRACT |
Nitric oxide (NO)
is known to produce some of its biological activity through
modification of cellular thiols. Return of cellular thiols to their
basal state requires the activity of the GSH redox cycle, suggesting
important interactions between NO signaling and regulation of cellular
redox status. Because continuous exposure to NO may lead to adaptive
responses in cellular redox systems, we investigated the effects of NO
on cellular GSH levels in vascular endothelial cells. Acute exposure (1 h) of cells to >1 mM
S-nitroso-N-acetyl-penicillamine (SNAP) led to depletion of GSH. On the other hand, chronic exposure to
lower concentrations of SNAP (
1 mM) led to a progressive increase in
cytosolic GSH, reaching fourfold above basal by 16 h. The mechanism may
involve an increase in GSH biosynthesis through effects on biosynthetic
enzymes or through increased supply of cysteine, the limiting
substrate. In this regard, we report that chronic exposure to SNAP led
to a concentration-dependent increase in cystine uptake over a time
course similar to that seen for elevation of GSH. The effect of SNAP on
cystine uptake was inhibitable by either cycloheximide or actinomycin
D, suggesting a requirement for both RNA and protein synthesis.
Furthermore, uptake was Na+
independent and was blocked by extracellular glutamate. Extracellular glutamate also blocked SNAP-mediated elevation of cytosolic GSH. Finally, in a coculture model, NO produced by cytokine-pretreated RAW
264.7 cells increased both GSH levels and cystine uptake in naive
endothelial cells. These findings strongly suggest that NO leads to
adaptive induction of the
x
c amino acid
transport system, increased cystine uptake, and elevation of
intracellular GSH levels.
cysteine; amino acid transport; nitrosative stress; oxidative
stress
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INTRODUCTION |
GLUTATHIONE is a critical intracellular reductant that
functions in protecting cells from free radicals, reactive oxygen
species, and toxic substances. The intracellular concentration of GSH
is in the range of 1-10 mM in many cells and is maintained by
controls on its biosynthesis. Several mechanisms are important. For
example, the rate-limiting enzyme
-glutamylcysteine synthase is
regulated by GSH levels (18, 26) and by oxidants (20) such that
depletion of GSH or the presence of oxidants increases its activity.
GSH synthesis is also dependent on the availability of the amino acid precursors glutamate, glycine, and cysteine (7, 8, 10, 18). Because
glutamate and glycine occur at relatively high intracellular
concentrations, cysteine availability largely determines GSH synthesis.
Therefore, intracellular GSH levels are dependent on cysteine levels in
the extracellular space and thus on transport of cysteine or cystine
into cells.
A number of mechanisms are available for transport of cystine or
cysteine equivalents (cysteinylglycine) into cells. Cysteine can be
taken up directly via the
Na+-dependent amino acid transport
system called the ASC system (4, 14, 29). In many cells, this is a
major route for supplying intracellular cysteine to maintain GSH
levels. However, because cystine levels are generally higher than
cysteine levels in extracellular fluids (and in cell culture media),
mechanisms for cystine uptake are also crucial for GSH biosynthesis.
Cystine is taken up by an amino acid transport system designated
x
c, which is
Na+ independent, inducible, and
exhibits a high degree of specificity for cystine and glutamate (7, 14,
19, 27, 33). Once inside a cell, cystine is rapidly reduced to
cysteine. In some cells, intracellular cysteine may result from the
activity of
-glutamyl transpeptidase utilizing extracellular GSH and
cystine as substrates (33).
Oxidant stress imposed by reactive oxygen species is known to increase
GSH levels as part of an adaptive response (7, 8, 19, 27). Although
regulation of
-glutamylcysteine synthase activity is certainly
involved after acute exposure (18, 20, 26), oxidants also induce
cystine uptake in some cells. For example, exposure of endothelial
cells (7, 19), V79 cells (21), and macrophages (27) to agents that
cause oxidant stress [H2O2,
arsenite, diethyl maleate (DEM), hyperoxia, and cadmium] leads to
increased activity of the x
c but
not the ASC uptake system. Induction of the
x
c system occurs over 6-12 h
in endothelial cells (6) or V79 cells (21) exposed to arsenite, leading
to a two- to threefold increase in GSH levels by 16 h.
Nitric oxide (NO) is well known to activate guanylate cyclase. In
addition to this signaling role, NO is also known to interact with
cellular redox systems, especially thiol groups. Thus NO exposure or
synthesis may impose a nitrosative stress similar to that imposed by
oxygen-derived species. Whether or not nitrosative stress produces
adaptive responses in cells is not well studied. However, exposure of
cells to NO protects them from apoptosis through induction of heat
shock proteins (13) or caspase 3 (9), and NO is known to react with and
modify cellular responses to oxygen-derived species (35, 36).
