Characterization of multiple cysteine and cystine transporters
in rat alveolar type II cells
Roy G.
Knickelbein,
Tamas
Seres,
Gregory
Lam,
Richard B.
Johnston Jr., and
Joseph B.
Warshaw
Department of Pediatrics, Yale University School of Medicine, New
Haven, Connecticut 06520
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ABSTRACT |
Cysteine availability is rate limiting for the
synthesis of glutathione, an important antioxidant in the lung. We used
rat alveolar epithelial type II cells to study the mechanism of
cysteine and cystine uptake. Consistent with carrier-mediated
transport, each uptake process was saturable with Michaelis-Menten
kinetics and was inhibited at 4°C and by micromolar levels of amino
acids or analogs known to be substrates for a specific transporter. A
unique system XAG was found that
transports cysteine and cystine (as well as glutamate and aspartate,
the only substrates previously described for system
XAG). We also identified a
second Na+-dependent cysteine
transporter system, system ASC, and two
Na+-independent transporter
systems, system xc for cystine and
system L for cysteine. In the presence of glutathione at levels
measured in rat plasma and alveolar lining fluid, cystine was reduced
to cysteine and was transported on systems ASC and
XAG, doubling the transport rate.
Cysteinylglycine, released from glutathione at the cell surface by
-glutamyl transpeptidase, also stimulated uptake after reduction of
cystine. These findings suggest that, under physiological conditions,
cysteine and cystine transport is influenced by the extracellular redox
state.
glutamate; alveolar; oxidant stress; lung
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INTRODUCTION |
ALVEOLAR TYPE I PNEUMOCYTES are very susceptible to
hyperoxic damage and are replaced by rapidly proliferating type II
cells that then differentiate into type I cells (33). However,
continued hyperoxia results in impairment of type II cell proliferation and differentiation that contributes to hyperoxic lung injury. Even
under normoxic conditions in vivo, inhibition of the synthesis of the
antioxidant glutathione (GSH) results in reactive
O2 metabolite-induced injury to
alveolar type II cells, suggesting that continual synthesis is
necessary to maintain adequate cellular GSH levels.
The availability of cysteine is the rate-limiting step in GSH synthesis
(13). The lung is dependent on an extracellular supply of cysteine that
is found in low (<10 µM) concentrations in plasma (31) or can be
supplied at the alveolar (apical) surface through the action of the
ectoenzyme
-glutamyl transpeptidase (GGT) that cleaves the
-peptide linkage of GSH to generate glutamate and cysteinylglycine
(CysGly; see Ref. 27). It is speculated that the dipeptide can then be
hydrolyzed to cysteine and glycine and that these amino acids are then
transported into the cell (27). Cystine and cysteine uptake have been
observed in type II cells (9, 20). Bukowski et al. (9) demonstrated
that 5 mM homocysteine or serine inhibited cysteine uptake, consistent with the presence of a system ASC transport process, that cystine uptake was inhibited by 5 mM homocysteate, consistent with transport by
system xc, and that cystine uptake
was stimulated in the presence of
Na+ by an as yet undetermined
transport process.
When a substrate is carried by more than one transport process,
alterations in transport (such as may occur during stress) cannot be interpreted effectively unless each individual process has
been characterized. Therefore, we explored cysteine and cystine transport by type II alveolar epithelial cells isolated from rat lung.
We found four distinct transport processes that are capable of carrying
cysteine and/or cystine. An
Na+-dependent aspartate and
glutamate transporter (system
XAG) was found to carry cysteine
and cystine. We confirmed the existence of
Na+-dependent cysteine transport
on system ASC. We also found evidence for two
Na+-independent systems, system L
for cysteine uptake and system xc
for cystine uptake. Physiological concentrations of GSH or CysGly were
capable of reducing cystine to cysteine that was then transported into
the cell. The relative importance of each transporter system for
cysteine and cystine uptake may vary, depending on the extracellular
redox status and the presence of certain amino acids.
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METHODS |
Materials.
Adult rats were purchased from Camm Research (Wayne, NJ).
L-[35S]cystine
and
L-[3H]glutamic
acid were purchased from NEN (Boston, MA).
L-[35S]cysteine
was prepared by preincubating
L-[35S]cystine
with 1 mM GSH, CysGly, or dithioerythritol (DTE) for at least 15 min.
