Inhibition of glutamine synthetase in a549 cells during hyperoxia.

High oxygen concentrations are used in the treatment of acute respiratory distress syndrome and hyaline membrane dis- ease. 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 Glnand Glncells. In hyperoxia, Gln � cells had greater survival then Glncells treated with MSO. Supplemental Gln could rescue cells in hyperoxia from the ef- fect of MSO, suggesting that GS, through the endogenous synthesis of Gln, could attenuate hyperoxic cell injury. In hy- peroxia, cells treated with 10-mM concentrations of Gln had increased survival compared with cells receiving 2-mM con- centrations. The higher concentration of Gln, however, did not decrease the percentage of cells undergoing necrosis. Hyperoxia exacerbates lung injury in patients with acute respiratory distress syndrome and in infants with bron- chopulmonary dysplasia (2). Excess oxygen molecules can overwhelm the cells' antioxidant mechanisms, leading to release of free oxygen radicals and subsequent growth ar- rest, injury, and cell death in the lung (3, 4). The lungs are a major source of glutamine (Gln) biosynthesis and gluta- mine 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 in- tegrity (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 epithe- lial 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 metabo- lized, with only 34% of it remaining unchanged in the intes- tinal venous blood (7). In type 2 alveolar cells, glutamine transport occurs predominantly through a high-affinity so- dium-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 demon- strated 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 neu- rons (12). GS induction has also been associated with cel- lular 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.