Furthermore, others have reported increases in GSH in cells exposed to
NO for 2 h (17), and recently, in preliminary experiments, we have
observed that exogenous NO leads to accumulation of GSH over an
extended time course. Because oxidative stress from oxygen-derived
species promotes an increase in GSH biosynthesis by inducing cystine
transport, we speculated that the accumulation of GSH in cells exposed
to NO occurred via a similar mechanism. In the present study, we present data that show that NO leads to elevation of intracellular GSH
in vascular endothelial cells through a mechanism that involves induction of cystine uptake. This adaptive response is potentially important in protecting cells from exposure to NO synthesized endogenously or NO produced in an inflammatory setting.
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MATERIALS AND METHODS |
Materials.
Fetal bovine serum (FBS) was obtained from HyClone (Logan, UT). All
other cell culture reagents were from GIBCO (Grand Island, NY). Tissue
culture plasticware was obtained from NUNC (Fisher Scientific, Raleigh,
NC), and Costar coculture plates were from Corning (Alton, MA).
S-nitroso-N-acetyl-penicillamine
(SNAP) was from Research Biochemicals International (Natick, MA).
L-[35S]cystine
was obtained from DuPont NEN (Boston, MA).
L-Cystine, glutamic acid, GSSG,
glutathione reductase, DEM, NADPH, and
N-acetyl-penicillamine were purchased
from Sigma Chemical (St. Louis, MO).
S-nitrosoglutathione (GSNO) was
synthesized as previously described (23). Enhanced chemiluminescense (ECL) reagents were from Amersham Life Science (Arlington Heights, IL).
Cell culture.
Vascular endothelial cells were isolated and grown in culture by
established methods as previously described (25). Briefly, bovine
aortic segments were cleaned and opened, and the endothelium was
removed by scraping. Isolates were plated on collagen-coated tissue
culture plasticware. Cells were grown in DMEM containing 10% FBS, 100 U/ml penicillin G sodium, 100 µg/ml streptomycin sulfate, and 250 ng/ml amphotericin B (complete DMEM) and incubated at 37°C in 7.5%
CO2 in air. Cells were removed
from flasks with trypsin-EDTA and seeded at 25% of confluent density
into fresh flasks.
Cells from the mouse monocyte-macrophage cell line RAW 264.7 were grown
in DMEM containing 10% FBS and antibiotic-antimycotic (see above) and
incubated at 37°C in 7.5% CO2
in air. Cells were passaged by treatment with trypsin-EDTA.
Exposure to NO.
Confluent monolayers of endothelial cells were treated with SNAP in
complete serum-containing DMEM for various periods of time and washed
with Hanks' balanced salt solution containing 10 mM HEPES (HBSS-HEPES;
pH 7.4) before GSH measurements or measurement of cystine uptake. To
determine the effect of NO produced by one cell on cystine uptake in
neighboring endothelial cells, we employed a coculture model. In this
model, RAW 264.7 cells were grown on the upper surface of the
collagen-coated membrane of the transferable well insert of a six-well
coculture tissue culture plate in complete DMEM. After reaching
confluence, RAW 264.7 cells were induced to express the inducible
isoform of NO synthase (iNOS) by incubating cells with a combination of
10 µg/ml lipopolysaccharide, 100 U/ml interferon-
, 1 ng/ml tumor necrosis factor-
, and 100 U/ml interleukin-1
in DMEM
containing 1% FBS for 24 h. Meanwhile, endothelial cells were grown to
confluence on the bottom of collagen-coated wells in separate plates
using DMEM as described above. To start a coculture experiment, inserts
containing RAW 264.7 cells expressing iNOS were rinsed twice with fresh
DMEM containing 1% FBS and then placed in wells containing confluent
endothelial cells that had also been rinsed with fresh DMEM containing
1% FBS. Cells were coincubated for 24 h, after which endothelial
extracts were prepared for glutathione measurement or endothelial cells
were used to measure cystine uptake. Under these conditions, RAW 264.7 cells produced ~5.3 nmol
NO · ml
1 · h
1.
Uptake assay.
Cystine uptake was determined using
[35S]cystine (6, 7).
Confluent endothelial cell monolayers of cells treated with SNAP or
cells from the coculture model were rinsed twice with warm HBSS-HEPES
and incubated for 60 min in the same buffer in room air at 37°C.