Either 1 mM GSH, CysGly, or DTE was also present in the
reaction mixture to prevent autooxidation of cysteine. Elastase was
obtained from Elastin Products (Owensville, MO). Rat immunoglobulin G
(IgG) and 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid
(BCH) were purchased from Calbiochem (La Jolla, CA). Dulbecco's modified Eagle's medium (DMEM) and Fungizone were obtained from GIBCO-BRL (Grand Island, NY). Fetal calf serum was purchased from Atlanta Biologicals (Norcross, GA). All other chemicals were obtained from Sigma Chemical (St. Louis, MO).
Isolation of type II cells.
Type II cells were isolated from the lungs of adult Sprague-Dawley rats
by utilizing a modification of the technique described by Dobbs et al.
(15). In brief, rats were anesthetized by injection of 120 mg/kg body
wt pentobarbital sodium intraperitoneally, and blood was cleared from
the lungs by perfusion through the heart. After lavage, the lungs were
filled with 1,125 units of elastase/lung and were incubated at 37°C
for 20 min. The released cells were collected from minced lung,
resuspended in DMEM, and plated on 75-mm petri dishes (1 dish/lung)
coated with 2.5 mg of IgG to remove lymphocytes and macrophages.
Nonadherent cells (1.0-1.5 × 106 cells/well) were then
incubated at 37°C in a humidified atmosphere of 90% air-10%
CO2 in 35-mm (6-well) tissue
culture plates. The incubation medium was 1.5 ml of DMEM containing
10% fetal bovine serum, 20 µg/ml streptomycin, 20 U/ml penicillin,
50 µg/ml gentamicin, and 1 µl/ml Fungizone. After incubation
overnight, the viability of attached cells (determined by trypan blue
exclusion) was 94.8 ± 1.7%. Greater than 95% of the cells were
found to be type II pneumocytes, as determined by a modification of the
Papanicolaou staining procedure (see Ref. 22).
Amino acid transport by type II cells.
Transport studies were performed in cells after overnight incubation in
six-well tissue culture plates. Immediately before transport, the
plates were removed from the CO2
incubator, and DMEM was replaced with
Na+-N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES) solution containing (in mM) 130 NaCl, 4.7 KCl, 2.5 CaCl2, 1.0 MgSO4, 1.2 KH2PO4,
20 HEPES, and 1.0 glucose (pH 7.4). For
Na+-free reactions, NaCl was
replaced with an equal concentration of choline chloride. Transport was
initiated by replacing the above solution with 1.0 ml of HEPES buffer
containing 0.14 µCi L-[3H]glutamate
or 0.9 µCi
L-[35S]cysteine
and cystine. Uptake was allowed to proceed at 37°C and then was
terminated by removing the media with suction and by washing four times
with ice-cold unlabeled HEPES buffer. Cells were lysed by adding 1 ml
of 0.1 N NaOH, 2%
Na2CO3,
and 0.02% sodium-potassium tartrate, and 3 h later, aliquots were
taken for protein analysis and determination of radioactivity using a
1214 Rackbeta liquid scintillation counter (LKB Wallac, Gaithersburg, MD).
Analysis of transport data.
When radiolabeled substrates were used to determine transport by cells
in culture, the resulting cell-associated radioactivity was a
combination of uptake, extracellular binding, and association with any
nonwashable extracellular fluid volume (ECFV). Therefore, control
experiments were performed at 4°C to inhibit transport. The
resulting radioactivity, consisting of extracellular binding and
trapping in the ECFV, was subtracted from uptake obtained at 37°C.
Addition of transport inhibitors to the reaction medium resulted in
accumulation of radioactivity similar to that seen by lowering the
temperature to 4°C.
Michaelis constants
(Km) were
calculated from the Lineweaver-Burk plot (see Ref. 32) of amino
acid uptake determined at five or more different concentrations. The
Ki, which is the
concentration of inhibitor that doubles the apparent
Km (or decreases
the affinity) of the substrate, was determined by using the Dixon plot
(see Ref. 32).
Bronchoalveolar lavage fluid.
