Introduction

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Glutathione (GSH), the most abundant intracellular nonprotein thiol, participates in many important biologic processes, such as metabolism of endogenous and exogenous compounds, cell proliferation, and regulation of gene expression. Perhaps the most important function of GSH is to detoxify various oxidants by directly scavenging free radicals or acting as a coenzyme in GSH peroxidase- or GSH-S-transferase-catalyzed reactions. The lung is a major target for oxidant damage due to its direct exposure to the atmosphere. GSH concentration in epithelial lining fluid (ELF) of the distal respiratory tract is much higher than in plasma, suggesting an important role of GSH in lung antioxidant defense (1). The importance of GSH in protecting the lung from oxidative damage caused by various chemicals has been well documented. For example, GSH has been shown to be involved in the protection of lung tissue against the toxicity of chrysotile (2), silica (7), endotoxin (6, 8), phosgene (9), and ozone (10, 11). It has been reported that GSH content is decreased in lung tissue and ELF from patients with various pulmonary diseases (1, 12- 14). These data suggest that GSH plays an important role in lung antioxidant defense and that altered GSH homeostasis may be involved in the pathogenesis of various pulmonary diseases. Understanding how lung epithelial cells maintain their intracellular GSH content during oxidative challenge could, therefore, provide important insights into the mechanism(s) by which the lung tissues protect themselves against oxidant injury and contribute to our understanding of potential factors that contribute to the pathogenesis of these pulmonary diseases.

There are several mechanisms by which cells maintain their intracellular GSH content; mainly, GSH redox cycling, de novo synthesis, and direct uptake. GSH redox cycling, catalyzed by GSH reductase, prevents the loss of GSH in the form of GSH disulfide that is generated during the reduction of various oxidants with GSH, by reducing GSH disulfide back to GSH. De novo GSH synthesis is a two-step reaction. The first step, which is also the rate-limiting step in de novo GSH synthesis, is the synthesis of -glutamylcysteine (-GC) from its constituent amino acids glutamic acid and cysteine, catalyzed by -GC synthetase (GCS) (glutamate-cysteine ligase, EC6.3.2.2). The second step is the synthesis of GSH from -GC and glycine, catalyzed by GSH synthetase (EC6.3.2.3). A few types of cells can directly import intact GSH from surrounding fluid; however, most cells, under both normal and oxidative stress conditions, depend on de novo synthesis to maintain their intracellular GSH content. Synthesis of GSH can also involve a salvage pathway in which -GC generated from the -glutamyl transpeptidase-catalyzed breakdown of extracellular GSH can be taken up and used by GSH synthetase to produce GSH. Nonetheless, previous studies from this laboratory have shown that de novo GSH synthesis appears to play the major role in maintaining GSH homeostasis in rat lung epithelial L2 cells (15). A large body of evidence suggests that regulation of GCS is a major determinant of GSH homeostasis not only for resting cells but also for oxidant-challenged cells (15). Knowledge of the mechanisms that regulate GCS expression in response to oxidant stress is therefore important for understanding how lung epithelial cells maintain their GSH content and defend themselves against oxidative challenge.

GCS is composed of two subunits, one heavy (GCS-HS) and one light (GCS-LS). GCS-HS possesses all the catalytic activity of the enzyme and can be feedback-inhibited by GSH, whereas GCS-LS has important regulatory functions. Although increasing evidence suggests that both subunits are essential for activity of the enzyme, the ratio of the two subunits varies markedly under both physiologic and pathologic conditions so that how regulation of induction of the two subunits contributes to maintenance of intracellular GSH is not well understood. Further, although accumulated data have indicated that the expression of two GCS subunits may be regulated independently under both physiologic and pathologic conditions, the signaling pathways involved in induction of either subunit by oxidants remains to be clarified.

In this study we examined the effects of the lipid peroxidation product 4-hydroxy-2-nonenal (4HNE) on GCS gene expression and activity in rat lung alveolar epithelial type II cells. Similar to previous observations in L2 cells, we demonstrate that there is a dose-dependent increase in GCS activity and protein content after treatment with 4HNE, which appears to be driven primarily by an increase in levels of messenger RNAs (mRNAs) for the GCS regulatory and catalytic subunits. Rat-lung epithelial L2 cells were used to further explore the potential signaling pathway involved in the induction of the two GCS subunits by 4HNE. The results demonstrate that induction of GCS-HS by 4HNE is mediated by activation of the extracellular regulated kinase (ERK) pathway whereas the induction of GCS-LS does not appear to involve either the ERK or p38^MAPK pathways. These findings indicate that upregulation of GCS is an important adaptive response of the alveolar epithelium to oxidative stress. These data also provide direct evidence that different signaling pathways are involved in the induction of the two GCS subunits by oxidants. Similar response to oxidant in both L2 cells and primary cultured alveolar epithelial type II (AT2) cells supports the use of L2 cells as a model for studying the regulation of GSH synthesis in response to oxidant injury in type II cells.

