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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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High oxygen concentrations are used in the treatment of
acute respiratory distress syndrome and hyaline membrane disease. Hyperoxia, however, can damage alveolar epithelial cells
through the release of free oxygen radicals. Supplemental
glutamine (Gln) has recently been shown to increase survival
of A549 cells, a distal epithelial cell line, during hyperoxia (1).
We found that supplemental Gln (Gln+) is essential for cell
growth in A549 cells. In room air, cells without supplemental
Gln (Gln
) survived with BCL-2 levels similar to those of Gln+
cells, but cell growth was minimal. We also evaluated the role
of glutamine synthetase (GS) in A549 cells during hyperoxia.
L-methionine sulfoximine (MSO), an irreversible inhibitor of
GS, was added to Gln+ and Gln cells. In hyperoxia, Gln
cells had greater survival then Gln cells treated with MSO.
Supplemental Gln could rescue cells in hyperoxia from the effect of MSO, suggesting that GS, through the endogenous
synthesis of Gln, could attenuate hyperoxic cell injury. In hyperoxia, cells treated with 10-mM concentrations of Gln had
increased survival compared with cells receiving 2-mM concentrations. The higher concentration of Gln, however, did not decrease the percentage of cells undergoing necrosis.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Hyperoxia exacerbates lung injury in patients with acute respiratory distress syndrome and in infants with bronchopulmonary dysplasia (2). Excess oxygen molecules can overwhelm the cells' antioxidant mechanisms, leading to release of free oxygen radicals and subsequent growth arrest, injury, and cell death in the lung (3, 4). The lungs are a major source of glutamine (Gln) biosynthesis and glutamine is released during periods of stress (5, 6). Recently it has been shown that Gln increases cell survival in A549 cells exposed to hyperoxia by protecting mitochondrial integrity (1).
Gln is the most abundant amino acid in the plasma. It has been shown to have a role in ammonium detoxification and amino acid homeostasis, as an energy source for epithelial cells, and as an osmolyte (6, 7). It has been shown that intestinal epithelial cells absorbed glutamine rapidly from the intestinal lumen and that glutamine is quickly metabolized, with only 34% of it remaining unchanged in the intestinal venous blood (7). In type 2 alveolar cells, glutamine transport occurs predominantly through a high-affinity sodium-dependent pathway (8). Gln has also been shown to protect endothelial cells from hydrogen peroxide oxidant stress by maintaining ATP levels in injured cells (9).
The purpose of this study is to investigate the role of Gln and glutamine synthetase (GS) in A549 cells in both room air and hyperoxia. GS is essential for endogenous synthesis of glutamine. Previous studies have demonstrated that GS is present in the lung (5, 10, 11). GS is also abundantly found in astrocytes and plays an important role in the transfer of glutamine from astrocytes to neurons (12). GS induction has also been associated with cellular differentiation in adipocytes (13). It is not known whether GS is involved in differentiation of type 2 to type 1 alveolar cells.
In this paper we will describe the effect of different concentrations of glutamine on A549 cells in both room air and 95% hyperoxia. We will also describe the effect of L-methionine sulfoximine (MSO), an irreversible inhibitor of GS, on cell growth and survival in A549 cells.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Cell Lines
A549 cells were obtained from American Type Culture Collection (ATTC, Rockville, MD). They were grown to 50% confluence in Ham's F-12 media (Cellgro) with 10% fetal bovine serum (FBS), and penicillin/streptomycin (100 U/ml). T25 flasks were seeded equally (100,000 cells) and cells were grown for 2 d in room air and 5% CO2 before being placed in hyperoxia and 5% CO2. Cells were 50% confluent after 2 d in room air. Cells were placed in either a modular incubator chamber (Billups-Rothenberg, del Mar, CA) and gassed with 95% and 5% CO2 daily or placed in room air and 5% CO2. Cells were fed and re-gassed daily. Cells were given different concentrations of supplemental glutamine ranging from 0-10 mM (Gibco/BRL, Gaithersburg, MD). Because all cells received 10% serum, cells given no supplemental Gln contained ~ 0.057 mM Gln concentrations in the cell media. Select cells were also given 3 mM MSO (L-methionine sulfoximine, Sigma M-5379; Sigma, St. Louis, MO), an irreversible inhibitor of GS, or 5 mM and 0.5 mM L-Buthionine-[S,R]-Sulfoximine (BSO). Cells were harvested at specific time periods. Viable cells were counted at each time point using trypan blue exclusion.