With the utilization of animals anesthetized for type II cell
isolation, the trachea was cannulated, and the lungs were filled with
10 ml of Krebs-Ringer buffer. After 1 min, the fluid was collected, and
the cells were removed by centrifugation at 14,000 revolutions/min
(Eppendorf 5415 C centrifuge) for 30 s. The supernatant was flash
frozen in liquid N2 and was stored
at
70°C until use. To quantify the amount of epithelial
lining fluid collected by bronchoalveolar lavage (BAL), the level of
urea in the BAL fluid was determined and was compared with undiluted
levels found in the plasma with the use of a commercially available kit
(Sigma Chemical; see Ref. 30).
GSH and glutathione disulfide determination.
The GSH and glutathione disulfide (GSSG) content were determined using
an enzymatic recycling assay based on GSH reductase as described by
Griffith (17). GSSG levels were determined after derivatization of
reduced GSH by 2-vinylpyridine.
Statistical methods.
All data shown are the means ± SE from
n experiments, each performed with
type II pneumocytes prepared from a different set of animals. To
determine whether an experimental manipulation had a significant effect
on uptake, the data were compared by using analysis of variance, and
then a Dunnett's test was used for the comparison of the control mean
with each other group mean.
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RESULTS |
Na+-dependent cotransport of
cystine and glutamate.
Because cystine and glutamate have been shown to share an
Na+-independent transporter
(system xc) in various cell
types, we compared cystine and cysteine transport with that of
glutamate in an Na+-containing
environment. In isolated type II pneumocytes, uptake of all three amino
acids was found to be stimulated by
Na+ (Fig.
1). The uptake rate of cysteine was much
greater than that of cystine or glutamate in either the presence or the
absence of Na+ (Fig. 1). As shown
in Table 1, seven different transporter
systems were candidates for uptake of these amino acids, based on
previous reports with other cell types (11, 18). Each transporter
system is characterized by its unique substrate specificity, dependence (or lack of dependence) on an inwardly directed
Na+ gradient as a driving force,
and selective inhibition by amino acids or amino acid analogs. As
substrates, these inhibitors prevent uptake of radiolabeled amino acids
that compete for the same system.

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Fig. 1.
Time course of amino acid uptake by isolated type II alveolar
epithelial cells. Freshly isolated cells were added to 6-well tissue
plates (106 cells/well in DMEM)
and were incubated in a 10% CO2
incubator overnight. Uptake of
[35S]cysteine and
[35S]cystine or
[3H]glutamate was
studied at 37°C in either the presence (130 mM NaCl) or absence
(130 mM choline chloride) of Na+
in media containing 1 µM cystine
(A), 1 µM cysteine
(B), or 5 µM glutamate
(C). When cysteine uptake was
studied, 1 mM cysteinylglycine (CysGly) was added to reaction mixture
to prevent autooxidation. Each point represents mean ± SE of
measurements done in duplicate in each of 3 separate cell preparations.
In some cases, SE is less than the width of symbol.
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Table 1.
Transporter systems for cysteine, cystine, or glutamate previously
identified in cells other than type II cells
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The similar dose response for cystine and glutamate uptake inhibition
by aspartate (Fig.
2A) or
glutamate (Fig. 2B) suggested that
the same transport system was responsible for the uptake of both
cystine and glutamate. Because the
Na+ stimulation of cystine
transport in type II cells (Fig. 1) has not been reported in other
cells, we studied the inhibitory effect of amino acids (or analogs)
known to compete for transport on a specific cystine or cysteine
carrier (see Table 1). As shown in Fig. 3,
the inhibitor profile was identical for glutamate and cystine uptake.
-Methylaminoisobutyric acid (MeAIB; an inhibitor of system A),
serine (substrate for system ASC), and arginine (substrate for systems
B0+ and
b0+) had no effect. Quisqualate
caused a 22-29% inhibition (Fig. 3), which is consistent with
inhibition of Na+-independent
cystine-glutamate or glutamate-glutamate exchange via system
xc (Table 1). Only the system
XAG-specific inhibitor L-aspartate-
-hydroxamate
(A
H) effectively inhibited either cystine or glutamate transport
(Fig. 3). One millimolar CysGly, which would block uptake on the
dipeptide carrier, had no effect on glutamate transport but increased
cystine transport fourfold. This selectivity suggested that the
transporter itself was not being modified. With the use of oxidized
CysGly [(CysGly)2], no stimulation was seen (Fig. 4), suggesting
that enhanced uptake was secondary to reduction of cystine to cysteine.