After two more rinses with HBSS-HEPES, the cells were incubated in 1 ml
of buffer containing
L-[35S]cystine
(1 µCi/60 µM) in room air at 37°C for 10 min. After removal of
the buffer, cells were rinsed four times with ice-cold HBSS-HEPES
buffer containing 600 µM cystine. Cells were lysed in 1% Triton
X-100 in HBSS-HEPES, and cystine uptake was measured by determining the
amount of 35S in an aliquot of the
lysate using a liquid scintillation counter. With these procedures,
cystine uptake was linear for at least 20 min. In experiments in which
the Na+ dependence of cystine
uptake was determined, Li+ (as
LiCl) was substituted for
Na+.
Glutathione assay.
Cells were rinsed twice with HBSS-HEPES and scraped into 0.5 ml of
ice-cold 1 M perchloric acid containing 2 mM EDTA and extracted for 30 min on ice. After centrifugation at 2,000 g in a microcentrifuge, the acid
extract was neutralized with 4 M KOH containing 0.6 M MOPS, and samples
were centrifuged to remove the potassium perchlorate precipitate. Total
glutathione levels (GSH and GSSG) were determined spectrophotometrically using the glutathione reductase-linked 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) recycling
assay (34). The assay conditions were 100 mM
KPO4 (pH 7.0), 100 µM DTNB, 300 µM NADPH, and 0.2 U/ml glutathione reductase. Each assay was
individually calibrated using known concentrations of GSH. All data
were normalized per milligram of cellular protein. Protein
concentrations were determined using the Bradford dye binding technique
with BSA as a standard (3).
Immunoblot procedures.
After incubation of RAW 264.7 cells with cytokines, cells on inserts
were lysed with boiling sample buffer containing 63 mM Tris (pH 6.8),
10% glycerol, and 2% SDS. After sonication, an aliquot was removed
for protein determination, and
-mercaptoethanol was added to a final
concentration of 5%. Extracts were boiled for 5 min at 100°C, and
equivalent amounts of extract prepared from treated and untreated cells
were loaded onto 8% SDS-polyacrylamide gels and resolved by SDS-PAGE.
Proteins were transferred from the gel to a nitrocellulose membrane by
electrophoretic elution in a buffer containing 25 mM Tris (pH 8.3), 130 mM glycine, 0.1% SDS, and 20% methanol for 12 h. Nonspecific binding
sites on the membrane were blocked with 5% nonfat dry
milk in 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20 at room
temperature with agitation for 1 h. The blot was probed with
an affinity-purified polyclonal anti-iNOS antibody raised against a
21-kDa protein fragment from the COOH-terminal region of mouse
macrophage iNOS (Transduction Laboratories, Lexington,
KY). The antibody was diluted 1:100,000 in blocking buffer
and incubated with the membrane for 1 h at room temperature with
agitation. The blot was washed five times with 10 mM Tris (pH 7.5), 100 mM NaCl, and 0.1% Tween 20 and incubated with agitation for 1 h with
rabbit anti-mouse IgG conjugated to horseradish peroxidase, diluted
1:2,000 in blocking buffer. The blot was rinsed five times, exposed to
ECL reagents according to the manufacturer's instructions, and exposed
to film.
Statistical analysis.
Group means were compared by one-way ANOVA and Student's
t-test.
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RESULTS |
In these studies, we examined the effects of nitrosative stress imposed
by incubation of vascular endothelial cells with SNAP. SNAP decomposes
slowly, presumably by homolytic decomposition to release NO. Metal
ions, especially copper ions, and visible light are known to catalyze
decomposition and may dramatically increase the rate of NO release
(28). The half-life is thus dependent on the conditions. We determined
the rate of decomposition of SNAP (measured as loss of the S-NO
absorbance at 335 nm) incubated with confluent endothelial cells in
DMEM containing 1% FBS under fluorescent light and found the half-life
to be 4 h. When vascular endothelial cells were exposed to SNAP, two
responses were seen. The nature of the response depended on the
concentration of SNAP and on the length of exposure. Acute exposure (1 h) to concentrations from 1 to 10 mM led to a fall in intracellular
glutathione (GSH and GSSG) concentrations (Fig.
1). The decline in GSH seen in response to a 1-h incubation with 5 mM SNAP was presumably the result
of ~0.8 µmol/ml NO released into solution (based on the half-life).
However, it is unlikely that the intracellular compartment is exposed
to all of the NO released, since NO produced in the medium may diffuse
into the atmosphere or may react with either O2 or metal ions both outside and
within the cell. Furthermore, because NO itself does not react
appreciably with cellular thiols, oxidation to more reactive species
such as NO+ is likely responsible
for the effects shown in Fig. 1. This acute response to NO exposure has
been previously reported by us and others (5, 11, 13, 25).

