Metallothionein, glutathione, and cystine transport in
pulmonary artery endothelial cells and NIH/3T3 cells
Irawan
Susanto1,2,
Shawn E.
Wright3,
Richard S.
Lawson1,
Charnae E.
Williams1, and
Susan M.
Deneke1
1 Division of Pulmonary
Diseases/Critical Care Medicine, Department of Medicine, The
University of Texas Health Science Center and
2 Audie L. Murphy Memorial
Veterans Hospital Division, South Texas Veterans Health Care System,
San Antonio, Texas 78284-7885; and
3 Pulmonary Medicine, St.
Joseph's Hospital and Medical Center, Phoenix, Arizona 85013-4223
 |
ABSTRACT |
Both glutathione
(
-glutamylcysteinylglycine; GSH) and the metalloprotein
metallothionein (MT) are composed of approximately one-third cysteine.
Both have antioxidant activity and are induced by oxidant stresses and
heavy metals. Intracellular cysteine levels may depend on uptake and
reduction of extracellular cystine. GSH synthesis can be limited by the
activity of the
cystine
transport system, which is induced by oxidants and other stresses. MT
is induced by treatments that also increase GSH levels and may compete
with GSH for intracellular cysteine. We investigated the induction of
MT and GSH and cystine transport in NIH/3T3 cells and bovine pulmonary
artery endothelial cells exposed to cadmium (Cd) or arsenite. Cd and
arsenite increased MT and GSH in both cells. Increases in MT and GSH
were accompanied by increases in cystine uptake. Inhibition of cystine
transport by glutamate decreased GSH levels and blocked Cd-induced GSH
increases in both cell types. MT levels were not significantly
affected, suggesting that MT synthesis is less sensitive to
intracellular cysteine levels than GSH synthesis.
oxidant stress; amino acid transport; antioxidants
 |
INTRODUCTION |
OXIDANT STRESS CAUSES cellular injury that may be
decreased if cellular antioxidant defenses are induced. Glutathione
(GSH) is an important component of antioxidant defenses in the lung and
other tissues. One of the factors regulating cellular GSH is the
availability of the cysteine substrate needed for synthesis (6, 12). In
the extracellular milieu, cystine (oxidized cysteine) is more abundant
and, therefore, is more readily available to the cells than cysteine
(3). In many cells, intracellular cysteine levels are regulated by the
rate of cystine uptake into the cells (3, 7). After transport into the
cell, cystine is reduced to cysteine (3).
Metallothionein (MT) is a metalloprotein that contains approximately
one-third cysteine residues and has been demonstrated in various
systems to have antioxidant activity (23, 24, 28). It has been
postulated that a primary role of MT is to limit the intracellular
concentration of heavy metals and thereby protect the cells from the
toxic effects of cadmium (Cd), copper, and mercury (28). This
metal-scavenging role of MT has been postulated to mediate the
induction of tumor resistance to anticancer drugs (17). MT has also
been found to be induced in a number of cell types and tissues in
response to many of the treatments that also increase cellular GSH
levels (23, 28). MT may thus potentially compete with GSH for cysteine
under conditions in which the availability of this amino acid is
limiting for GSH synthesis.
Cellular GSH levels have been correlated with changes in cystine
transport in isolated endothelial cells, smooth muscle cells, macrophages, fibroblasts, and brain cells (3, 4, 6, 7, 10-15,
19-22). Cystine transport in these cells occurs primarily through
an inducible transport system designated as
by Bannai (3). Intracellular
glutamate (Glu) acts as a countertransport agent, facilitating uptake
of extracellular cystine (5). This system, which is sodium independent
and inhibited by extracellular Glu or homocysteate, is inducible by a
variety of stresses including exposure to metals, sulfhydryl reagents,
and oxidants. Deneke and co-workers (10-14) have previously
reported that cystine transport in bovine pulmonary artery endothelial
cells (BPAEC) is primarily through the
system (15, 26).