This is supported by the fact that 1 mM of the thiol reducing agents
GSH,
N-acetyl-L-cysteine (NAC), and DTE had the same effect as 1 mM CysGly (Fig. 4). At the same
time, a physiologically relevant concentration of 30 µM CysGly or 60 µM GSH doubled cystine uptake (Fig. 4).

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Fig. 2.
Inhibition of glutamate and cystine uptake by aspartate or glutamate.
Two-minute uptake of 5 µM
[3H]glutamate or 1 µM [35S]cystine was
studied in the presence of varying concentrations of aspartate
(1-50 µM; A) and glutamate
(1-200 µM; B). Results are
expressed as percentage of control
Na+-dependent uptake (means ± SE of experiments done in duplicate in each of 3 separate cell
preparations).
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Fig. 3.
Effect of amino acids and amino acid analogs on 2-min uptake of 5 µM
[3H]glutamate or 1 µM [35S]cystine.
Inhibitor concentration was 1 mM except for quisqualate (200 µM) and
L-aspartate- -hydroxamate
(A H; 400 µM). MeAIB, -methylaminoisobutyric acid. Results
are expressed as percentage of control
Na+-dependent uptake (means ± SE of experiments done in duplicate in each of 3 separate
cell preparations). Significantly different from control
(P < 0.01).
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Fig. 4.
Two-minute uptake of 1 µM
[35S]cystine by type
II cells during exposure to various reducing agents.
(CysGly)2, cysteinylglycine
disulfide, oxidized form of CysGly; GSH, glutathione; NAC,
N-acetyl-L-cysteine;
Hcy, homocysteine; DTE, dithioerythritol. Data points represent means ± SE of duplicate measurements in each of 3 separate cell
preparations. Control is transport in the absence of reducing agents.
Significantly different from control:
* P < 0.05;
P < 0.01.
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Na+-dependent
transport of cysteine and cystine.
Na+-dependent cysteine transport
is known to occur via system ASC, which also carries alanine and serine
(18). If all Na+-dependent
cysteine transport occurs via system ASC, uptake should be totally
inhibited by 1 mM serine. With the use of 1 mM CysGly to
reduce cystine to cysteine, uptake was found to be only partially inhibited by serine (Fig. 5). Because A
H
blocked Na+-dependent cystine
uptake (Fig. 3), we investigated the effect of this inhibitor on
Na+-dependent cysteine uptake.
A
H (400 µM) also inhibited cysteine uptake by ~50%, and a
combination of serine and A
H completely inhibited
Na+-dependent cysteine uptake
(Fig. 5).

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Fig. 5.
Characterization of CysGly-stimulated
[35S]cysteine and
[35S]cystine uptake (2 min). CysGly (1 mM) was added to uptake media to reduce cystine to
cysteine, and uptake was compared in the presence of 400 µM A H, 1 mM Ser, or both. Data points represent means ± SE of duplicate
measurements in each of 3 separate cell preparations. Cyst(e)ine,
cysteine and cystine. Control is uptake in the absence of CysGly.
Significantly different from control:
* P < 0.05;
P < 0.01.
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To determine whether cysteine and cystine compete with aspartate and
glutamate for transport by system
XAG, the ability of each amino
acid to inhibit uptake was studied (Table
2).
Na+-dependent uptake of
radiolabeled cystine, glutamate, and cysteine were inhibited by low
micromolar concentrations of unlabeled glutamate or aspartate. Because
cysteine reduces
[35S]cystine to
cysteine, the Ki
for cysteine inhibition of cystine transport could not be accurately
determined. Although 500 µM cystine significantly inhibited uptake of
the other three amino acids (~50%), its low solubility prevented
Ki determination.
Transport on the putative XAG
transporter was further characterized by the determination of the
affinity for each amino acid found to be a substrate (Table
3). Two-minute
Na+-dependent uptakes were studied
at various external concentrations, and the
Km was determined
from a Lineweaver-Burk plot of the data. Because cysteine was found to
be transported by two distinct
Na+-dependent transport processes
(Fig. 5), the Km
for XAG was determined under
conditions that inhibited system ASC (i.e., addition of 1 mM serine to
the medium). Likewise, the
Km for cysteine
on system ASC was determined in experiments in which transport on
system XAG was inhibited with 400 µM A
H. Although cystine uptake also occurred via system
XAG, its affinity was very low.
Na+-independent
transport of cystine and glutamate.