Annexin V Binding
Cells were treated with 2, 5, and 10 mM Gln+ and placed in 95% hyperoxia and 5% CO2 for annexin V binding studies to determine viable, apoptotic, and late apoptotic/necrotic cells at 48, 72, and 96 h. The TACS Annexin V-FITC (cat# TA4638; R&D Systems, Minneapolis, MN) kit was used following the manufacturer's protocol. Briefly, cells were washed, trypsinized, and stained with propidium iodide and annexin-V-FITC. Suspended cells were then sorted and counted using a FACScan (Becton Dickinson, Franklin Lakes, NJ) and analyzed with the program Mod Fit LT (Verity, Topshan, ME) to determine the percentage of viable, apoptotic, or late apoptotic/necrotic cells. Cells which stained only with annexin V were considered early apoptotic cells, cells that stained with both propidium iodide and annexin V were considered late apoptotic or necrotic cells, and cells that stained with neither annexin V or propidium iodide were considered viable cells.
Western Blots in Cells
Whole-cell lysates were solubilized in 2% SDS and equal amounts of protein were loaded per lane as determined using the Biorad DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Lysates were run on a 12% SDS-polyacrylamide gel and transferred to nitrocellulose. Western blot analysis was performed using, cyclin B1 polyclonal antibody (1:200 dilution, sc-752; Santa Cruz Biotechnology Inc., Santa Cruz, CA), anti Bcl-2 monoclonal antibody (65111A; PharMingen, San Diego, CA), proliferating cell nuclear antigen (PCNA) monoclonal antibody (sc-56; Santa Cruz Biotechnology Inc.) or glutamine synthetase polyclonal antibody (sc-6640; Santa Cruz Biotechnology Inc.). The monoclonal antibodies were used at concentrations of 1-2 µg/ml as recommended in 5% blotto, phosphate-buffered saline (PBS) and 0.05% Tween overnight at 4°C. The blots were washed three times in PBS-Tween, incubated with a mouse Ig, horseradish peroxidase-linked whole antibody (1:500 dilution) (RPN 2108; Amersham, Arlington Heights, IL), or peroxidase-labeled antirabbit antibody (1:2,000 dilution) for 1 h then washed and developed using chemiluminescence (ECL) (RPN 2106; Amersham).
5-Bromo-2-Deoxyuridine Staining
Gln+ cells (2 and 10 mM) in 95% hyperoxia were incubated with 5- bromo-2-deoxyuridine (BRDU) labeling reagent for 60 min. Antibody staining was then performed as per the Amersham cell proliferation kit (RPN20).
Glutathione Quantification
Measurements of total glutathione were performed on A549 cells containing 2 and 10 mM Gln in 95% hyperoxia-treated cells for 72 h. Cells were counted and equal numbers of cells from both groups were processed for total glutathione content. Cells were processed as recommended using the Total Glutathione Quantification kit (cat.# T419-10; Dojindo Molecular Technologies Inc, Gaithersburg, MD). The change in absorbance at 405 nm was measured at 20 min and the concentration of glutathione (GSH) was calculated against a standard curve.
