Expression, localization, and functional evaluation of CFTR in bovine corneal endothelial cells

Abstract

HCO${}_3^{-}$ -dependent fluid secretion by the corneal endothelium controls corneal hydration and maintains corneal transparency. Recently, it has been shown that mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the corneal endothelium; however, protein expression, functional localization, and a possible role in HCO${}_3^{-}$ transport have not been reported. Immunoblotting for CFTR showed a single band at ∼170 kDa for both freshly isolated and primary cultures of bovine corneal endothelial cells. Indirect immunofluorescence confocal microscopy indicated that CFTR locates to the apical membrane. Relative changes in apical and basolateral chloride permeability were estimated by measuring the rate of fluorescence quenching of the halide-sensitive indicator 6-methoxy-N-ethylquinolinium iodide during Cl⁻influx in the absence and presence of forskolin (FSK). Apical and basolateral Cl⁻ permeability increased 10- and 3-fold, respectively, in the presence of 50 μM FSK. FSK-activated apical chloride permeability was unaffected by H₂DIDs (250 μM); however, 5-nitro-2-(3-phenylpropyl-amino)benzoic acid (NPPB; 50 μM) and glibenclamide (100 μM) inhibited activated Cl⁻fluxes by 45% and 30%, respectively. FSK-activated basolateral Cl⁻ permeability was insensitive to NPPB, glibenclamide, or furosemide but was inhibited 80% by H₂DIDS. HCO${}_3^{-}$ permeability was estimated by measuring changes in intracellular pH in response to quickly lowering bath [HCO${}_3^{-}$ ]. FSK (50 μM) increased apical HCO${}_3^{-}$ permeability by twofold, which was inhibited 42% by NPPB and 65% by glibenclamide. Basolateral HCO${}_3^{-}$ permeability was unaffected by FSK. Genistein (50 μM) significantly increased apical HCO${}_3^{-}$ and Cl⁻ permeability by 1.8- and 16-fold, respectively. When 50 μM genistein was combined with 50 μM FSK, there was no further increase in Cl⁻ permeability; however, HCO${}_3^{-}$ permeability was reduced to the control level. In summary, we conclude that CFTR is present in the apical membrane of bovine corneal endothelium and could contribute to transendothelial Cl⁻ and HCO${}_3^{-}$ transport. Furthermore, there is a cAMP-activated Cl⁻ pathway on the basolateral membrane that is not CFTR.