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Articles in PresS, published online ahead of print November 27, 2001
Am J Physiol Cell Physiol, 10.1152/ajpcell.00384.2001
Submitted on August 9, 2001
Accepted on November 25, 2001
1 School of Optometry, Indiana University, Bloomington, IN, USA
* To whom correspondence should be addressed. E-mail: jbonanno{at}indiana.edu.
HCO3-dependent fluid secretion by the corneal endothelium controls corneal hydration and maintains corneal transparency. Recently it was shown that mRNA for the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is expressed in corneal endothelium, however, protein expression, functional localization and possible role in HCO3- transport have not been reported. Immunobloting for CFTR showed a single band at approximately 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 MEQ 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 50mM FSK. FSK-activated apical chloride permeability was unaffected by H2DIDs (250µM), however NPPB (50 µM) and glibenclamide (100µM) inhibited activated Cl- fluxes by 45% and 30%, respectively. FSK-activated basolateral Cl- permeability was insensitive to NPPB, glibenclamide or furosemide, but inhibited 80% by H2DIDS. HCO3- permeability was estimated by measuring changes in pHi in response to quickly lowering bath [HCO3-]. 50µM FSK increased apical HCO3- permeability by 2 fold, which was inhibited 42% by NPPB and 65% by Glibenclamide. Basolateral HCO3- permeability was unaffected by FSK. 50µM genistein significantly increased apical HCO3- and Cl- permeability by 1.8 and 16 fold, respectively. When 50µM genistein was combined with 50µM forskolin, there was no further increase in Cl- permeability, however HCO3- permeability was reduced to control level. In sum, we conclude that CFTR is present in the apical membrane of bovine corneal endothelium and could contribute to transendothelial Cl- and HCO3- transport. Furthermore, there is a cAMP activated Cl- pathway on the basolateral membrane that is not CFTR.
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