	Materials and Methods

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Chemicals

4HNE was purchased from Cayman Chemical Co. (Ann Arbor, MI) and TRIzol Reagent, an RNA isolation solution, was from Life Science Technologies (Grand Island, NY). PD98059 and SB202198 were from Calbiochem Co (La Jolla, CA). QuickHyb solution and salmon sperm DNA were from Stratagene (La Jolla, CA). -Glutamylglutamic acid was purchased from Bachem (Torrance, CA), and polyvinylidene fluoride (PVDF) membranes were from Millipore Company (Bedford, MA). Enhanced chemiluminescence (ECL) reagent and Hyperfilm ECL film were from Amersham (Buckinghamshire, UK). Microcon-10 tubes were from Amicon Company (Beverly, MA). All high-performance liquid chromatography (HPLC) solvents were Baker Analyzed HPLC-grade reagents from VWR Scientific (San Diego, CA). All chemicals used were at least analytical grade.

Type II Cell Isolation and Culture

Rat alveolar type II cells were isolated from adult male Sprague- Dawley rats by disaggregation with elastase (2.0 to 2.5 U/ml) (Worthington Biochemical, Freehold, NJ) followed by panning on immunoglobulin G-coated bacteriologic plates as previously described (18, 19). Enriched type II cells were resuspended in defined serum-free medium consisting of Dulbecco's modified Eagle's medium and Ham's F12 nutrient mixture in a 1:1 ratio (DME-F12) (Sigma Chemical, St. Louis, MO), supplemented with 1.25 mg/ml bovine serum albumin (Collaborative Research, Bedford, MA), 10 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid, 0.1 mM nonessential amino acids, 2.0 mM glutamine, 100 U/ml sodium penicillin G, and 100 µg/ml streptomycin. Cell purity (> 90%) was assessed by staining freshly isolated cells for lamellar bodies with tannic acid, and cell viability (> 90%) was measured by trypan blue dye exclusion. After counting, the cells were diluted with F12 K medium containing 10% fetal bovine serum (FBS) and seeded on 100-mm² plates at a density of 1 × 10⁷ cells/100 mm². The cells were cultivated overnight at 37°C, 5% CO₂, and then treated with 4HNE for 3 to 12 h for Northern analysis or for 16 h for enzyme activity and Western blotting analyses.

L2 Cell Culture and Treatment with Inhibitors

L2 cells, originally derived from type II pneumocytes of adult rat lungs, were obtained from the American Type Culture Collection (Rockville, MD) and grown in flasks with Ham's F-12 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin at 5% CO₂ and 37°C. 4HNE was dissolved in ethanol; inhibitors of mitogen-activated protein kinase (MAPK) pathways PD98059 and SB202198 were dissolved in dimethyl sulfoxide (DMSO). The final concentration of ethanol and DMSO in medium was 0.1%. L2 cells, when about 90% confluent, were treated with PD98059 or SB202198 at the concentrations indicated in RESULTS and the figure captions for 30 min and then with 20 µM of 4HNE for 3 h. The cells were washed once with 1× phosphate-buffered saline (PBS) after treatment and harvested with a cell scraper in 1× PBS for RNA isolation.