Statistical Analysis
Statistical calculations were performed using the SPSS 8.0 statistical package for Windows (Chicago, IL) Differences in measured variables between experimental and control groups were determined using comparison of the means using a one-way ANOVA and Student's t test (two-tailed, unequal variance). Statistical difference was accepted at P < 0.05.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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The Effect of Gln and MSO on Cell Viability and Cell Growth in A549 Cells in Room Air
A549 cells were grown in room air and 5% CO2 for up to
96 h. Cells were divided into four groups: 0.5-mM Gln+
cells, 2-mM Gln+ cells, Gln cells, and Gln MSO+ cells
(Figure 1). There was a significant difference in cell
growth at 48, 72, and 96 h between the Gln+ cells and the
Gln cells (P < 0.001). Both the 0.5 mM and 2 mM Gln+
cells grew in room air with no significant difference in cell
growth between the two groups. The Gln cells, however,
remained viable, but did not proliferate. The Gln
MSO+ treated cells were similar to the Gln cells remaining viable but not proliferating in room air. These
findings suggest that exogenous Gln is essential for cell
growth but not cell viability in A549 cells in room air.
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Bcl-2 protein levels were then measured as a marker
for cell survival in the four different groups of cells. Bcl-2,
like Bcl-XL, is an anti-apoptotic gene that resides in the
mitochondrial membrane and inhibits mitochondrial
membrane permeability (MMP) (14). Inhibition of MMP
prevents apoptosis. We found that both Gln+ and Gln
cells expressed bcl-2 protein to a similar degree in room
air (Figure 2). Protein levels of cyclin B1 were then measured as a marker of cell proliferation. Cyclin B1 accumulates in S phase and complexes with cdc2. Activation of the
cyclin B1/cdc2 complex is necessary for initiation of mitosis (15). In the absence of cyclin B1, mitosis does not occur. Furthermore, transcription of cyclin B1 is negatively
regulated by p53 (16). We found that cyclin B1 protein levels were decreased at 48 and 72 h in Gln and Gln
MSO+ cells, in contrast to 0.5-mM or 2-mM Gln+ cells, in
which cyclin B1 levels remained the same.
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The Effect of Gln and MSO on Cell Viability and Cell Growth in A549 Cells in 95% Hyperoxia
A549 cells were then placed in 95% oxygen and 5% CO2
for up to 96 h. Cells were divided into four Gln+ treatment groups: 0.5-mM Gln+, 2-mM Gln+, 0.5-mM Gln+
MSO+, and 2-mM Gln+ MSO+ cells, and one Gln
treatment group.
Cell survival was measured by counting cells using trypan blue exclusion at 48, 72, and 96 h (Figure 3A). At 48 h
the 0.5-mM Gln+ MSO+ cells were significantly less than
the 2-mMGln+ cells (P < 0.006). At 72 h of hyperoxia, the
0.5-mM Gln+ MSO+ and Gln cells were significantly
less than the 0.5-mM Gln+, 2-mM Gln+, and 2-mM Gln+
MSO+ cells (P < .002). At 96 h the Gln cells were significantly less than the 0.5-mM Gln+, 2-mM Gln+, and
2-mM Gln+ MSO+ cells (P < 0.001) and the 0.5-mM Gln+
MSO+ cells were significantly less than the 2-mM Gln+
and 2-mM Gln+ MSO+ cells (P < 0.03).
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To determine if GS protein was detectable and may be
acting as a source of endogenous glutamine, Western blot
analysis using a GS antibody was performed. GS protein
was found to be present in A549 cells (Figure 3B). Gln
MSO+ cells were then placed in hyperoxia and found to
have marked cell death, and few viable cells in contrast
to the Gln+ cells (Figure 3C). Bcl-2 protein levels were
also found to be maintained in the Gln+ cells but not in
Gln cells after 72 h of hyperoxia (Figure 4).
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These findings suggest that GS may be an important source of endogenous Gln to A549 cells during hyperoxic stress. Furthermore, hyperoxic cells treated with 2 mM Gln+ MSO+ had no significant difference in cell survival at 96 h compared with 2 mM Gln + cells, demonstrating that exogenous glutamine can rescue cells inhibited by MSO.