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Fig. 1.
Effect of
S-nitroso-N-acetyl-penicillamine
(SNAP) on intracellular GSH levels. Confluent monolayers of vascular
endothelial cells were incubated at 37°C with 0, 1, 5, or 10 mM
SNAP for 1 h in DMEM (1 ml total volume) containing 10% serum and 1%
antibiotics. Cells were rinsed and treated with perchloric acid to
precipitate protein, and acid extracts were used to measure
intracellular GSH + GSSG concentrations. Data are expressed as means ± SE in nmol GSH/mg protein and represent 3 similar independent
experiments with triplicate observations in each experiment.
* Significantly different from control,
P < 0.05.
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In contrast to these effects resulting from acute exposure to
millimolar concentrations, chronic exposure to lower concentrations of
SNAP (those that do not deplete GSH) led to a time-dependent elevation
of intracellular GSH (Fig. 2). The increase
in GSH was first evident at 6 h, continued to increase to about four
times the control levels, and remained elevated for 16-18 h. The
effect of SNAP on GSH levels was concentration dependent (Fig.
3), reaching a maximum at 1 mM. At higher
concentrations (5 mM), cells appeared to be damaged (some were
detached). Incubation with
N-acetyl-penicillamine did not produce
a similar increase in GSH. In fact, at higher concentrations
(
1 mM N-acetyl-penicillamine), GSH
levels were lower than those in untreated cells. Long-term
incubation with other nitrosothiols (GSNO) also led to a rise
in cytosolic GSH levels (Fig.
3B). In contrast to the effect of
SNAP, the effect of GSNO was still evident at 5 mM, perhaps due
to the longer half-life of GSNO (slower NO release) and lack of overt
signs of injury (no detachment).

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Fig. 2.
Time course for SNAP-induced elevation of GSH levels. Confluent
monolayers of vascular endothelial cells were treated with 1 mM SNAP in
DMEM (2 ml) containing 10% serum and 1% antibiotics at 37°C.
Incubations were stopped at times indicated, cells were rinsed and
treated with perchloric acid to precipitate protein, and acid extracts
were used to measure intracellular GSH + GSSG. Data are expressed as
means ± SE in nmol GSH/mg protein and represent 3 similar
independent experiments with triplicate observations in each
experiment.
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Fig. 3.
Effect of S-nitrosothiols on GSH
levels in endothelial cells. Confluent monolayers of vascular
endothelial cells were incubated in DMEM containing 1% fetal bovine
serum (FBS) and 1% antibiotics with various concentrations of either
SNAP or S-nitrosoglutathione (GSNO)
for 16 h at 37°C. Cells were then rinsed with ice-cold Hanks'
balanced salt solution-10 mM HEPES (HBSS-HEPES; pH 7.4) and treated
with perchloric acid, and acid extracts were used to measure
intracellular GSH + GSSG. A:
incubation with SNAP or
N-acetyl-penicillamine (NAP).
B: incubation with GSNO or GSSG. Data
are expressed as means ± SE in nmol GSH/mg protein;
n = 4 measurements.
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Others have seen increases in intracellular GSH levels after exposing
cells to arsenite, DEM, or
H2O2
(6, 7, 19, 21). In each case, the rise in GSH was dependent on
induction of cystine transport. Untreated confluent monolayers of
bovine aortic endothelial cells transport cystine at a nearly linear
rate (180 pmol · 106
cells
1 · 10 min
1) for 20 min (Fig.
4A). As
reported by others, when cells were incubated for 24 h with the
thiol-modifying agent DEM (125 µM), the rate was significantly
increased (Fig. 4A). Similarly, when endothelial cells were incubated for 24 h with SNAP, we observed a
concentration-dependent increase in the rate of cystine uptake (Fig.
4B). Incubation with 1 mM SNAP
increased the rate of cystine uptake by approximately twofold. These
data are the first demonstration that NO or its redox derivatives
increase cystine uptake in endothelial cells.

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Fig. 4.
Stimulation of cystine uptake by SNAP. Confluent monolayers of
endothelial cells were treated with SNAP for 24 h in DMEM (2 ml)
containing 10% serum and 1% antibiotics at 37°C. To measure
cystine uptake, cells were rinsed and incubated in HBSS-HEPES
containing 1 µCi
[35S]cystine (60 µM)
for 10 min. After times indicated, cells were rinsed 4 times with
ice-cold HBSS-HEPES buffer containing 600 µM cystine and lysed in 1%
Triton X-100 in HBSS-HEPES, and cystine uptake was measured by
determining amount of 35S in an
aliquot of lysate using a liquid scintillation counter.
