Oxidant stress induces gamma-glutamylcysteine synthetase and glutathione synthesis in human bronchial epithelial NCI-H292 cells
| Autor: | Phillip J Thompson, Neil L. A. Misso, David Neil Watkins, Sangeeta Ray |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
chemistry.chemical_classification
Reactive oxygen species biology Immunology Inflammation Glutathione medicine.disease_cause Molecular biology Enzyme assay chemistry.chemical_compound Menadione chemistry Biochemistry biology.protein medicine Immunology and Allergy medicine.symptom Glyceraldehyde 3-phosphate dehydrogenase Oxidative stress Intracellular |
| Zdroj: | Clinical & Experimental Allergy. 32:571-577 |
| ISSN: | 0954-7894 |
| Popis: | BACKGROUND The bronchial epithelium is exposed to reactive oxygen species (ROS) derived from cigarette smoke, air pollutants and activated leucocytes. Glutathione (GSH) prevents ROS-mediated loss of cell function, tissue injury and inflammation, and its synthesis is regulated by gamma-glutamylcysteine synthetase (gamma-GCS). However, the capacity of bronchial epithelial cells to adapt to oxidative stress and the mechanisms involved are not known. OBJECTIVE To investigate the effects of oxidative stress on the regulation of GSH synthesis in human bronchial epithelial (NCI-H292) cells. METHODS NCI-H292 cells were exposed to menadione and intracellular GSH concentrations were measured by spectrophotometry. gamma-GCS activity was measured by HPLC assay and changes in gamma-GCS mRNA by Northern blotting. RESULTS Exposure to menadione (MQ, 10-200 microm, 30-120 min) decreased total cellular GSH content, measured immediately after exposure to MQ. However, GSH content measured 6-12 h after withdrawal of the oxidant stress (MQ, 50 microm, 30 min), increased c. two fold over baseline levels (P < 0.001). gamma-GCS activity measured 6 h (21.7 +/- 3.4 nmol/min/mg, SD, n = 5, P < 0.01) or 12 h (23.2 +/- 4.6, P < 0.001) after MQ treatment was also significantly increased compared with untreated cells (12.8 +/- 1.0). Similarly, gamma-GCS mRNA expression increased 1.3-1.6-fold relative to GAPDH mRNA, 3-6 h after MQ treatment. The MQ-induced increase in gamma-GCS mRNA expression was completely inhibited by actinomycin D. CONCLUSIONS Bronchial epithelial (NCI-H292) cells respond rapidly and sensitively to oxidant stress, and this adaptive response is mediated by increased gamma-GCS mRNA transcription and enzyme activity. |
| Databáze: | OpenAIRE |
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