Cell Survival in BSO-Treated A549 Cells in Hyperoxia
MSO is an effective irreversible inhibitor of GS but it can
also reversibly inhibit 
-glutamylcysteine synthetase (-GS). -GS is the rate-limiting enzyme in GSH synthesis (18). A
decrease in cellular GSH could result in decrease cell survival in hyperoxia due to increase oxidative stress. MSO,
however, is only 1% as potent as BSO in inhibiting -GS
(19). To determine if MSO has a significant effect on cell
survival through -GS inhibition, cell survival of Gln+
MSO+ cells in hyperoxia was compared with Gln+ BSO+
cells in hyperoxia. In room air, BSO has been shown to be
an effective inhibitor of - GS without causing toxicity of
cells in culture (20). We found that Gln+ cells with 5 mM BSO+ had no significant toxicity in room air. At 48, 72, and 96 h of hyperoxia, 2 mM Gln+ BSO+ treated cells had
significantly more cell death when compared with 2 mM
Gln+ MSO+ cells and 2 mM Gln+ cells in hyperoxia
(Figures 5A and 5B) and BSO-treated cells in hyperoxia
could not be rescued by 10 mM Gln+. Furthermore, 2-mM
Gln+ and 2-mM Gln+ MSO+ cells had similar protein
expression of bcl-2 in hyperoxia, in contrast to 2-mM Gln+
BSO+ cells, which had no detectable bcl-2 protein (Figure
5C). These results demonstrate that MSO has little effect
on cell survival in hyperoxia when cells are supplemented
with exogenous Gln, in contrast to cells treated with BSO,
which show a profound decrease in cell numbers. This suggests that MSO-treated cells have minimal -GS inhibition in hyperoxia compared with BSO-treated cells.
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Cell Survival and Proliferation in A549 Cells Supplemented with 2, 5, and 10 mM Concentrations of Gln
Because 2 mM Gln+ cells had increased survival in hyperoxia compared with 0.5 mM Gln+ cells, we were interested in determining whether higher concentrations of Gln would confer further protection against cell death in hyperoxia. Therefore, A549 cells were treated with 2, 5, and 10 mM concentrations of Gln, and cell survival was determined by trypan blue exclusion. The 10-mM Gln+ cells had significantly more cells surviving at 96 h of hyperoxia then the 2-mM Gln+ cells (P < 0.003) (Figure 6A). Osmolality of the media was similar between the three groups (2 mM: 326 mOsm; 5 mM: 321 mOsm; and 10 mM: 326 mOsm).
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As a marker for cell proliferation we measured proliferating cell nuclear antigen (PCNA) levels after 48 h of hyperoxia in the 0.5-, 2-, and 10-mM Gln+ cells and found that PCNA protein expression was greater in the 10-mM Gln+ cells compared with the 0.5- and 2-mM Gln+ cells (Figure 6B). BRDU staining was then performed on cells receiving 2 mM and 10 mM Gln+ exposed to 95% hyperoxia for 48 h, and increased BRDU staining was found in the 10-mM Gln+ cells compared with the 2-mM Gln+ cells (Figure 6C). Finally, as an additional marker of proliferation, cyclin B1 protein levels were measured in 2-mM and 10-mM Gln+ cells exposed to hyperoxia. Protein expression by western blot analysis was markedly less at 72 h in the 2-mM Gln+ cells compared with the 10-mM Gln+ cells, suggesting less growth impairment in the 10-mM Gln+ cells (Figure 6D).
We then measured GSH levels in 2-mM and 10-mM Gln+ cells exposed to hyperoxia. This was done to determine if 10-mM Gln+ cells had less GSH depletion in hyperoxia than 2-mM Gln+ cells, accounting for the greater survival of the 10-mM Gln+ cells in hyperoxia. The GSH levels were significantly higher in the 10-mM Gln + cells compared with the 2-mM Gln+ cells (P < 0.02) (Figure 7).