In other cells, Na+-independent
transport of cystine and glutamate has been shown to occur on system
xc, a transporter that utilizes
the outwardly directed glutamate gradient to drive cystine into the
cell (2). To determine whether system
xc was responsible for the
Na+-independent uptake shown in
Fig. 1, a 5-min transport was determined with or without inhibitors
specific for system xc
(quisqualate) or system XAG
(A
H). Both Na+-independent
cystine and glutamate uptake were inhibited by excess external
unlabeled cystine or glutamate and by quisqualate but not by A
H
(Fig.
6A).
This demonstrated the specificity of A
H to inhibit glutamate and
cystine on system XAG and of
quisqualate to inhibit transport on system
xc.

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Fig. 6.
Effect of transport inhibitors on
Na+-independent uptake of 1 µM [35S]cystine,
5 µM [3H]glutamate,
or 1 µM
[35S]cysteine.
A: 5-min uptake of cystine or
glutamate was determined in media in which choline chloride was used in
place of NaCl in the presence or absence of 400 µM A H, 200 µM
quisqualate, 500 µM cystine, or 500 µM glutamate.
B: 2-min uptake of
[35S]cysteine was
studied in media where choline chloride was used in place of NaCl in
the presence or absence of 400 µM A H, 100 µM quisqualate, or 1 mM 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH).
CysGly (1 mM) was added to reaction mixture to prevent autooxidation of
cysteine. Data points represent means ± SE of duplicate
measurements in each of 3 separate cell preparations and are expressed
as percentage of control (i.e., transport in the absence of competing
amino acid or amino acid analog). Significantly different
from control (P < 0.01).
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Na+-independent
transport of cysteine.
Na+-independent transport of
cysteine was also observed in type II cells (Fig. 1). As shown in Fig.
6B, this transport was not influenced
by the inhibitor of
Na+-independent cystine transport
(quisqualate). A
H, which inhibits Na+-dependent cysteine transport,
caused a slight but significant inhibition of
Na+-independent cysteine
transport. This inhibition may represent the small cysteine uptake
occurring on system XAG even in
the absence of Na+. An
Na+-independent transporter that
carries a number of amino acids, including cysteine, has been described
in fibroblasts. This transporter, known as system L, is specifically
inhibited by BCH (18). As shown in Fig.
6B, type II cell transport of cysteine
in the absence of Na+ was
inhibited by BCH, indicating the participation of system L in
Na+-independent cysteine uptake.
Effect of GSH and GSSG on cysteine and cystine transport.
As shown in Fig. 4, physiological levels of GSH can enhance cystine
transport via the reduction to cysteine. To further investigate the
physiological relevance of this observation, we determined GSH and GSSG
levels in rat plasma and BAL fluid. The GSH and GSSG levels were found
to be 49.8 ± 7.5 and 4.5 ± 1.4 µM, respectively, in plasma
and 336 ± 3.2 and 135 ± 6.3 µM, respectively, in
BAL. Cystine transport was found to be increased 80 and 179% when
studied in the presence of the GSH and GSSG concentrations found in
plasma or BAL, respectively (Fig.
7A).
This stimulatory effect was secondary to the reduction of cystine to
cysteine by GSH (but not by GSSG). Na+-independent cystine uptake was
also stimulated (2-fold) by using concentrations of GSH and GSSG found
in plasma and BAL (Fig. 7B).

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Fig. 7.
Effect of physiological levels of GSH and glutathione disulfide (GSSG)
on cysteine and cystine uptake by type II cells. Uptake of
[35S]cystine (1 µM)
was determined in the presence of GSH and GSSG (GSH + GSSG), GSH, or
GSSG found in plasma (50 µM GSH and 5 µM GSSG) or bronchoalveolar
lavage (BAL) fluid (336 µM GSH and 135 µM GSSG).
A: 2-min cystine transport studied in
the presence of Na+.
B: 2-min
Na+-independent transport of
cystine determined when NaCl was replaced with choline chloride.
Results are from duplicate measurements in each of 3 separate cell
preparations and are expressed as percentage of control for both
Na+-containing and
Na+-free experiments.
Significantly different from control:
* P < 0.05;
P < 0.01.