A: time course of cystine uptake in
control and with diethyl maleate (DEM; 125 µM). Data are expressed as
counts/min (cpm) of
[35S]cystine taken up
into cells per microgram of protein (means ± SE,
n = 6).
B: cystine uptake after 24-h
incubation of endothelial cells with SNAP at indicated concentrations.
Data are expressed as multiples of control value and represent 3 similar independent experiments with triplicate observations in each
experiment. Basal uptake was 71.17 ± 2.31 cpm · µg
protein 1 · 10 min 1 (mean ± SE), which
is equivalent to 370 pmol · 106
cells 1 · 10 min 1. * Significantly
different from control, P < 0.05.
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In subsequent experiments, we examined the mechanisms for increased
uptake and characterized the transport system. The increase in cystine
transport activity was observed as early as 4 h after exposure to 1 mM
SNAP and continued to be elevated for at least 24 h (Fig.
5). The mechanism for increased cystine
uptake appeared to require both RNA and protein synthesis, since either
actinomycin D or cycloheximide blocked the response to SNAP (Fig.
6). As such, the effect of SNAP appears to
be the induction of expression of a cystine transport system. The time
course for NO-mediated effects on cystine uptake is similar to that
seen for NO-stimulated increases in cellular GSH reported in Fig. 2 and
is consistent with a role for induced cystine uptake in the elevation
in GSH.

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Fig. 5.
Time course for SNAP-stimulated cystine uptake. Confluent monolayers of
endothelial cells were treated with 1 mM SNAP in DMEM (2 ml) containing
10% serum and 1% antibiotics at 37°C. After times indicated,
cells were rinsed twice with HBSS-HEPES and cystine uptake was
determined as described in Fig. 4. Data are expressed as cpm
[35S]cystine/µg
protein (means ± SE, n = 6).
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Fig. 6.
Effect of cycloheximide or actinomycin D on SNAP-stimulated cystine
uptake. Confluent endothelial cells were incubated in buffer containing
1 mM SNAP and either cycloheximide (CHX; 1 µg/ml) or actinomycin D
(AD; 2.5 µg/ml) for 8 h in DMEM containing 10% serum and 1%
antibiotics at 37°C. Cells were then rinsed as described in Fig. 4,
and cystine uptake was determined. Data are expressed as multiples of
control value (means ± SE, n = 6).
Control value for this experiment was (mean ± SE) 29.3 ± 0.5 cpm · µg
protein 1 · 10 min 1, which is equivalent
to ~173
pmol · 106
cells 1 · 10 min 1. * Significantly
different from control, P < 0.001. + Significantly different
from SNAP-treated group, P < 0.001.
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We further characterized SNAP-induced cystine uptake by examining the
Na+ dependence and effect of
glutamate. As shown in Fig. 7, substituting Li+ for
Na+ in the medium had little
effect on the extent of cystine uptake (compared with control without
Na+) induced following a 24-h
exposure to SNAP, suggesting that stimulated uptake is primarily
Na+ independent.
Na+-independent cystine uptake is
characteristic of the x
c system.
The cystine transport system is also known to have a high affinity for
glutamate and exchanges intracellular glutamate for extracellular
cystine. Addition of glutamate to the extracellular medium would be
expected to effectively block cystine transport. As shown in Fig.
8, this is indeed the case in endothelial
cells, since addition of 5 mM glutamate to the medium completely
blocked the increase in cystine transport induced by a 24-h incubation with 1 mM SNAP. Others have shown that
-glutamyl transpeptidase, which is also inhibited by glutamate and is
Na+ independent, participates in
cystine uptake in some cells (33). We have examined the potential role
of this enzyme by investigating the effects of acivicin, a specific
-glutamyl transpeptidase inhibitor, on SNAP-induced cystine uptake.
Acivicin (5 mM) did not significantly alter cystine uptake following
treatment of cells with SNAP (Fig. 9),
suggesting that
-glutamyl transpeptidase is not responsible
for the glutamate-sensitive cystine uptake we have measured.
Taken together, these data strongly suggest that NO increases cystine
uptake by inducing some critical component of the
x
c transport system.

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Fig. 7.
Effect of Na+ on SNAP-induced
cystine uptake. Confluent endothelial cells were treated with 1 mM SNAP
in DMEM containing 10% serum and 1% antibiotics at 37°C as
described in Fig. 4. After 24 h, cells were rinsed and cystine uptake
was measured in Na+-complete
buffer or buffer in which Li+
replaced Na+. Data are expressed
as cpm
[35S]cystine/µg
protein (means ± SE, n = 6).