In this study, we investigated the induction of MT as it relates to GSH
levels and cystine transport activities after exposure to either Cd or
the sulfhydryl reagent sodium arsenite in NIH/3T3 cells and BPAEC. We
hypothesized that induction of MT and GSH synthesis requires increased
cellular uptake of cystine and that blocking of cystine uptake could
interfere with the ability of the cell to respond to various stresses
by increasing either MT or GSH levels.
 |
MATERIALS AND METHODS |
Chemical reagents.
Dulbecco's phosphate-buffered saline (PBS), RPMI-1640, Dulbecco's
modified Eagle's medium (DMEM), Fungizone, penicillin-streptomycin, and trypsin-EDTA were purchased from Life Technologies
(GIBCO BRL, Grand Island, NY). Diethyl
maleate (DEM), disulfiram (DSF), sodium meta-arsenite (Ars),
L-cysteine,
L-cystine,
L-glutamic acid, fetal calf
serum (FCS), and substrates for the GSH assay were purchased from Sigma
(St. Louis, MO). Radiolabeled
[14C]cystine was
obtained from Amersham (Arlington Heights, IL). Lithium chloride was
obtained from Mallinckrodt (Paris, KY).
Cell cultures.
BPAEC were isolated by collagenase treatment of fresh pulmonary
arteries and characterized as previously described (26). Cells were
seeded at a density of 4-8 × 104/35-mm2
dish and grown to confluence (>1 × 106 cells/dish) in RPMI-1640
supplemented with 10% FCS, penicillin-streptomycin, and Fungizone as
previously described (26). Cells were grown in 21%
O2-5%
CO2-balance
N2 at 37°C. Media were changed
every 24-48 h before experimental exposures. NIH/3T3 cells
(American Type Culture Collection, CRL 1658) were maintained in DMEM
supplemented with 10% FCS, penicillin-streptomycin, and Fungizone.
Treatments.
Exposures to Cd, Ars, DSF, or DEM were begun after cells reached
confluence. Treatments lasted 16-24 h, at which time the inducing
agents were rinsed off and MT, GSH, or cystine uptake experiments were
begun. Glu, used to inhibit cystine uptake, was added at the same time
as Cd.
Cell counts.
Cell counts were performed on the same plates as those used for GSH
measurements or on replicate plates for MT or cystine uptake assays
using a calibrated Coulter counter as described previously (11, 14).
Cell sizes were determined with a Coulter channelizer.
GSH assay.
For GSH measurements, cells were harvested with trypsin-EDTA. An
aliquot of the cells was treated with 10% perchloric acid, sonicated,
centrifuged, and immediately frozen for later GSH assay by the method
of Tietze (29) as described by Akerboom and Sies (1).
Amino acid uptake.
Amino acid uptake studies were performed as previously described (10).
[14C]cystine was used
for all uptake experiments. BPAEC or 3T3 cell monolayers were washed
four times with warm PBS containing 14 mM glucose and incubated for 60 min in PBS-glucose. After two more rinses, labeled
L-cystine was added to each dish
at a concentration of 1 mCi/ml. The total cystine concentration was
0.06 mM in PBS-glucose. Cells were incubated for 10 min, followed by
four rinses. The supernatant was aspirated, and the cells were
dissolved in 1 ml of 1% Triton X-100. A 0.5-ml aliquot was counted in
Ecolite (ICN; Costa Mesa, CA), using a
-counter.
Cellular MT determination.
Intracellular MT was measured in cell lysates using a modification of
109Cd-labeled hemoglobin binding
assay (16). Cells were scraped into 400 ml of
CdCl2 in
tris(hydroxymethyl)aminomethane (Tris) buffer and then subjected to
repeated freeze-thaw cycles and sonicated. After 10 min of
centrifugation, 25 ml of the supernatant were diluted to 200 ml using a
Tris-Cd solution, and an equal volume of
109CdCl2
was added. The sample was vortexed briefly and allowed to sit for 10 min before 100 ml of hemoglobin were added. The mixture was heated for
4 min and centrifuged, and the supernatant was removed. Hemoglobin
binding was repeated once, and then the supernatant was counted using a
-counter.
