Glutamine is an important mitochondrial substrate implicated in the protect
ion of cells from oxidant injury, but the mechanisms of its action are inco
mpletely understood. Human pulmonary epithelial-like (A549) cells were expo
sed to 95% O-2 for 4 days in the absence and presence of glutamine. Cell pr
oliferation in normoxia was dependent on glutamine, and glutamine deprivati
on markedly accelerated cell death in hyperoxia. Glutamine significantly in
creased cellular ATP levels in normoxia and prevented the loss of ATP in hy
peroxia seen in glutamine-deprived cells. Mitochondrial membrane potential
as assessed by flow cytometry with chloromethyltetramethylrosamine was incr
eased by glutamine in hyperoxia-exposed A549 cells, and a glutamine dose-de
pendent increase in mitochondrial membrane potential was detected. Glutamin
e-supplemented, hyperoxia-exposed cells had a higher O-2 consumption rate a
nd GSH content. Electron and fluorescence microscopy revealed that, in hype
roxia, glutamine protected cellular structures, especially mitochondria, fr
om damage. In hyperoxia, activity of the tricarboxylic acid cycle enzyme al
pha -ketoglutarate dehydrogenase was partially protected by its indirect su
bstrate, glutamine, indicating a mechanism of mitochondrial protection.