Molecular regulation of lung endothelial glutamine synthetase expression.

Abstract

The lungs play a crucial role in maintaining amino acid homeostasis by exporting glutamine. Lung glutamine release is increased markedly in patients with sepsis, and in rat models injection of endotoxin causes up-regulation of glutamine synthetase (GS), the principal enzyme of glutamine synthesis. To investigate the molecular regulation of this response in the lung microvasculature we studied the effects of several hormones and cytokines that mediate the septic response on the expression of GS in rat microvascular pulmonary endothelial cells (MPECs). MPECs were grown to confluence and incubated with the synthetic glucocorticoid dexamethasone, prostaglandins, cytokines, or activated complement C5a. Cellular lysates were prepared and total cellular RNA was extracted, hybridized with a GS complementary DNA derived probe, and normalized to reduced glyceraldehyde-phosphate dehydrogenase. GS protein content was determined by Western blotting with a GS antibody. Of the compounds tested, only dexamethasone caused a marked increase (tenfold or greater) of GS messenger RNA and protein levels in MPECs. Dexamethasone-induced accumulation of GS messenger RNA was rapid, dose-dependent, and maximal after 4 hours of exposure. GS protein levels were maximal after 8 hours and remained elevated for at least 48 hours. The dose of dexamethasone sufficient to induce 50% of maximal GS messenger RNA and protein level increase was approximately 10 nmol/L. The dexamethasone-induce increase of GS messenger RNA level was completely blocked by the glucocorticoid receptor antagonist RU38486 and by the transcriptional inhibitor actinomycin D but was not inhibited by the translational inhibitor cycloheximide. Glucocorticoids augment GS expression in rat lung microvascular endothelial cells in a manner consistent with a direct transcriptional response via glucocorticoid receptors. Other septic response mediators had minimal effect on GS expression. Induction of GS expression by adrenocorticoids is likely to contribute to the marked ability of the lungs to augment glutamine production during septic states.