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

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

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 gamma -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

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

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 gamma -glutamyl transpeptidase (GGT) that cleaves the gamma -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.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

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

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. alpha -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-beta -hydroxamate (Abeta 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-beta -hydroxamate (Abeta H; 400 µM). MeAIB, alpha -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). dagger  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; dagger  P < 0.01.

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 Abeta H blocked Na+-dependent cystine uptake (Fig. 3), we investigated the effect of this inhibitor on Na+-dependent cysteine uptake. Abeta H (400 µM) also inhibited cysteine uptake by ~50%, and a combination of serine and Abeta 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 Abeta 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; dagger  P < 0.01.

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.

                              
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Table 2.   Ki for amino acids transported by the type II cell system XAG transporter

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 Abeta H. Although cystine uptake also occurred via system XAG, its affinity was very low.

                              
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Table 3.   Km of cysteine and cystine transporters in type II cells

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 (Abeta H). Both Na+-independent cystine and glutamate uptake were inhibited by excess external unlabeled cystine or glutamate and by quisqualate but not by Abeta H (Fig. 6A). This demonstrated the specificity of Abeta 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 Abeta 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 Abeta 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). dagger  Significantly different from control (P < 0.01).

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). Abeta 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; dagger  P < 0.01.

    DISCUSSION
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Abstract
Introduction
Methods
Results
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References

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.

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 Abeta 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 Abeta 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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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