* Significantly different from paired control (Cont),
P < 0.05. + Significantly different
from Na+-free control,
P < 0.05.
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Fig. 8.
Effect of glutamate (GLU) on SNAP-induced cystine uptake. Confluent
endothelial cells were treated with SNAP (1 mM) in DMEM containing 10%
serum and 1% antibiotics at 37°C as described in Fig. 4. Cystine
uptake was determined in presence or absence of glutamate (5 mM) in
uptake assay buffer. Data are expressed as multiples of control (means ± SE, n = 6).
* Significantly different from control,
P < 0.001. + Significantly different
from SNAP-treated group, P < 0.001.
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Fig. 9.
Effect of acivicin on SNAP-induced cystine uptake. Confluent
endothelial cells were treated with SNAP (1 mM) in DMEM containing 10%
serum and 1% antibiotics at 37°C as described in Fig. 4. Cystine
uptake was determined in presence or absence of acivicin (5 mM) added
to uptake assay buffer. Data are expressed as multiples of control
(means ± SE, n = 3).
* Significantly different from control,
P < 0.001.
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As shown above, exposure to low levels of SNAP (
1 mM) increased GSH
levels over a time course similar to that seen for induction of the
cystine transport system. This suggests that increased cystine uptake
participates in increased intracellular GSH levels. In the following
experiments, we explored this possibility using glutamate in the medium
bathing cells to block the newly expressed x
c uptake system. As shown in Fig.
10, addition of 5 mM glutamate to the
medium completely prevented the SNAP-induced increase in intracellular
GSH, providing strong evidence that cystine uptake played at least a
partial role in elevation of GSH levels.

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Fig. 10.
Effects of glutamate on increase in intracellular GSH stimulated by
SNAP. Confluent endothelial cells were treated with 1 mM SNAP, 1 mM
SNAP + 5 mM glutamate, or 5 mM glutamate alone for 17 h in DMEM
containing 10% serum and 1% antibiotics at 37°C. Data are
expressed as means ± SE in nmol GSH/mg protein
(n = 6). * Significantly
different from control, P < 0.05. + Significantly different
from SNAP-treated group, P < 0.05.
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Finally, we determined whether NO produced by one cell type could
affect GSH levels and cystine uptake in a neighboring cell. To examine
this, we used RAW 264.7 rat monocytes grown on the membrane insert of a
Transwell apparatus. These cells were pretreated with a cytokine
mixture to induce iNOS. We have shown in other studies that the
combination of endotoxin and interferon-
used in these studies leads
to expression of iNOS within 4 h. Levels of protein expression reached
maximal levels by ~24 h and remained elevated for at least 48 h (Fig.
11A).
RAW 264.7 cells treated in this manner synthesized NO at a linear rate
(5.3 nmol · ml
1 · h
1 · insert
1).
The inserts were rinsed and then placed in plates in which endothelial
cells had been grown to confluence on the bottom of the well. The
distance separating the two cell types was ~1 mm in the coculture
apparatus, necessitating diffusion of NO (or some NO-related product)
produced by the RAW 264.7 cells over this distance to affect the
endothelial cell monolayer. As shown in Fig.
11B, RAW 264.7 cells producing NO
increased GSH levels in the endothelial cell monolayer and induced
cystine uptake in these cells (Fig.
11C). Addition of aminoguanidine to
inhibit iNOS during the coincubation of RAW 264.7 cells with
endothelial cells prevented both the increase in GSH levels and
induction of cystine uptake (Fig. 11,
B and
C). Aminoguanidine has no effect on
SNAP-induced cystine uptake (data not shown). These data suggest that
NO produced endogenously by one cell type may induce cystine transport
and elevate GSH levels in neighboring cells.

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Fig. 11.
Effect of NO production by RAW 264.7 cells on endothelial cell (EC)
cystine uptake and intracellular GSH content.
A: RAW 264.7 cells were preincubated
for times indicated in DMEM containing 1% FBS and 1% antibiotics in
presence or absence of 10 µg/ml lipopolysaccharide, 100 U/ml
interferon- , 1 ng/ml tumor necrosis factor- , and 100 U/ml
interleukin-1. Cells were rinsed with HBSS-HEPES and scraped into
sample buffer as described. After resolution of proteins by SDS-PAGE
and transfer to nitrocellulose, inducible NO synthase (iNOS) was
visualized using a specific iNOS antibody.