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Annexin V binding was then measured to determine if the increased survival in the 10-mM Gln+ group compared with 2-mM Gln+ cells was secondary to a difference in the percentages of viable, apoptotic, or necrotic cells (Figure 8). At 96 h, cells in room air (RA) had significantly more viable cells then the hyperoxic treated cells in any group (RA and 2 mM, P < 0.03; RA and 5 mM, P < 0.02; RA and 10 mM, P < 0.01). Also, at 96 h the RA cells had significantly less necrotic cells than the 10-mM-treated cells (P < 0.03). At 96 h the percentage of cells undergoing necrosis was 21.7% (± 8.9%), 25.3% (± 10.1%), and 29.1% (± 13.1%) in the 2 mM, 5 mM, and 10 mM Gln+- treated cells, respectively, compared with the room air cells, which had 6.6% (± 1.1%) cells undergoing necrosis. In all hyperoxic treated cells the percentage of necrotic cells increased significantly from 48 to 96 h (2 mM, P < 0.01; 5 mM, P < 0.004; 10 mM, P < 0.01) and the percentage of viable cells decreased significantly (2 mM, P < 0.04; 5 mM, P < 0.03; 10 mM, P < 0.05).
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P < 0.03). n = 6 for the hyperoxic treated cells and n = 3 for the room air cells. Error bars represent standard deviations. Comparison of the means was done
by one-way analysis of variance.
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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In our study we have shown that A549 cells require Gln for growth and that Gln enhances cell survival under hyperoxic conditions. We have also found that cells not supplemented with exogenous Gln and exposed to hyperoxia had further decreases in cell number when treated with MSO, an irreversible inhibitor of glutamine synthetase. When MSO-treated cells were supplemented with Gln, however, the effect of MSO was attenuated. Supplementation of A549 cells with higher concentrations of Gln (10 mM) during exposure to hyperoxia further attenuated cell death; however, it did not reduce the percentage of cells undergoing necrosis by annexin V binding.
In this study we have found that A549 cells require supplemental Gln for growth. The requirement for exogenous
Gln in A549 cells was also recently shown by Ahmad and
coworkers (1), who demonstrated that Gln was an essential substrate for cells during hyperoxia, and protected the
mitochondria from oxidative stress. In room air we found
that A549 cells, receiving no supplemental Gln, remained
viable over a 96-h period with stable levels of bcl-2; however, growth was static and protein levels of cyclin B1, a
cyclin necessary for progression of the cell through mitosis, were decreased. Although GS protein was present in
A549 cells, when it was inhibited by MSO there was no
significant difference in cell viability or cell growth between Gln and Gln MSO+ cells in room air. These
findings suggest that Gln is not an essential substrate for
survival of A549 cells in room air; however, a critical concentration of Gln is necessary for A549 cells to proliferate. Furthermore, although GS protein is present in A549 cells,
endogenous Gln production through GS is not sufficient
for cell growth of A549 cells.
In 95% hyperoxia, Gln cells had significantly fewer
cells surviving then Gln+ cells. This may be secondary to
hyperoxia directly inhibiting GS activity, or hyperoxia may
be decreasing new protein synthesis in the cell. Furthermore, in contrast to room air cells, Gln MSO+ cells in
hyperoxia had far fewer cells surviving than Gln cells.
The reason for the decrease in cell survival in the Gln
MSO+ cells may be multifactorial. GS may be acting
through a direct or indirect mechanism to attenuate hyperoxic cell injury. Inhibition of GS by MSO may decrease
the availability of Gln to the hyperoxic cell, resulting in increased cell death in the absence of exogenous Gln. The
need for exogenous Gln in hyperoxia would be consistent
with the conclusions of Ahmad and coworkers that Gln is
used as an alternative substrate in A549 cells in hyperoxia (1). In IEC-6 cells, a rat intestinal crypt cell line, cell proliferation was decreased in cells receiving 10 mM MSO (21). The effect of MSO in blocking cell proliferation was rescued by supplemental Gln. The concentration of exogenous Gln needed, however, was in excess of the amount of
exogenous Gln needed in the absence of MSO, suggesting
that exogenous Gln is processed less efficiently then endogenous Gln in proliferating rat intestinal cells.
