Improved oxygenation promotes CFTR maturation and trafficking in MDCK monolayers

• Published in: American Journal of Physiology: Cell Physiology 2001-01, Vol.280 (1), p.C135-C145
• Main Authors: Bebok, Zsuzsanna, Tousson, Albert, Schwiebert, Lisa M, Venglarik, Charles J
• Format: Article
• Language: English
• Subjects: Animals; Cell Culture Techniques - methods; Cell Differentiation - drug effects; Cell Differentiation - physiology; Cell Membrane - drug effects; Cell Membrane - metabolism; Cell Polarity - drug effects; Cell Polarity - physiology; Cells, Cultured - cytology; Cells, Cultured - drug effects; Cells, Cultured - metabolism; Colforsin - metabolism; Colforsin - pharmacology; Culture Media - pharmacology; Cystic Fibrosis - metabolism; Cystic Fibrosis - pathology; Cystic Fibrosis - physiopathology; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - metabolism; Dogs; Hypoxia - metabolism; Hypoxia - pathology; Hypoxia - physiopathology; Oxygen - pharmacology; Protein Transport - physiology; RNA, Messenger - metabolism
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.2001.280.1.c135

Departments of 1  Medicine, 4  Physiology and Biophysics, and 3  Cell Biology and 2  Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294-0005 Culturing airway epithelial cells with most of the apical media removed (air-liquid interface) has been shown to enhance cystic fibrosis transmembrane conductance regulator (CFTR)-mediated Cl secretory current. Thus we hypothesized that cellular oxygenation may modulate CFTR expression. We tested this notion using type I Madin-Darby canine kidney cells that endogenously express low levels of CFTR. Growing monolayers of these cells for 4 to 5 days with an air-liquid interface caused a 50-fold increase in forskolin-stimulated Cl current, compared with conventional (submerged) controls. Assaying for possible changes in CFTR by immunoprecipitation and immunocytochemical localization revealed that CFTR appeared as an immature 140-kDa form intracellularly in conventional cultures. In contrast, monolayers grown with an air-liquid interface possessed more CFTR protein, accompanied by increases toward the mature 170-kDa form and apical membrane staining. Culturing submerged monolayers with 95% O 2 produced similar improvements in Cl current and CFTR protein as air-liquid interface culture, while increasing P O 2 from 2.5% to 20% in air-liquid interface cultures yielded graded enhancements. Together, our data indicate that improved cellular oxygenation can increase endogenous CFTR maturation and/or trafficking. cystic fibrosis; cellular polarization; hypoxia; cell culture methods