B and
C: confluent EC were coincubated for
20 h in DMEM containing 10% serum and 1% antibiotics with RAW 264.7 cells that had been induced to express iNOS. EC were rinsed, and either
extracts were prepared as described in Fig. 2 and used to determine
GSH + GSSG levels (B) or,
in separate experiments, EC were used to determine cystine uptake as
described in Fig. 4 (C). In some
experiments, 4 mM aminoguanidine (AG) was added to DMEM during
coincubation to inhibit iNOS in RAW 264.7 cells. Data are expressed as
means ± SE for triplicate determinations. * Significantly
different from control, P < 0.05.
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DISCUSSION |
NO is thought to regulate cellular function by interacting with
critical thiols, leading to formation of both protein and small-molecular-weight nitrosothiols (1, 12, 30, 31). This mechanism
has been proposed to be involved in NO-mediated activation of
p21ras (15) and
Ca+-dependent
K+ channels (2), inactivation of
caspases (9, 22), and regulation of ryanodine receptor activity (32,
37). In addition, acute exposure of cells to NO is associated with a
transient decline in cellular GSH levels (5, 11, 25), presumably due to
conversion of GSH to GSNO (5). It should be pointed out that these
effects are not mediated by NO directly, since NO is a weak oxidant (1, 31). Rather, other redox forms, especially
NO+ or species with
NO+-like activity, produced
through reaction of NO with transition metal ions, heme iron, or
molecular oxygen are proposed to be intermediates (1, 31). These
reactive NO-derived species are potent oxidants and pose a nitrosative
stress to cells by compromising thiol redox status. In this regard, the
GSH redox cycle has been shown to be an important protective mechanism
that rapidly reverses NO-mediated protein modification and GSH
depletion. For example, we have shown that the GSH redox cycle is
required for reversal of NO-mediated glyceraldehyde-3-phosphate
dehydrogenase inhibition in intact cells (23, 24) and in the recovery
of GSH levels in cells exposed to exogenous sources of NO (25). In
fact, both glutathione reductase and GSH levels are critical and
necessary for restoring the native reduced protein thiol (24, 25).
Through this mechanism, the GSH redox cycle is likely important in
regulating cellular signaling mechanisms used by NO and provides a
mechanism for restoring the basal state after proteins are modified (25). Similarly, the GSH redox cycle protects cells from oxidative stress imposed by oxygen-derived species. Chronic exposure of cells to
oxidants leads to an array of adaptive responses. Given the importance
of the GSH redox cycle, it is not surprising that one important
component of the adaptive response is elevation of intracellular GSH
levels. In many cells, this has been shown to occur through a mechanism
that involves induction of cystine transport (7, 14, 27, 33). Because
it is possible that cells respond to nitrosative and oxidative stress
in similar ways, we have examined the effects of NO on cellular GSH
levels and shown for the first time that NO, like reactive oxygen
species, leads to an adaptive increase in GSH though a mechanism that, at least in part, requires induction of cystine transport.
Although acute exposure to NO leads to GSH depletion,
chronic exposure leads to elevation of cellular GSH. In the present study, we show that 1 h of incubation with 5 mM SNAP leads to a 40%
fall in GSH. Similar reductions in GSH levels have been reported by us
and others in cells exposed to a variety of NO donors, including GSNO
and spermine NO (25). In one study, it was suggested that the rapid
fall in GSH in cells exposed to NO resulted from conversion of GSH to
GSNO intracellularly (5). In this report, GSNO was identified as the
NaBH4-reducible component of
cellular extracts. In recent work by us (25), we report that GSNO and
spermine deplete GSH in endothelial cells by promoting rapid formation
of glutathionyl protein mixed disulfides. Because protein mixed
disulfides are rapidly reduced by
NaBH4 to release free GSH, it
seems likely that the presence of these mixed disulfides can account
for GSNO identified previously by others (5) and is the likely
explanation for the fall in GSH reported in the present study.
In early experiments, we had observed that, after the initial decline
in GSH, levels recover and begin to exceed the concentrations present
in control cells. The extent of the initial decline is concentration
dependent such that low concentrations of NO donor (
1 mM SNAP in the
present study) lead to a rise in GSH without evidence of depletion. The
increase in GSH was significant by 6 h. Several mechanisms may be
involved in elevation of cellular GSH levels. These include increased
biosynthesis of GSH or inhibition of GSH export. We have not observed
any measurable GSH export from our endothelial cells and thus can rule
out this mechanism, leaving us with the possibility that GSH
biosynthesis is increased. GSH biosynthesis is dependent on the
activity of the rate-limiting enzyme,
-glutamylcysteine synthetase,
and the availability of cysteine (18, 26). The activity of
-glutamylcysteine synthetase is regulated by GSH, which competes
with glutamate at the glutamate binding site in the catalytic domain
and serves as a potent inhibitor of the enzyme. As levels of
intracellular GSH fall due to mixed disulfide formation, conjugation,
or export,
-glutamylcysteine synthetase is activated. GSH also
appears to maintain inhibition of the enzyme by keeping an allosteric
thiol reduced (20). However, because GSH depletion is not necessarily
seen in cells in which GSH levels rise and because GSH levels are above
control levels for an extended period, GSH depletion and subsequent
activation of
-glutamylcysteine synthetase seems to be an unlikely
explanation for increased biosynthesis. Direct effects of NO redox
species on
-glutamylcysteine synthetase have not been documented,
although NO appears to induce the expression of this enzyme in lung
epithelial cells (16). Possibly this ability to increase levels of
enzyme protein may explain the increase in GSH. However, data showing that the NO-induced increase in GSH is inhibitable by extracellular glutamate, a competitive inhibitor of cystine uptake, suggest that
induction of the x
c transport
system is quantitatively more important.