Measurement of GCS Activity

GCS activity was measured by analyzing -GC production by HPLC as previously described (20). Briefly, the cells were harvested using a rubber policeman in 1× PBS and then sonicated briefly in 0.3 ml of lysis solution (0.1 M Tris-HCl [pH 8.2], 150 mM KCl, 20 mM MgCl₂, and 2 mM ethylenediaminetetraacetic acid [EDTA]) containing 2 µg/ml each of leupeptin and aprotinin, and 50 µg/ml of phenylmethylsulfonyl fluoride. The sonicated samples were centrifuged at 105,000 rpm at 4°C for 30 min. To remove endogenous inhibitors, acceptors, and amino acids, the supernatant was further centrifuged in Microcon-10 tubes at 12,000 rpm for 30 min at 4°C. The concentrates were washed three times with lysis solution and centrifuged under the same conditions for 30 min each time. Final concentrates were tested for their protein content, using a BCA kit supplied by Pierce (Rockford, IL) (21) and used for enzyme activity analysis. The reaction was initiated by adding protein to the prewarmed (37°C) incubation mixture, which contained 20 mM L-glutamic acid, 5 mM cysteine, 5 mM dithiothreitol (DTT), 10 mM adenosine triphosphate, 0.1 M Tris-HCl (pH 8.2), 150 mM KCl, 20 mM MgCl₂, 2 mM EDTA, and 0.04 mg/ml acivicin. Final protein concentration in the reaction mixture was between 0.1 and 1 mg/ml. After 30 min of incubation, 5% sulfosalicylic acid was added to stop the reaction and 0.4 M N-ethylmorpholine/40 mM KOH was added to bring the pH of the reaction mixture to 8.5. A total of 5 mM monobromobimane was then added. The derivatization was carried out in the dark for 20 min at room temperature. Sulfosalicylic acid (10%) was added again after derivatization and the derivatized thiols were analyzed by HPLC using standard curves generated with known amounts of -GC and GSH as described previously. GCS activity was reported as nmol -GC/min/mg protein.

Western Blotting Analysis of GCS Proteins

The cell samples were prepared as described for the analysis of GCS activity and the amounts of GCS catalytic and regulatory subunit proteins were determined by Western analysis as described previously (20). Briefly, the supernatant (containing 50 µg protein) was mixed with 1 vol of 2× DTT gel loading buffer (0.125 M Tris-HCl [pH 6.5], 20% glycerol, 4% sodium dodecyl sulfate [SDS], 0.0025% pyronin Y, and 15.4 mg/ml DL-DTT) and subjected to 10% SDS-polyacrylamide gel electrophoresis. After electrophoresis, proteins were transferred to a PVDF membrane (Millipore). The membranes were then blocked with 5% nonfat milk at room temperature for 30 min and incubated with rabbit polyclonal antirat GCS antibodies at 4°C overnight. Heavy-subunit antibodies were raised against a 19-amino acid GCS-HS peptide (amino acids 295-313, NH₂-CRWGVISASVDDRTREERG-COOH) conjugated to carrier keyhole limpet homocyanin. Light-subunit antibodies were raised against a holoenzyme purified from rat hepatocytes and further purified from the GCS-LS serum (20), according to Meyer and colleagues (22, 23). After washing with Tris-buffered saline containing 0.05% Tween 20 the membranes were incubated with an appropriately diluted goat-antirabbit antibody, tagged with horseradish peroxidase, at room temperature for 1 h and then, with an ECL (ECL+Plus) reagent mixture for 5 min after additional washing. Film exposure was carried out at room temperature with a Hyperfilm ECL film (Amersham).

Northern Hybridization Analysis of GCS mRNAs

GCS-HS (804 base pairs [bp]) and GCS-LS (1,001 bp) cDNA probes were generated by reverse transcription and polymerase chain reaction amplification of rat kidney RNA and L2 cell RNA, respectively (16, 24). Probes were labeled with [-³²P] deoxycytidine triphosphate using a random-primer DNA labeling kit from Life Technologies (Gaithersburg, MD). GCS mRNA levels were determined by Northern analysis as described previously (20). Briefly, total RNA was extracted from the cells after treatment using TRIzol Reagent (Life Technologies) according to the protocol provided by the manufacturer. A total of 20 µg RNA from each sample was resolved on a 1.2% agarose gel and transferred onto a Nylon membrane. Hybridization was carried out with GCS-LS complementary DNA (cDNA), GCS-HS cDNA, or 18S cDNA probes at 60°C for 2 h using Quikhyb solution (Stratagene). After hybridization, the membranes were washed twice with 2× sodium chloride-sodium citrate (SSC) buffer/0.1% SDS for 15 min at room temperature, then with 0.1× SSC/0.1% SDS for 15 min at 50°C. The membranes were scanned and radioactivity was quantitated by an Instantimager (Packard Instrument Co., Meriden, CT).

Statistics

Data are expressed as means ± standard error of the mean (SEM) and evaluated by one-way analysis of variance. Statistical significance was determined by Fisher LSD test. P < 0.05 was considered significant.