Another mechanism in which GS may help attenuate hyperoxic injury may be through the induction of heat shock protein-70 (HSP70). Intestinal epithelial cells that were treated with Gln were more resistant to oxidant and heat injury through the induction of HSP70 (21). Wong and colleagues also found that induction of HSP70 in A549 cells protected cells from hyperoxia, possibly through the attenuation of ATP depletion and lipid peroxidation (22).
GS may have several other important roles in the lung. Earlier studies in rats that were injected intravenously with 13N ammonia, showed that the lungs may convert up to 30% of circulating ammonia to Gln (23). Furthermore, studies in bats have suggested that GS may protect the lungs against modest levels of inhaled ammonia (24). Second, in vivo induction of GS from the lungs may contribute to Gln release into the circulation during times of stress. Both Souba (25) and Ardawi (26) have shown that the lung is capable of releasing large amounts of Gln into the pulmonary bed in response to sepsis. Austgen and coworkers also found that early sepsis was associated with a net release of Gln from the lungs, but late sepsis was associated with an increase in GS activity and Gln balance, suggesting an increase in Gln consumption by injured cells in the lung (27). Involvement in cell differentiation is yet another function of GS. GS has been shown to induce differentiation of adipocytes (28), and intestinal epithelial cells treated with MSO show less cell differentiation (29). It is unknown whether GS is involved in cell differentiation of type 2 to type 1 epithelial cells in the lung.
MSO irreversibly inhibits GS (30). Richman and associates also reported that MSO inhibits -glutamylcysteine synthetase (-GCS), therefore inhibiting GSH synthesis. Inhibition of -GCS by MSO, however, unlike
that of GS, is reversible (30) and MSO inhibits -GCS
100 times less than buthionine sulfoximine (BSO) (19).
Potentially the decrease survival in Gln MSO+ cells
exposed to hyperoxia could be secondary to glutathione
depletion in the cell. To help clarify this, we compared
survival of Gln+ MSO+ cells with Gln+ BSO+ cells in
hyperoxia. Concentrations of BSO in this study were
nontoxic to cells in room air. The Gln+ BSO+ cells had
significantly fewer cells surviving than the Gln+ MSO+ cells. This experiment demonstrated that in hyperoxia,
BSO-treated cells, unlike MSO-treated cells, could not
be rescued by exogenous Gln. This suggests that the primary effect of MSO in Gln+ MSO+ cells is inhibition of
GS and not depletion of GSH.
Bcl-2 protein levels were decreased in A549 cells treated with BSO. This suggests that the presence of bcl-2 is associated with increase cell survival during hyperoxia and that GSH may support bcl-2 protein levels in hyperoxia. In another model, Kane and coworkers found that cells overexpressing bcl-2 had higher levels of GSH and greater survival compared with control cells when treated with BSO (31). This suggests that bcl-2 increases cell survival during oxidative stress by decreasing levels of reactive oxygen species in the cell. In hyperoxia we found that cells treated with 10 mM Gln had higher GSH levels after 72 h than cells treated with 2 mM Gln, which suggests that exogenous Gln also protects against oxidative stress by supporting GSH levels in the cell. It has been shown that Gln is taken up into adult alveolar epithelial cells through a Na-dependent amino acid cotransport system (32). It is possible that this cotransport system is altered during hyperoxia-induced oxidative stress and that higher amounts of exogenous Gln are needed for adequate uptake of Gln into the cell. Measurements of intracellular Gln levels in conjunction with GSH levels would be useful in helping to clarify whether increasing the amount of exogenous Gln has a direct effect on intracellular Gln and GSH levels.
