Dependence of cAMP meditated increases in Cl- and HCO(3)- 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 HCO(3)(-) 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 HCO(3)(-) permeability and steady-state HCO(3)(-) 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 approximately 80% in CFTR siRNA treated cultures. Forskolin (10 microM) 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 HCO(3)(-) permeability was increased 2-fold by 10 microM forskolin, which was reduced to control level in siRNA treated cultures. Similarly, there was no effect on baseline apical HCO(3)(-) permeability by knocking down CFTR expression. The steady-state apical-basolateral pH gradient (DeltapH) at 4h in control cultures was increased approximately 2.5-fold by forskolin. In CFTR siRNA treated cells, the baseline DeltapH was similar to control, however forskolin did not have a significant effect. We conclude that forskolin induced increases in apical HCO(3)(-) permeability in bovine corneal endothelium requires CFTR. However, CFTR does not have a major role in determining baseline apical chloride or HCO(3)(-) permeability.

Figure 1

Western blot for CFTR and βactin using protein from control, siControl, and CFTR siRNA treated (20 and 5 nM) cultured bovine corneal endothelial cells. Representative of three experiments.

Figure 2

Apical Cl^- Permeability in control and CFTR siRNA treated cells. Cells were depleted of Cl^-, loaded with the halide sensitive fluorescent dye MEQ and perfused on basolateral and apical sides with Cl^- free ringer’s solution. Relative apical Cl^- permeability is measured as the initial rate of MEQ fluorescence quenching upon addition of Cl^- to the apical perfusing solution. A. Representative experiments showing the change in MEQ fluorescence relative to the starting fluorescence value in response to addition of chloride on the apical side in the absence and presence of 10 μM forskolin for siControl and CFTR siRNA treated cultures. B. Bar graph summarizes the Cl^- permeability data (n=8). *, mean value significantly different compared to control (p<0.05); error bars show Standard Deviation.

Figure 3

Apical HCO₃^- Permeability in control and CFTR siRNA treated cells. A. Cells were perfused in bicarbonate-rich ringer’s on both sides. Where indicated the apical ringer’s was changed to low bicarbonate (LB) in the absence and then presence of 10 μM forskolin. Upper trace: siControl; Lower trace: CFTR siRNA. Rates (dpHi/min) are indicated adjacent to the trace for these experiments. B. Bar graph summarizes the HCO₃^- permeability data (n=8). *, mean value significantly different compared to control (p<0.05); error bars show Standard Deviation.

Figure 4

Effect of CFTR siRNA knockdown on steady-state ΔpH (apical-basolateral pH) after 4 hours. Light bars unstimulated cells; Dark bars 10 μM forskolin stimulation. *significantly different from unstimulated (p<0.05, n=6); error bars show Standard Deviation.