Calculations and statistics.
MT levels, GSH levels, and rates of cystine uptake were expressed per
106 cells for both cell types.
Significant differences between the various groups were determined for
paired samples by Student's t-test or
for multiple samples by analysis of variance with the post hoc
Scheffé's test for groups with significant differences.
 |
RESULTS |
Effects of Cd on cells.
BPAEC and 3T3 cells were exposed to 5 mM
CdCl2 for 24 h. MT and GSH levels
and rates of cystine uptake were assayed after the exposure and were
compared with untreated controls. The results are summarized in Table
1. In BPAEC, Cd exposure resulted in MT
levels that were 302 ± 60% of control levels and GSH levels that
were 239 ± 27% of control levels. Exposure of 3T3 cells to Cd
resulted in a similar percent increase in MT (353 ± 13% of control) but lower increases in GSH (137 ± 4.8% of control).
Cystine uptake was modestly induced in BPAEC to 145 ± 16% of control. Cystine uptake in 3T3 cells was also slightly induced.
Basal cystine transport activity in 3T3 cells was similar to cystine
uptake in control BPAEC (Table 2); however,
both basal MT and GSH levels were higher in the 3T3 cells. The cystine
transport systems in both cells were found to be predominantly sodium
independent (Table 2), characteristic of the
system.
Effects of Ars, DEM, and DSF on cells.
Exposure of the cells to low levels of thiol reactive agents including
Ars, DSF, or DEM for 6-24 h has previously been reported to induce
cystine transport activity and increase GSH levels in cells utilizing
the
cystine transport system (3, 6, 7). DEM at higher levels has been used as an effective agent to
deplete GSH in these cells, but, as previously shown, induction of
-mediated cystine transport
activity occurs at concentrations low enough that no measurable GSH
depletion occurs (3, 11). Both Ars and DEM have been shown to be very effective at inducing MT synthesis in vivo (2, 8). We wanted to
determine the relative effects of thiol reactive agents and Cd on MT
levels and GSH levels in our cells. In the experiments reported here,
exposure to 2.5 µM Ars increased MT levels in both 3T3
cells and BPAEC; however, Ars was not as effective as Cd at inducing MT
in either cell type (Table 3). Deneke (10)
has previously reported that 24 h of exposure of BPAEC to low levels of
Ars resulted in increases in both GSH levels and cystine transport in
these cells. The data in Table 3 confirm these previous observations. These increases were also seen in 3T3 cells exposed to Ars (Table 3).
In attempting to determine the effect of DEM on MT, GSH, and cystine
transport levels in 3T3 cells, we found that 3T3 cells were extremely
sensitive to DEM. DEM treatments at levels that induced cystine
transport in BPAEC completely killed the 3T3 cells. DSF, another
sulfhydryl reactive compound, has been reported to increase cystine
transport and GSH in BPAEC (13). DSF (25 µM) also caused significant
increases in intracellular MT (229 ± 73% of control) and cystine
transport (294 ± 6% of control) in 3T3 cells. Changes in GSH in
DSF-treated 3T3 cells were not significant (117 ± 25% of
control).
As we have noted, when 3T3 cells were exposed to DEM, the DEM was very
toxic at levels well below those that have previously been reported to
be nontoxic in BPAEC and type II cells (9, 11, 20). We have determined
that 3T3 cells are extremely susceptible to GSH depletion by DEM (Fig.
1). Essentially 100% of the total cell GSH
was depleted by exposure to 50 µM DEM for 4 h. In contrast, it has
previously been reported that treatment of BPAEC or type II lung
epithelial cells with 50 µM for 2-4 h depleted >10% of the
total cellular GSH (3, 11, 19). The fact that DEM depletes GSH primarily through a reaction catalyzed by glutathione transferase (12) suggests that the 3T3 cells have a significantly higher level of
transferase activity than BPAEC or other cells discussed in the
literature (2, 9, 15, 19, 22).

