Apical and basolateral CO2-HCO3- permeability in cultured bovine corneal endothelial cells

Apical and basolateral CO2-HCO, permeability in cultured bovine corneal endothelial cells. Am. J. Physiol. 277 (Cell Physiol. 46): C545-C553, 1999.-Corneal endothelial function is dependent on HCO3- transport. However, the relative HCO3- permeabilities of the apical and basolateral membranes are unknown. Using changes in intracellular pH secondary to removing CO2-HCO3-; (at constant pH) or removing HCO3- alone (at constant CO2) from apical or basolateral compartments, we determined the relative apical and basolateral HCO, permeabilities and their dependencies on Na+ and Cl-. Removal of CO2-HCO3- from the apical side caused a steady-state alkalinization (+0.08 pH units), and removal from the basolateral side caused an acidification (-0.05 pH units). Removal of HCO3- at constant CO2 indicated that the basolateral HCO3- fluxes were about three to four times the apical fluxes. Reducing perfusate Na+ concentration to 10 mM had no effect on apical flux but slowed basolateral HCO3- flux by one-half. In the absence of Cl-, there was an apparent increase in apical HCO3- flux under constant-pH conditions; however, no net change could be measured under constant-CO2 conditions. Basolateral flux was slowed similar to 30% in the absence of Cl-, but the net flux was unchanged. The steady-state alkalinization after removal of CO2-HCO3-, apically suggests that CO2 diffusion may contribute to apical HCO3- flux through the action of a membrane-associated carbonic anhydrase. Indeed, apical CO2 fluxes were inhibited by the extracellular carbonic anhydrase inhibitor benzolamide and partially restored by exogenous carbonic anhydrase. The presence of membrane-bound carbonic anhydrase (CAIV) was confirmed by immunoblotting. We conclude that the Na+-dependent basolateral HCO3- permeability is consistent with Na+-nHCO(3)(-), cotransport. Changes in HCO3-, flux in the absence of Cl- are most likely due to Na+-nHCO(3)(-), cotransport-induced membrane potential changes that cannot be dissipated. Apical HCO3- permeability is relatively low, but may be augmented by CO2 diffusion in conjunction with a CAIV.