Supply of intracellular cysteine is known to limit GSH synthesis in
many cases (7, 8, 10, 18). Cysteine itself is transported by the ASC
amino acid transport system (4, 14, 29). This uptake mechanism is
Na+ dependent and has a high
capacity for cysteine transport (4, 14, 29). However, cysteine is
oxidized to cystine outside cells, and cystine levels are generally
higher than cysteine levels in the extracellular environment. Thus
mechanisms for cystine uptake are important in maintaining
intracellular GSH levels. A specific transport system designated
x
c, which is
Na+ independent and exchanges
extracellular cystine for intracellular glutamate, has been described
in a number of cells (7, 14, 19, 27, 33). Although the individual
components have not been identified, this uptake system has been
demonstrated to be activated by oxidant stress, sulfhydryl modifying
reagents, arsenite, and cadmium in vascular endothelial cells,
macrophages, and smooth muscle cells (7, 14, 19, 27, 33). The increase
in cystine uptake stimulated by NO requires RNA and protein synthesis,
becomes evident by ~6 h, and remains elevated for at least 24 h. This time course is consistent with the increase in GSH levels seen in cells
exposed to SNAP. Although it had been suggested that depletion of GSH
is involved in stimulation of cystine uptake (19), the effect we report
with NO occurs in the absence of a detectable fall in cellular GSH
levels. NO induction of cystine uptake appears to be mediated by the
x
c transport system, since it is
Na+ independent and inhibitable by
extracellular glutamate.
In some cells,
-glutamyl transpeptidase may also participate in
cysteine uptake. This protein is a cell surface enzyme that forms
-glutamyl amino acids (
-glutamylcysteine for example) and
transports them into the cell, where cysteine may be released by
peptidases. In studies with pancreatic duct cells (33), cystine uptake
was found to be mediated by both the
x
c transport system and
-glutamyl transpeptidase (which accounted for 40-50% of
cystine uptake). Uptake by this
-glutamyl transpeptidase is also
Na+ independent and inhibitable by
glutamate. However, because
-glutamyl transpeptidase is specifically
blocked by acivicin, this compound can be used to distinguish between
the two possibilities. In our studies, acivicin had no effect on
NO-induced cystine uptake, suggesting that in endothelial cells
induction of the x
c system is the
primary mechanism. Furthermore, as mentioned above, induction of
cystine uptake by NO fully explains the increase in cellular GSH levels
stimulated by NO, since extracellular glutamate completely prevented
this effect, and suggests the requirement of the
x
c system.
Induction of cystine uptake and elevation of intracellular GSH by NO
donors suggest that NO produced by one cell might alter GSH metabolism
in a neighboring cell. In our studies, NO produced by rat monocytes
increased GSH levels and cystine uptake in endothelial cells after 20 h
of coculture. This is important for several reasons. First, it suggests
that endogenous NO production can induce cystine uptake and elevate GSH
levels in cells. It also suggests that NO production by one cell can
mediate adaptive responses in neighboring cells. Thus, in a setting
where iNOS is induced and produces NO over an extended period, cells
adapt by increasing their GSH levels and ability to reverse NO-mediated
changes in cellular thiols (25). This is likely to be an important
mechanism whereby tissue cells resist the potentially damaging effects
of NO produced during inflammation.
 |
ACKNOWLEDGEMENTS |
This work was supported by National Heart, Lung, and Blood
Institute Grants HL-54149, HL-42444, and HL-51183.
 |
FOOTNOTES |
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. R. Whorton,
C138B Levine Science Research Center, Dept. of Pharmacology and Cancer
Biology, Duke University Medical Center, Durham, NC 27710 (E-mail:
awho{at}duke.edu).
Received 31 August 1998; accepted in final form 18 December 1998.
 |
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