Role of carbonic anhydrase IV in corneal endothelial HCO3- transport.
OBJECTIVE
Carbonic anhydrase activity has a central role in corneal endothelial function. The authors examined the role of carbonic anhydrase IV (CAIV) in facilitating CO(2) flux, HCO(3)(-) permeability, and HCO(3)(-) 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 CO(2) fluxes and HCO(3)(-) permeability were determined by measuring pH(i) changes in response to altering the CO(2) or HCO(3)(-) gradient across the apical membrane. Basolateral to apical (B-to-A) HCO(3)(-) 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 DeltapH (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 microM benzolamide and CAIV siRNA reduced apparent apical CO(2) flux by approximately 20%; however, they had no effect on HCO(3)(-) permeability or HCO(3)(-) 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 CO(2) flux.


CONCLUSIONS
CAIV does not facilitate steady state cell-to-apical CO(2) flux, apical HCO(3)(-) permeability, or B-to-A HCO(3)(-) flux. Steady state pH changes, however, suggest that CAIV may have a role in buffering the apical surface.
Sun
Xing Cai
XC
Indiana University, School of Optometry, Bloomington, Indiana 47405, USA.
Li
Jinhua
J
Cui
Miao
M
Bonanno
Joseph A
JA
D000818:Animals; D001485:Basement Membrane / Q000378:metabolism; D001574:Benzolamide / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism*; D001692:Biological Transport; D002245:Carbon Dioxide / Q000378:metabolism; D030741:Carbonic Anhydrase IV / Q000037:antagonists & inhibitors / Q000502:physiology*; D002257:Carbonic Anhydrase Inhibitors / Q000494:pharmacology; D002417:Cattle; D002463:Cell Membrane Permeability; D002478:Cells, Cultured; D004728:Endothelium, Corneal / Q000166:cytology / Q000201:enzymology*; D019084:Fluorescent Antibody Technique, Indirect; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007425:Intracellular Membranes; D018613:Microscopy, Confocal; D034741:RNA, Small Interfering / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027982:Sodium-Bicarbonate Symporters / Q000378:metabolism; D014162:Transfection
D001639:Bicarbonates; D002257:Carbonic Anhydrase Inhibitors; D034741:RNA, Small Interfering; D027982:Sodium-Bicarbonate Symporters; D002245:Carbon Dioxide; D030741:Carbonic Anhydrase IV; D001574:Benzolamide