Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC187
Secretion by the human airway epithelial cell line Calu-3 depends on basolateral chloride loading by AE2
J. Huang1, J. Shan1, S. L. Alper2, J. W. Hanrahan1,3
1. Physiology, McGill University, Montreal, Quebec, Canada. 2. Medicine, Harvard Medical School and Molecular and Vascular Medicine Unit and Renal Unit, Beth Israel Deaconess Medical Center, Boston, Massachusetts, United States. 3. Research Institute, McGill University Health Centre, Montreal, Quebec, Canada.
The anion exchanger type 2 (AE2) carries out Na+-independent Cl-/HCO3- exchange in non-epithelial cells and at the basolateral membrane of many epithelia, however its role in transport across airway epithelial cells is poorly understood. We have identified the splice variants AE2a and AE2b2 in the Calu-3 cell line, a commonly used model for human airway submucosal gland serous cells. To assess their physiological role, we targeted both variants using lentivirus-mediated RNA interference to generate a stable AE2 knock-down Calu-3 cell line (AE2-KD). AE2 mRNA expression was reduced by 87% according to real-time PCR (n=6), and Western blots revealed ~89% reduction in AE2 protein expression. Stimulation of fluid secretion by cpt-cAMP (200 μmoles/L) and forskolin (10 μmoles/L) was reduced by 60% in AE2-KD cell monolayers when compared with control monolayers (AE2-KD: 36.6±0.6 μL/72h vs Control: 95.8±5.3 μL/72h, n=6, p< 0.0001). Residual secretion by AE2-KD cells was 4-fold more sensitive to basolateral bumetanide (AE2-KD: ~39% inhibition, Control: 10% inhibition; n=6), suggesting a larger fraction of the basolateral Cl- entry was mediated by the Na+-K+-2Cl cotransporter NKCC1. Forskolin-stimulated transepithelial currents calculated under open circuit conditions were also attenuated in AE2-KD cells (1.15±0.27 μEq cm-2 h-1 vs 2.18±0.19 μEq cm-2 h-1, n=3, p<0.0001). pH-stat measurements of basolateral HCO3- flux in the absorptive direction (produced by imposing an apical-to-basolateral HCO3- gradient and permeabilizing the apical membrane with 360μg/mL nystatin) indicated that basolateral Cl-/HCO3- exchange in AE2-KD cells was negligible (0.01±0.01 μEq cm-2 h-1) compared to control Calu-3 cells (0.48±0.08 vs , n=3, p<0.0001). Thus AE2 appears to be the predominant anion exchanger mediating basolateral HCO3- flux in these cells. Finally, suppressing AE2 levels increased the level of cystic fibrosis transmembrane conductance regulator CFTR protein by 70% without altering expression of NKCC1 or the sodium bicarbonate cotransporter NBC1 (n=3), perhaps in response to alterations in intracellular pH. These results demonstrate that AE2 mediated Cl-/HCO3- exchange contributes to basolateral Cl- entry and plays an important role in anion and fluid secretion by Calu-3 airway epithelial cells.
Where applicable, experiments conform with Society ethical requirements