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DISCUSSION |
To meet the specialized needs of each cell type, numerous amino acid
transporters have developed, each with its own substrate specificity,
inducibility, and cellular distribution (11). A number of these systems
are known to transport cystine or cysteine (Table 1). Analysis of the
transport processes involved in cysteine and/or cystine uptake
in rat type II alveolar epithelial cells is consistent with the
existence of the four distinct transport proteins shown in Fig.
8. They include two
Na+-dependent carriers (systems
XAG and ASC) and two
Na+-independent carriers (systems
L and xc). Others previously
demonstrated that system ASC, which is known to transport many
different amino acids, is present in lung type II cells (9, 19), but
cysteine transport by system ASC was not characterized. The
transporter systems utilized by amino acids or amino acid analogs
important for this study are shown in Fig. 8.

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Fig. 8.
Na+-dependent and -independent
transporters for cysteine and cystine in rat alveolar epithelial type
II cells. Letters in circles are the established names for each
transporter system. Presence of
Na+ indicates that transport on
this carrier is driven by the Na+
gradient. Amino acids and amino acid analogs shown at each transporter
are those used in this study and do not necessarily represent all
substrates for each system. Those compounds in italics and parentheses
were used as the specific inhibitor of the corresponding transporter
and had little or no effect on the other 3 carriers.
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After uptake,
[35S]cysteine and
[35S]cystine can be
utilized for protein or GSH synthesis. Although cellular metabolism of
cysteine will not directly affect the interpretation of transport data, the potential efflux of both labeled and unlabeled GSH could. Therefore, cysteine kinetics were determined in both control cells and
cells pretreated with 0.5 mM acivicin and 0.5 mM
L-buthionine-[S,R]-sulfoximine (BSO) to prevent the release of cysteine from GSH by inhibiting GGT and
GSH synthesis, respectively. Pretreatment with these inhibitors did not
significantly alter the
Km of cysteine
for either system ASC or system
XAG (data not shown). However,
maximal uptake rate was decreased, consistent with acivicin competing
for transport as previously described (7). Therefore, BSO and acivicin
were not used in cysteine and cystine transport experiments discussed in this manuscript. In other preliminary experiments, we found that
after loading type II cells with
[35S]GSH, only 6.8%
of the radiolabel had effluxed after 2 min. During 2-min uptake
studies, [35S]cysteine
and [35S]cystine must
enter the cell and GSH must be synthesized before efflux can occur.
Therefore, the error due to 35S
efflux would be much less than 6.8% and was not taken into
consideration in the calculations presented in this study.
In other types of cells, cysteine has been shown to be transported
dominantly by the Na+-dependent
transport system ASC (11, 18). Cystine transport, however, was believed
to occur exclusively in an
Na+-independent manner on either
system xc or system
b0+ in mammalian cells (2, 4, 5,
11). Because cysteine is readily oxidized to cystine, these
Na+-independent transporters were
found to be necessary for maintaining intracellular cysteine levels
required for GSH metabolism (4). Therefore, it was unexpected that,
besides transport of cysteine, the transport of cystine in type II
cells was also markedly stimulated by the presence of
Na+ (Fig. 1; see Ref. 9).
The transport of glutamate (another precursor of GSH) has been shown in
numerous cell types to occur in both
Na+-dependent and
Na+-independent manners (4,
11). Glutamate uptake has been observed in L2 cells (which are
clonally derived from alveolar epithelial cells), but
Na+-dependent and
Na+-independent transport was not
differentiated (28). As shown in Fig. 1,
Na+ stimulated the uptake of
glutamate in type II cells. In all tissues in which
Na+-dependent glutamate transport
has been described, it occurs on system
XAG, which has a high affinity and
specificity for glutamate and aspartate (11, 18). Bukowski et al. (9)
recently described Na+-dependent
cystine transport in type II cells that was inhibited to a similar
degree by 5 mM glutamate (53%) and aspartate (59%). Cysteine uptake
was also inhibited by 5 mM glutamate and aspartate (35 and 33%,
respectively). On the basis of these results, we hypothesized that
cysteine and cystine may be transported by a promiscuous system
XAG-like transport process. Using
kinetic analysis, we showed the existence of such a transport process,
which was inhibitable by A
H (Figs. 3 and 5) and had a high affinity
for glutamate, aspartate, and cysteine and a low affinity for cystine (Tables 2 and 3). This putative system XAGlike
transport process on type II cells, in contrast to system
XAG defined in all other tissues,
can carry cysteine and cystine (Fig. 8). Recently, Dowd et al. (16)
injected rat brain cRNA encoding one of the system XAG transporter isoforms
(excitatory amino acid carrier) into Xenopus oocytes. The resulting
Na+-dependent glutamate transport
was inhibited by L-aspartate
(Ki = 2 µM)
and, consistent with our findings, by cysteine
(Ki = 4.9 µM).
