Mitochondrial Oxidative Stress in the Lungs of Cystic Fibrosis Transmembrane Conductance Regulator Protein Mutant Mice

Abstract

Cystic fibrosis is a fatal genetic disorder involving dysfunction of the cystic fibrosis transmembrane regulator protein (CFTR) resulting in progressive respiratory failure. Previous studies indicate that CFTR regulates cellular glutathione (GSH) transport and that dysfunctional CFTR is associated with chronic pulmonary oxidative stress. The cause and the source of this oxidative stress remain unknown. The current study examines the role of the mitochondria in CFTR-mediated pulmonary oxidative stress. Mitochondrial GSH levels and markers of DNA and protein oxidation were assessed in the lung mitochondria from CFTR-knockout mice. In addition, in vitro models using human CFTR-sufficient and -deficient lung epithelial cells were also employed. Mitochondrial GSH levels were found to be decreased up to 85% in CFTR-knockout mice, and 43% in human lung epithelial cells deficient in CFTR. A concomitant 29% increase in the oxidation of mitochondrial DNA, and a 30% loss of aconitase activity confirmed the existence of a mitochondrial oxidative stress. Flow cytometry revealed significantly elevated levels of cellular reactive oxygen species (ROS) in CFTR-deficient human lung cells. These studies suggest that dysfunctional CFTR leads to an increase in the level of ROS and mitochondrial oxidative stress. This oxidative stress, however, appears to be a consequence of lower mitochondrial GSH levels and not increased oxidation of GSH. Further studies are needed to determine how CFTR deficiency contributes to mitochondrial oxidative stress and the role this plays in CFTR-mediated lung pathophysiology.

Organelle redox of CF and CFTR-corrected airway epithelia

“…Mitochondrial GSH levels have also been reported to be lower in both lung cells from CFTR knock out mice and nonCF mice and also in cultured human CF (IB3) vs. CFTR-corrected (C38) cells (59). In contrast, the present data showed no differences in mitochondrial redox potentials between CF and CFTR-corrected cells.…”

“…Hypoxia like that observed for CF airway epithelial cell cultures is known to increase mitochondrial production of ROS (50,15,16,51), and recent measurements of mitochondrial GSH and DNA oxidation have given indications that CF mitochondria are oxidized compared to nonCF (17). However, experiments using roGFP1 here showed that adenoviral expression of CFTR did not alter redox regulatory properties of the cell surface, mitochondria, cytosol or ER.…”

“…It is possible that differences in mitochondrial redox potentials between CF15 and CFTR-corrected CF15 cells were too small to measure with our method -assuming mitochondrial [GSH] = 10 mM in non-CF or CFTR-corrected cells and using data in Fig. 2 from Day et al (59) and the Nernst equation, we calculate that mitochondrial redox potentials would have been more oxidized by only 6-8 mV in IB3 (CF) compared to C38 (CFTR-corrected) cells. Alternatively, it is also possible that the CF15 cells have different mitochondrial GSH transport mechanisms compared to the IB3-C38 cells.…”

“…Because hypoxia can lead to increased production of ROS by mitochondria (15,16), it might be expected that this effect would oxidize the mitochondrial matrix in CF cells compared to normal. Recent experiments have shown substantial reductions in glutathione levels measured in mitochondria and cytosol from CF cells, indicative of excess oxidation in CF (17).…”

Glutathione Transport Is a Unique Function of the ATP-binding Cassette Protein ABCG2

“…The cells were lysed with a sonicating probe (Cole-Parmer Instruments, Vernon Hill, IL), and lactate dehydrogenase activity was determined in both media and lysate samples. To measure toxicity, the percent of total lactate dehydrogenase release was utilized as described previously (22). Cell lysate protein values were used to normalize GSH values and were determined spectrophotometrically with the Coomassie Protein Assay kit according to the manufacturer's protocol (Pierce).…”