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Xing Cai Sun, Jinhua Li, Miao Cui, Joseph A. Bonanno; Role of Carbonic Anhydrase IV in Corneal Endothelial HCO3− Transport. Invest. Ophthalmol. Vis. Sci. 2008;49(3):1048-1055. doi: 10.1167/iovs.07-1188.
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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 pHi 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 ΔpH (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.
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