It is generally assumed that extracellular cysteine is rapidly oxidized
to cystine and then is transported into the cell (2, 13). Yet, >90%
of the GSH found in rat plasma (26) and human BAL fluid (10) is in the
reduced form. Therefore, we hypothesized that the extracellular fluid
surrounding type II cells would provide a reductive environment capable
of reducing cystine to cysteine. Consistent with this, we found that a
physiological concentration of GSH (60 µM in rat plasma; see Ref. 26)
partially reduced cystine to cysteine followed by a twofold increase in
uptake (Fig. 4). Similar results were seen with 30 µM CysGly (Fig.
4). CysGly is released by the GGT-mediated extracellular hydrolysis of
GSH (2, 23, 27). The alveolar surface of rat type II cells has been
shown to express GGT (21, 23). In rats, the CysGly level was found to
be ~50% of GSH excreted into biliary fluid. Inhibition of GGT
activity on the bile duct epithelial cells caused a 60% decrease in
CysGly levels in biliary fluid (25). Because we found 336 µM GSH in
alveolar lavage fluid, consistent with the concentration in human BAL
fluid (10), the in vivo CysGly levels are likely greater than the 30 µM found to be reductive in our study. The above results suggest
that, in the microenvironment around type II cells, GSH and CysGly
concentrations are sufficient to keep cysteine in the reduced form.
Although the plasma does not come in direct contact with type II cells,
the GSH and GSSG content may represent levels found along the
basolateral surface. With the use of the GSH and GSSG levels we found
in plasma or BAL fluid, cystine was reduced to cysteine, resulting in
increased transport (Fig. 7, A and
B). System ASC has been shown to
occur in most cells, including red blood cells (35) and endothelial cells (14); therefore, we speculate that plasma levels of cystine are
significantly higher than those of cysteine not because this is an
oxidizing environment but rather because the high affinity and rate of
cysteine transport processes result in efficient scavenging of the
amino acid in its reduced form. The distribution of the system
XAG transport process capable of
carrying cysteine is not yet known.
Total reduction of cystine to cysteine with 1 mM CysGly, GSH, NAC, or
DTE resulted in a fourfold increase in
Na+-dependent uptake (Fig. 4),
consistent with previous observations in type II cells in which the
rate of cysteine uptake was found to be much greater than that of
cystine (9, 20). No stimulation was seen when studied in the presence
of inhibitors of Na+-dependent
cysteine transport (Fig. 5). Similar results were obtained in type II
cells in which 1 mM GSH was used to reduce cystine (14) and also in
endothelial cells in which 1 mM NAC was used to reduce cystine (29).
NAC has been used in an attempt to increase GSH levels in vivo in
conditions such as acquired immunodeficiency syndrome or therapeutic
administration of high levels of
O2 in which endogenous levels
become pathologically low (6, 34). The action of NAC has been
attributed to the generation of free cysteine (by the deacylation of
NAC) that then participates in GSH synthesis (29). Our results are
consistent with NAC directly reducing cystine to cysteine (29) because
any increase in unlabeled free cysteine (by deacylation) would compete
with radioactive cysteine, resulting in an inhibition of uptake. This
also suggests that NAC is not a substrate for system ASC. Homocysteine,
which is both a reducing agent and a substrate for system ASC, does inhibit uptake (Fig. 4), which is consistent with published results (29).
The Km for
cysteine (16.9 µM) on system ASC (determined in the presence of A
H
to inhibit system XAG; see Fig. 8)
was lower than the 50-150 µM reported for other cell types (18)
but was similar to the 29 µM seen when the system ASC gene
(ASCT1) was expressed in
Xenopus oocytes (1). In vivo, cysteine
transport on system ASC may be limited by competition of the various
amino acids, including serine (Table 1 and Fig. 8), known to be
substrates for this carrier (2, 11, 18). However, the affinity for cysteine was found to be greater than that of other amino acids transported by system ASC (1, 18), which may give it a competitive edge. Furthermore, it has been demonstrated in other cells that intracellular levels of amino acids transported by system ASC trans-stimulate uptake of
extracellular amino acids by this carrier (4). Therefore, the actual in
vivo transport of cysteine by type II cell system ASC is difficult to
predict. In some cells, cysteine has also been shown to be transported
by system A (11, 18). The failure of MeAIB to inhibit
Na+-dependent cysteine transport
(Fig. 3) demonstrates that system A, which is known to be present in
type II cells (8), is not utilized for cysteine transport.
