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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

Immunocytochemical localization of Na⁺-HCO₃^– cotransporters and carbonic anhydrase dependence of fluid transport in corneal endothelial cells

²Ophthalmology and ³Physiology and Cellular Biophysics, Columbia University, New York, New York 10032; and ¹Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103

Submitted 2 December 2003 ; accepted in final form 6 February 2004

In corneal endothelium, there is evidence for basolateral entry of HCO₃^– into corneal endothelial cells via Na⁺-HCO₃^– cotransporter (NBC) proteins and for net HCO₃^– flux from the basolateral to the apical side. However, how HCO₃^– exits the cells through the apical membrane is unclear. We determined that cultured corneal endothelial cells transport HCO₃^– similarly to fresh tissue. In addition, Cl^– channel inhibitors decreased fluid transport by at most 16%, and inhibition of membrane-bound carbonic anhydrase IV by benzolamide or dextran-bound sulfonamide decreased fluid transport by at most 29%. Therefore, more than half of the fluid transport cannot be accounted for by anion transport through apical Cl^– channels, CO₂ diffusion across the apical membrane, or a combination of these two mechanisms. However, immunocytochemistry using optical sectioning by confocal microscopy and cryosections revealed the presence of NBC transporters in both the basolateral and apical cell membranes of cultured bovine corneal endothelial cells and freshly isolated rabbit endothelia. This newly detected presence of an apical NBC transporter is consistent with its being the missing mechanism sought. We discuss discrepancies with other reports and provide a model that accounts for the experimental observations by assuming different stoichiometries of the NBC transport proteins at the basolateral and apical sides of the cells. Such functional differences might arise either from the expression of different isoforms or from regulatory factors affecting the stoichiometry of a single isoform.

confocal microscopy; cryosections; stoichiometry; pH titration; chloride channels

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