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Fig. 1.
Diethyl maleate (DEM) was added in increasing concentrations to NIH/3T3
cells, and intracellular levels of glutathione (GSH) were measured
after 4 h. Values are means ± SD for 4 replicate plates.
|
|
Effects of inhibition of cystine uptake on MT and
GSH levels.
Glu has been shown to be an effective competitive inhibitor of cystine
transport by the
system in a
number of cell types (7, 12). When 5 mM Glu was added to 3T3 cells, GSH
levels were decreased to <25% of control levels by 24 h (Fig. 2). Increases in GSH after exposure to Cd
were also prevented by the presence of Glu. In contrast, MT levels in
the control cells were not significantly blocked by Glu nor was the
induction of MT synthesis by Cd prevented. Similar effects were seen in BPAEC (Fig. 3). GSH levels were also
significantly depleted by exposure to Glu for 24 h, and Cd-induced
increases in GSH were substantially blocked by Glu. MT levels were not
significantly affected by Glu in either control or Cd-treated BPAEC.

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Fig. 2.
CdCl2 (5 µM) and/or
glutamic acid (pH 7.4; 5 mM) was added to 3T3 cells. After 24 h, cells
were harvested, and assays for metallothionein (MT) and GSH were
performed on each group. Values are means ± SD for 10 individual
plates from 2 separate experiments. Cd, cadmium. * Means were
less than values for equivalent groups without
glutamate.
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Fig. 3.
CdCl2 (5 µM) and/or
glutamic acid (pH 7.4; 5 mM) was added to bovine pulmonary artery
endothelial cells (BPAEC). After 24 h, cells were harvested, and assays
for MT and GSH were performed on each group. Values are means ± SE
for 10 individual plates from 2 separate experiments. * Means
were less than values for equivalent groups without glutamate.
|
|
 |
DISCUSSION |
We have shown that both GSH and MT levels can be increased in BPAEC and
3T3 cells after exposure to Cd and Ars. The magnitude of the increases
varies between the cell types. GSH level expressed as a percentage of
control level is increased significantly more in BPAEC than in 3T3
cells exposed to either Ars or Cd. Cystine uptake was also increased,
at least to some extent, in both cell types. Increases in cystine
uptake in BPAEC tended to be higher than in 3T3 cells, but the
differences were not statistically significant.
Our data (Table 2) show that 3T3 cells also have higher basal levels
(2- to 3-fold) of both MT and GSH per cell. We have noted, however,
that the 3T3 cells are somewhat larger than the BPAEC. We have
determined using cell size measurements obtained by our Coulter counter
channelizer that the 3T3 cells had an average volume of 1.95 × 10
3 compared with 1.16 × 10
3 fl/cell for
BPAEC. This could account for some, but not all, of the
difference in total GSH and MT expressed per cell.
It has previously been reported that
, the oxidant-inducible and
sodium-independent transport system for cystine, is common to a variety
of cells from various species (3-7, 10-15). In addition to
BPAEC, this system is found in murine peritoneal macrophages, human
diploid fibroblasts, bovine smooth muscle cells, and fibroblasts
isolated from rat lungs. Cystine uptake increases in these cells
occurred in parallel with GSH increases, indicating that increased
cystine uptake might be used to provide the necessary intracellular
cysteine for GSH synthesis when the cells were subjected to various
stresses.
The NIH/3T3 cells appear to have a cystine transport system that is
similar to the
system. The 3T3
cells responded qualitatively similarly to sulfhydryl active agents, as
did other cell types. One exception is the response of the cells to
DEM. The 3T3 cells were very sensitive to DEM. It was toxic to these
cells at levels well below those commonly used to deplete GSH in other
cell types. Depletion of GSH in 3T3 cells occurred at levels well below
those previously reported for fibroblasts and endothelial cells (see
Fig. 1; Refs. 3, 4, 7, 11, 20).