In all tissues previously studied, cystine was found to be transported
in an Na+-independent manner by
system xc or system
b0+ (11, 18). The failure of 1 mM
arginine to inhibit cystine uptake (Fig. 3) suggests that type II cells
do not have system b0+, consistent
with Bertran et al. (5), who saw no signal in lung on a Northern blot
analysis for rat b0+ amino acid transporter
mRNA. In contrast, 200 µM quisqualate, which
specifically inhibits system xc
(Table 1 and Fig. 8), significantly inhibited
Na+-independent cystine uptake in
type II cells (Fig. 6A). Similarly, Bukowski et al. (9) reported that 5 mM homocysteate inhibited cystine
uptake, which is consistent with the presence of system xc. In contrast, quisqualate had
no effect on Na+-independent
cysteine transport, demonstrating that cystine was totally reduced by 1 mM CysGly or GSH (Fig. 6B).
Na+-independent cysteine uptake
was inhibited by BCH in type II cells (Figs. 1 and
6B). This is consistent with uptake
on system L, which has been described in other tissues (11, 18). With
the use of in vitro conditions,
Na+-independent transport of
cysteine and especially cystine represents a minor component of total
uptake (Fig. 1). In vivo transport of cystine by system
XAG, however, is unlikely because
of its low affinity for the carrier
(Km > 500 µM).
Na+-dependent transport relies on
an Na+ gradient maintained by
Na+-K+-ATPase.
During oxidant stress, the decrease in intracellular ATP levels (24)
would negatively influence
Na+-dependent transport systems.
Under these conditions, the
Na+-independent systems
xc and L would be increasingly
important. This is especially true for system
xc during oxidant stress in which
a decrease in the GSH-to-GSSG ratio would decrease the ability of
extracellular fluids to keep cysteine in its reduced form. System
xc has been shown to be
upregulated in several types of cells, including fibroblasts (3), after
in vitro exposure to hyperoxia. Brodie and Reed (7) showed that
cellular GSH levels in A549 lung carcinoma cells are directly
correlated with cystine transport rate. Horton et al. (20) demonstrated
that transport of methionine in type II cells was similar to the
transport of cysteine and much greater than that of cystine. Yet no GSH
synthesis was detected in cells first depleted of GSH followed by
incubation with methionine. These results indicate that the
cystathionine pathway (transsulfuration of methionine to cysteine) is
relatively inactive in type II cells. In contrast, both extracellular
cysteine and cystine were readily utilized for GSH synthesis in type II cells (20), emphasizing the importance of cysteine and cystine transport.
In conclusion, 1) cysteine uptake by
type II pneumocytes is consistent with transport on two
Na+-dependent systems (ASC and
XAG) as well as on the
Na+-independent transport
system L; 2) cystine is transported in both an
Na+-independent and -dependent
manner, consistent with uptake via systems
xc and
XAG, respectively, and we
speculate that, under physiological conditions, transport occurs mainly
on system xc; 3) at the extracellular surface of
type II cells, physiological levels of GSH are sufficient to reduce
cystine to cysteine, thereby enhancing uptake; and
4) based on these observations, we
hypothesize that the importance of each of these transport processes
for cysteine and cystine uptake will vary depending on the amino acid
composition and the overall redox state of the transporter
microenvironment.
 |
ACKNOWLEDGEMENTS |
We thank Dr. George Lister for assistance in analysis of data.
 |
FOOTNOTES |
This work was supported by National Institutes of Health Grants P50
HL-46488 and AI-24782 and by the March of Dimes Birth Defects
Foundation.
Address for reprint requests: R. G. Knickelbein, Yale Univ. School of
Medicine, Dept. of Pediatrics, 333 Cedar St., PO Box 208064, New Haven,
CT 06520-8064.
Received 16 October 1996; accepted in final form 3 September 1997.
 |
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