	Results

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4HNE Increases GCS Activity in Type II Cells

Previous data from this laboratory have shown that 4HNE, a lipid peroxidation product, induces expression of both GCS subunits in L2 cells. To see whether type II cells respond to oxidant challenge in the same way we measured the effect of 4HNE on GCS activity in primary rat AT2 cells. GCS activity was determined after AT2 cells were treated with 5 or 10 µM 4HNE for 16 h, the conditions which have been shown previously to increase GCS activity in L2 cells. 4HNE increased GCS activity in AT2 cells in a concentration-dependent manner. As shown in Figure 1, the activity of GCS was significantly increased by 30 and 46% after the cells were treated with 5 and 10 µM 4HNE for 16 h, respectively, compared with a vehicle-treated control group (P < 0.05).

View larger version (33K): [in this window] [in a new window]   Figure 1.   4HNE increased GCS activity in rat lung type II cells. Freshly isolated rat lung type II cells were treated with 0, 5, and 10 µM 4HNE for 16 h after overnight plating. GCS activity was measured using an HPLC assay as described in MATERIALS AND METHODS. Results are expressed as means ± standard error (SE) of four determinations. *Significantly different from control (P < 0.05).

Effect of 4HNE on the Amounts of GCS Subunit Proteins in Type II Cells

The amounts of both GCS subunit proteins were analyzed by Western blotting to determine whether the increased GCS activity in type II cells caused by 4HNE was accompanied by an increase in GCS protein content. As shown in Figure 2, the amount of GCS regulatory subunit protein was significantly increased by 117 and 174% after the cells were treated with 5 and 10 µM 4HNE, respectively, for 16 h compared with a vehicle-treated control group (P < 0.05). The amount of GCS catalytic subunit protein was only slightly (20%) increased in type II cells after treatment with 10 µM 4HNE for 16 h (P < 0.05), whereas there was no significant changes with 5 µM 4HNE. This pattern of induction of GCS proteins by 4HNEi.e, a disproportionate increase in the amount of the regulatory subunit relative to the catalytic subunitis similar to that previously reported in L2 cells.

View larger version (46K): [in this window] [in a new window]   Figure 2.   Effect of 4HNE on GCS subunit protein content in rat lung type II cells. Freshly isolated rat lung type II cells were treated with 0, 5, and 10 µM 4HNE for 16 h after overnight plating. The amounts of GCS catalytic and regulatory subunit proteins were measured by Western analyses as described in MATERIALS AND METHODS. The results are expressed as means ± SE of four determinations. *Significantly different from control (P < 0.05).

Effect of 4HNE on the GCS mRNA Content in Type II Cells

The effect of 4HNE on GCS mRNA content was examined to see whether the increase in protein content was the result of increased levels of either or both GCS-subunit mRNAs. The results show that the amount of GCS regulatory subunit mRNA increased dramatically in a concentration-dependent manner after type II cells were treated with 5 or 10 µM 4HNE, peaking at 6 h after exposure. As shown in Figure 3, the amount of GCS regulatory subunit mRNA increased by 220 and 397%, respectively, after 3 h; by 280 and 410%, respectively, after 6 h; and by 166 and 290%, respectively, after 12 h treatment with 5 and 10 µM 4HNE. The induction of GCS catalytic subunit mRNA was much less than that of the regulatory subunit. A significant increase in the amount of GCS catalytic subunit mRNA was observed only after treatment with 10 µM 4HNE for 6 h. A bigger increase in the amount of GCS regulatory subunit mRNA compared with that of GCS catalytic subunit mRNA parallels the pattern of increase in GCS subunit proteins.

View larger version (64K): [in this window] [in a new window]   Figure 3.   Effect of 4HNE on expression of GCS mRNA in rat lung type II cells. Freshly isolated rat lung type II cells were treated with 0, 5, and 10 µM 4HNE for 3, 6, or 12 h after overnight plating. Total RNA was isolated and Northern hybridization was performed as described in MATERIALS AND METHODS. 18S was used to normalize the amount of sample loading. The results are expressed as means ± SE of two or three samples. *Significantly different from control (P < 0.05).

