Exocytosis is not involved in activation of Cl⁻ secretion via CFTR in Calu-3 airway epithelial cells

Abstract

Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl−channel, which mediates transepithelial Cl− transport in a variety of epithelia, including airway, intestine, pancreas, and sweat duct. In some but not all epithelial cells, cAMP stimulates Cl− secretion in part by increasing the number of CFTR Cl− channels in the apical plasma membrane. Because the mechanism whereby cAMP stimulates CFTR Cl− secretion is cell-type specific, our goal was to determine whether cAMP elevates CFTR-mediated Cl− secretion across serous airway epithelial cells by stimulating the insertion of CFTR Cl− channels from an intracellular pool into the apical plasma membrane. To this end we studied Calu-3 cells, a human airway cell line with a serous cell phenotype. Serous cells in human airways, such as Calu-3 cells, express high levels of CFTR, secrete antibiotic-rich fluid, and play a critical role in airway function. Moreover, dysregulation of CFTR-mediated Cl− secretion in serous cells is thought to contribute to the pathophysiology of cystic fibrosis lung disease. We report that cAMP activation of CFTR-mediated Cl− secretion across human serous cells involves stimulation of CFTR channels present in the apical plasma membrane and does not involve the recruitment of CFTR from an intracellular pool to the apical plasma membrane.

E3KARP Mediates the Association of Ezrin and Protein Kinase A with the Cystic Fibrosis Transmembrane Conductance Regulator in Airway Cells

“…Interestingly, CFTR expression at the apical membrane was not homogenous in Calu-3 epithelia (Fig. 8D), which is consistent with the previous findings of Loffing et al (19). E3KARP was present also at the apical membranes, although lower levels appeared also to be present in the lateral membranes (Fig.…”

“…The results from these protein interaction assays suggest that E3KARP should be found in proximity to ezrin and CFTR, which are localized at the apical membrane domain of polarized airway epithelia (8,19). By using laser-scanning confocal immunofluorescence microscopy, we found that ezrin and CFTR are expressed predominantly at the apical membrane domain of polarized Calu-3 cells (Fig.…”

Myosin VI Regulates Endocytosis of the Cystic Fibrosis Transmembrane Conductance Regulator

“…Cell Lines and Cell Culture-Calu-3 cells were obtained from the American Type Culture Collection (Manassas, VA) and cultured in tissue culture flasks (Costar, Corning Corporation, Corning, NY) at 37°C in minimum Eagle's medium containing 50 units/ml of penicillin, 50 g/ml of streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, and 10% fetal bovine serum in a 5% CO 2 /95% air incubator at 37°C as described previously (48). Calu-3 cells were also grown on Transwell permeable supports (24-mm diameter, 0.4-m pore size; Corning Corporation) in air-liquid interface culture for 14 to 21 days (48).…”

“…Calu-3 cells were also grown on Transwell permeable supports (24-mm diameter, 0.4-m pore size; Corning Corporation) in air-liquid interface culture for 14 to 21 days (48). Under these conditions, Calu-3 cells become polarized (48). Sodium n-butyrate (5 mM) was added to the Calu-3 cell culture medium 48 h before experiments to stimulate CFTR expression.…”

“…Sodium n-butyrate treatment did not affect the expression of endogenous myosin VI and Dab2. HEK293 cells (American Type Culture Collection) were seeded in tissue culture flasks coated with Vitrogen plating medium containing Dulbecco's modified Eagle's medium (JRH Biosciences, Lenexa, KS), human fibronectin (10 g/ml; Collaborative Biomedical Products, Bedford, MA), 1% Vitrogen 100 (Collagen, Palo Alto, CA), and bovine serum albumin (10 g/ml; Sigma) (48) and cultured at 37°C in Dulbecco's modified Eagle's medium supplemented with 50 g/ml of streptomycin, 50 units/ml of penicillin, and 2 mM L-glutamine in a 5% CO 2 /95% air incubator at 37°C. HEK293 cells stably expressing wt-CFTR were a generous gift of Dr. Neil Bradbury (University of Pittsburgh School of Medicine, Pittsburgh, PA) (49).…”