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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% O2 produced
similar improvements in Cl current and CFTR protein as
air-liquid interface culture, while increasing
PO2 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
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