Effects of PD98059 and SB202198 on the Induction of GCS Gene in Rat Lung Epithelial L2 Cells

PD98059, a MAPK inhibitor, specifically blocks the signal transduction between MAPK/ERK kinase-1 and ERK, whereas SB202198 is a specific inhibitor for p38^MAPK pathway in MAPK activation cascade. PD98059 and SB202198 were used in this study to examine the potential pathway by which 4HNE induced the expression of both GCS subunits. As previously shown, treatment of L2 cells with 20 µM 4HNE for 3 h induced a marked increase in mRNA of both GCS subunits with a relatively greater increase in the regulatory subunit. The quantity of 50 µM PD98059 almost completely blocked the induction of GCS-HS by 4HNE but had no significant effect on the induction of GCS-LS in L2 cells (Figure 4). On the other hand, SB202198, up to the concentration of 37.5 µM, had no significant effect on the induction of either subunit of GCS by 4HNE (Figure 5).

View larger version (51K): [in this window] [in a new window]   Figure 4.   Effect of PD98059 on the induction of GCS mRNA by 4HNE in L2 cells. L2 cells were pretreated with PD98059 (50 µM) or DMSO for 30 min, then treated with 4HNE (20 µM) or ethanol for 3 h. Values are means ± SEM of two or three samples. Lower panel: a, significantly different from ethanol control; b, significantly different from PD98059 control; c, significantly different from 4HNE.

View larger version (48K): [in this window] [in a new window]   Figure 5.   Effect of SB202198 on the induction of GCS mRNA by 4HNE in L2 cells. Top panel: L2 cells were pretreated with different concentrations of SB202198 or DMSO for 30 min and then treated with 20 µM 4HNE or ethanol for 3 h. Middle panel: L2 cells were treated with 25 µM SB202198 or DMSO for 30 min and then treated with 20 µM 4HNE or ethanol for 3 h. Bottom panel: summary of the data from top and middle panels. Values are means ± SEM of two or three samples. In bottom panel: a, significantly different from ethanol control; b, significantly different from SB202198 control.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

GSH plays an important role in lung antioxidant defense. In this study we demonstrated that upon challenge by 4HNE, rat AT2 cells increased their capacity to synthesize GSH, reflected by an increase in GCS activity. The increase in GCS activity induced by 4HNE in type II cells was accompanied by an increase in mRNA and protein content for both GCS subunits, with a predominant increase in the level of the GCS regulatory subunit. Further evaluation of the signaling pathways involved was made with L2 cells. These studies indicate that the increase in GCS-HS, but not GCS-LS, appears to be mediated by activation of the ERK pathway, supporting the notion that the two GCS subunits are differentially regulated. Inasmuch as 4HNE is a major lipid peroxidation product, the increase in GCS expression observed in response to 4HNE may reflect a general adaptive response of lung epithelial cells to oxidative challenge. These results suggest that GSH plays an important role in lung antioxidant defense and that an increase in GSH biosynthesis may be an important mechanism by which lung epithelial cells increase their intracellular GSH content during oxidative stress.

GCS is composed of two subunits, one catalytic and one regulatory. On the basis of studies using recombinant GCS proteins it has been proposed that GCS-HS has all of the catalytic activity of the holoenzyme and can be feedback-inhibited by GSH. GCS-LS, on the other hand, does not have any catalytic activity but has important regulatory functions (25, 26). It has been suggested by Huang and coworkers that without the regulatory subunit, the catalytic subunit may not have any activity under physiologic conditions due to the low glutamate and high GSH concentrations in vivo (25). Although there is increased evidence that the regulatory subunit is critical to GCS activity in vivo, the exact function of the regulatory subunit is still not clear. Interestingly, enzyme activity, which was measured at a saturating glutamate concentration (20 mM) without GSH, was increased after the cells were treated with 5 µM 4HNE for 16 h, although only the regulatory subunit was increased under this condition. These data suggest that the regulatory subunit may have additional functions than previously believed. Tipnis and associates (27) also reported that GCS activity was increased when purified GCS regulatory subunit protein was directly added to HeLa cell extracts or when COS cells were transiently transfected with the regulatory subunit cDNA. After transient transfection, the increase in GCS activity was accompanied by an increase in GSH content without any change in the amount of the catalytic subunit. Because the activity of GCS was measured using a saturating glutamate concentration (60 mM) without addition of GSH to the reaction mixture, these results suggest that the regulatory subunit modifies GCS activity by some mechanisms other than decreasing Km for glutamate as suggested by Huang and colleagues (25, 26). Further, a recent report from our laboratory has shown that in human immunodeficiency virus type 1 Tat-transgenic mice, GSH depletion in liver and erythrocytes was accompanied only by a decrease in the amount of GCS regulatory subunit (28). The sensitivity of the enzyme to inhibition by GSH was increased in liver and erythrocytes from Tat-transgenic mice compared with that from control mice, which was consistent with the one function of the regulatory subunit proposed by Huang and coworkers; an increase in the Ki for GSH. However, the Km for glutamate was not different between control and Tat-transgenic mice. These results confirm an important role for the regulatory subunit in regulating GCS activity, but it also raises further questions about the precise mechanism whereby the regulatory subunit modulates GCS activity. Additional studies are needed to further delineate the function of the GCS regulatory subunit. Nevertheless, the current study indicates that the GCS regulatory subunit may play a more important role in maintaining GSH homeostasis during oxidative stress because expression of this subunit is more dramatically induced than is the catalytic subunit upon oxidative challenge.