Both BPAEC and 3T3 cells appear to be dependent on cystine transport
for the synthesis of GSH in control cells as well as in cells exposed
to Cd (Figs. 2 and 3). Glu is unlikely to inhibit GSH synthesis
directly. Glu itself is a substrate for GSH synthesis and actually has
been reported to increase GSH synthesis in vitro by blocking feedback
inhibition of
-glutamylcysteine synthetase by GSH (18). Treatment of
3T3 cells with Glu depleted GSH to a greater extent than similar
treatment of BPAEC. This suggests various possibilities. For example,
GSH turnover in 3T3 cells may be more rapid than that in BPAEC; the
cystine transport system in 3T3 cells may be more sensitive to
competitive inhibition by Glu; or basal levels of cysteine may be lower
in 3T3 cells than in BPAEC, and thus GSH synthesis might be more
dependent on continuing cystine uptake.
In contrast, the synthesis of MT in either cell type was not
significantly inhibited by Glu in either Cd-treated or untreated cells.
The fact that MT and GSH presumably compete for the same cysteine pool
suggests that available intracellular cysteine is preferentially used
for formation of the cysteinyl-tRNA complex required for protein
synthesis and is only secondarily available for the non-tRNA-mediated
two-step enzymatic synthesis of GSH from Glu, cysteine, and glycine.
From the data in Tables 1 and 2, one can compare the total amounts of
cysteine in MT and GSH in control and Cd-treated cells in the two cell
types. (Calculations are based on the fact that cysteine is
approximately one-third of the total weight for both GSH and MT.) Total
cysteine in intracellular MT is ~5.2% of total cysteine in GSH in
control BPAEC and 6.5% of the total GSH cysteine in cells treated with
CdCl2. In 3T3 cells, however, MT
contained ~11.7% of the cysteine in GSH for control cells and up to
32% of the total amount of GSH cysteine after treatment with
CdCl2. Thus cysteine utilization
for MT synthesis is unlikely to interfere significantly with GSH
requirements for BPAEC but may significantly compete with GSH synthesis
for available cysteine in the 3T3 cells, particularly in cells in which
MT synthesis is induced by Cd or other agents.
The competition for available cysteine is demonstrated even more
strongly in Fig 2. When Cd stress is combined with inhibition of
cystine uptake in the 3T3 cells, the GSH levels are actually depleted,
whereas MT levels are increased. This results in the total cell
cysteine in MT being approximately twice the total cell cysteine in
GSH. This type of situation may have in vitro analogs. Generally, in
liver and other tissues, a much larger fraction of the cysteine pool is
incorporated into GSH than into MT. In livers of rats at birth,
however, the total amount of cysteine in MT is nearly equal to that in
GSH (27). Whether the low GSH levels in newborn animals reflect an
immaturity of the mechanisms by which cells utilize extracellular
cysteine sources or whether they reflect deficiencies in extracellular
cysteine and its precursors was not determined.
Reports from in vivo studies confirm that decreasing available cysteine
in adult animals can result in lower GSH levels but not in lower levels
of MT. Fasting has been reported to deplete liver GSH in rats up to
50% (25). In these studies, MT levels were not correspondingly reduced
but actually elevated, perhaps because of oxidant stress resulting from
inadequate GSH levels. Similar results have been reported for rats fed
a sulfur-amino acid-deficient soya-based diet (27). Our data confirm at
the cellular level that MT synthesis is less affected than GSH
synthesis by treatments that reduce intracellular cysteine
availability.
 |
FOOTNOTES |
Address for reprint requests: I. Susanto, Div. of Pulmonary
Diseases/Critical Care Medicine, Dept. of Medicine, The Univ. of Texas
Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio,
TX 78284-7885.
Received 18 July 1997; accepted in final form 13 November 1997.
 |
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