Gln has been shown to protect endothelial cells exposed to hydrogen peroxide by stabilizing ATP levels (33), and ATP levels in A549 cells exposed to hyperoxia have been shown to be maintained in cells receiving supplemental Gln (7). Maintaining cellular ATP levels has been associated with altering the balance between apoptosis and necrotic cell death in favor of apoptotic cell death (34). We observed that 10-mM Gln+ cells had significantly more cells surviving when compared with 2-mM Gln+ cells after 96 h of hyperoxia. We used annexin V binding to determine if altering the concentration of Gln in the media altered the balance of cells undergoing apoptosis and necrosis during hyperoxic exposure. We found no decrease in the percentage of cells undergoing necrotic cell death in 10-mM Gln+ cells compared with 2-mM and 5-mM Gln+ cells at 48, 72, or 96 h of hyperoxia. At 96 h of hyperoxia the percentage of necrotic cells ranged from 21.7-29.1% in the 2-, 5-, and 10-mM Gln+ cells compared 6.6% in the room air cells. Therefore, we found (as did others) that the primary mode of cell death from hyperoxia in A549 cells was necrosis (35, 36). The percentage of cells undergoing necrotic cell death in our experiments, however, may have been underestimated because only adherent cells were counted.
Adaptation or cross-tolerance to ammonia toxicity may explain the protective effect of high concentrations of Gln against hyperoxia. Gln acts as an osmolyte in the cell and it has been proposed that cerebral toxicity from hyperammonemia is secondary to astrocyte swelling from increase synthesis of Gln through the GS pathway. Cell swelling from hyperammonemia can be partially alleviated by MSO (37). The mechanism by which this occurs is unclear; however, if high concentrations of Gln exist outside the cell, less cellular Gln may be synthesized in the cell through the GS pathway. This could lead to increased tolerance to ammonia toxicity through less cellular production of Gln.
In summary, we have shown that supplemental Gln attenuates cell injury from hyperoxia in A549 cells and that
exogenous Gln is necessary for growth of A549 lung epithelial cells in room air. GS is present in A549 cells but its
role is unclear. Cells with GS, in the absence of supplemental Gln, do not proliferate in room air or survive well
in hyperoxia. The addition of MSO to Gln cells, however, results in much fewer cells surviving in hyperoxia
when compared with Gln cells, suggesting a role for GS
during hyperoxic stress. The additive injurious effect of
MSO on cells without supplemental Gln in hyperoxia does
not appear to be a direct result of glutathione depletion
because supplemental Gln can rescue A549 cells from the
effect of MSO in hyperoxia but not in cells treated with
BSO. Finally, we found that high concentrations of supplemental Gln can improve survival of A549 cells in hyperoxia, possibly through the maintenance of GSH levels in
the cell. The high concentrations of Gln, however, do not
decrease the percentage of cells undergoing necrosis.
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Footnotes |
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Address correspondence to: Dr. Sharon McGrath-Morrow, Department of Pediatric Pulmonary, Johns Hopkins Hospital, Park 316 N. Wolfe St., Baltimore, MD 21287-2533. E-mail: smorrow{at}jhmi.edu
(Received in original form June 28, 2001 and in revised form February 13, 2002).
Abbreviations: L-buthionine-[S,R]-sulfoximine, BSO; bromodeoxyuridine, BRDU; enhanced chemiluminescence, ECL; fetal bovine serum, FBS; glutamine, Gln; Gln synthetase, GS; glutathione, GSH; heat shock protein-70, HSP70; mitochondrial membrane permeability, MMP; L-methionine sulfoximine, MSO; phosphate-buffered saline, PBS; proliferating cell nuclear antigen, PCNA.
Acknowledgments:
The authors would like to thank Dr. Saul Brusilow and Dr.
Lee Choo-Kang for their insight and useful discussions. This work was supported by a NIH-KO8 award #HL03624
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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