Although both subunits can be induced by various chemicals or agents, there is increasing evidence that the mechanisms responsible for the induction of these two subunits are different (20, 29). The expression of the two GCS subunits under physiologic conditions may also be differentially regulated inasmuch as the ratios of the two GCS subunit mRNAs vary among different tissues (30). Consistent with this, Tat protein has been shown to downregulate expression of the regulatory but not the catalytic subunit (28). In the present study we demonstrate that 4HNE dramatically increases the expression of the regulatory subunit but has only a small effect on the catalytic subunit, further supporting the notion that different mechanisms may be involved in the regulation of the expression of the two GCS subunits. A major mechanism through which signals from environmental stimuli are transmitted to the nucleus involves activation of kinases related to the MAPK superfamily. Three families of MAPKs have been identified: the ERK family, the p38^MAPK family, and the c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) family. Although the picture is changing, in general it is thought that ERKs respond primarily to mitogen stimulation whereas the p38^MAPK and JNK/SAPK families are involved mainly in responses of the cells to inflammatory cytokines and oxidants. Although the signaling pathway through which oxidants activate GCS gene expression remains to be clarified, the results from our study strongly suggest that the ERK pathway was involved in the induction of GCS-HS, but not GCS-LS, by 4HNE. PD98059 almost completely blocked the induction of GCS-HS and had no effect on the induction of GCS-LS by 4HNE. In contrast, the induction of GCS-LS involved neither the ERK nor the p38^MAPK pathway because neither PD98059 nor SB202198 had any effect on the induction of GCS-LS by 4HNE in these cells. These data clearly indicate that different mechanisms (signaling pathways) are involved in the induction of the two GCS subunits by 4HNE. 4HNE has been shown to be able to activate JNK and p38^MAPK pathways in rat liver epithelial RL34 cells (32). One of the substrates of JNK/SAPK is c-Jun, a component of transcription factor activator protein-1, which has been identified in the promoter regions of both GCS subunits in humans and has been suggested to be involved in the induction of GCS gene expression by various oxidants. Whether the JNK/SAPK pathway is involved in the induction of GCS-LS by 4HNE needs further investigation.

The rat alveolar epithelial L2 cell line was first isolated and cloned from adult rat alveolar epithelial cells using clonal selection techniques (33, 34). Because the isolation of type II cells is technically difficult whereas L2 cells are commercially available and easy to culture, L2 cells have been widely used as a model for lung type II cells in various studies. Cell lines, which are generally created by transfecting cells with viral DNA or treating cells with a chemical to immortalize them, commonly show altered properties compared with the original cells. Therefore, it is important to know whether a cell line maintains the same metabolic function, phenotype, or responsiveness as the parental cells when extrapolating the in vitro results to the in vivo situation. Although L2 cells are not virally or chemically transformed, they are nevertheless immortalized cells and as such, the degree to which they retain the metabolic function or responsiveness of parental lung epithelial type II cells needs to be established. Douglas and colleagues reported that L2 cells maintained the shape of type II alveolar pneumonocytes and synthesized lecithin in the same fashion as whole lung tissue (33, 34). Monteil and associates also reported that L2 cells retained the same phenotype and functions as type II cells, including differentiation, synthesis of various endogenous compounds, and expression of specific receptors (35). The present study indicates that L2 cells retain the same responsiveness to oxidant challenge as parental lung epithelial type II cells with regard to increasing their GSH synthesis capacity. The mechanisms for increasing GSH synthesis upon oxidative challenge are preserved in this cell line compared with type II cells, supporting the use of L2 cells as model for studying the responsiveness of lung type II cells to oxidative challenge.