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Role of Carbonic Anhydrase IV in Corneal Endothelial HCO₃^– Transport

From Indiana University, School of Optometry, Bloomington, Indiana.

PURPOSE. Carbonic anhydrase activity has a central role in corneal endothelial function. The authors examined the role of carbonic anhydrase IV (CAIV) in facilitating CO₂ flux, HCO₃^– permeability, and HCO₃^– flux across the apical membrane.

METHODS. Primary cultures of bovine corneal endothelial cells were established on membrane-permeable filters. Apical CAIV was inhibited by benzolamide or siRNA knockdown of CAIV. Apical CO₂ fluxes and HCO₃^– permeability were determined by measuring pH_i changes in response to altering the CO₂ or HCO₃^– gradient across the apical membrane. Basolateral to apical (B-to-A) HCO₃^– flux was determined by measuring the pH of a weakly buffered apical bath in the presence of basolateral bicarbonate-rich Ringer solution. In addition, the effects of benzolamide and CAIV knockdown on steady state pH (apical-basolateral compartment pH) after 4-hour incubation in DMEM were measured.

RESULTS. CAIV expression was confirmed, and CAIV was localized exclusively to the apical membrane by confocal microscopy. Both 10 µM benzolamide and CAIV siRNA reduced apparent apical CO₂ flux by approximately 20%; however, they had no effect on HCO₃^– permeability or HCO₃^– flux. The steady state apical-basolateral pH gradient at 4 hours was reduced by 0.12 and 0.09 pH units in benzolamide- and siRNA-treated cells, respectively, inconsistent with a net cell-to-apical compartment CO₂ flux.

CONCLUSIONS. CAIV does not facilitate steady state cell-to-apical CO₂ flux, apical HCO₃^– permeability, or B-to-A HCO₃^– flux. Steady state pH changes, however, suggest that CAIV may have a role in buffering the apical surface.