Dependence of cAMP meditated increases in Cl− and HCO3− permeability on CFTR in bovine corneal endothelial cells

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) is present on the apical membrane of corneal endothelial cells. Increasing intracellular [cAMP] with forskolin stimulates an NPPB and glibenclamide-inhibitable apical Cl− and HCO3− permeability [Sun, X.C., Bonanno, J.A., 2002. Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells. Am. J. Physiol. Cell Physiol. 282, C673-C683]. To definitively determine that the increased permeability is dependent on CFTR, we used an siRNA knockdown approach. Apical Cl− and HCO3− permeability and steady-state HCO3− flux were measured in the presence or absence of forskolin using cultured bovine corneal endothelial cells that were transfected with CFTR siRNA or a scrambled sequence control. CFTR protein expression was reduced by ∼80% in CFTR siRNA treated cultures. Forskolin (10μM) increased apical chloride permeability by 7-fold, which was reduced to control level in siRNA treated cells. CFTR siRNA treatment had no effect on baseline apical chloride permeability. Apical HCO3− permeability was increased 2-fold by 10μM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO3− permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (ΔpH) at 4h in control cultures was increased ∼2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline ΔpH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO3− permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO3− permeability.