Apical and basolateral CO2- HCO 3 − permeability in cultured bovine corneal endothelial cells

Abstract

Corneal endothelial function is dependent on[Formula: see text] transport. However, the relative[Formula: see text] permeabilities of the apical and basolateral membranes are unknown. Using changes in intracellular pH secondary to removing CO2-[Formula: see text](at constant pH) or removing [Formula: see text]alone (at constant CO2) from apical or basolateral compartments, we determined the relative apical and basolateral [Formula: see text] permeabilities and their dependencies on Na+ and Cl−. Removal of CO2-[Formula: see text]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[Formula: see text] at constant CO2 indicated that the basolateral[Formula: see text] 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[Formula: see text] flux by one-half. In the absence of Cl−, there was an apparent increase in apical [Formula: see text] flux under constant-pH conditions; however, no net change could be measured under constant-CO2 conditions. Basolateral flux was slowed ∼30% in the absence of Cl−, but the net flux was unchanged. The steady-state alkalinization after removal of CO2-[Formula: see text]apically suggests that CO2diffusion may contribute to apical[Formula: see text] 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[Formula: see text] permeability is consistent with Na+-[Formula: see text]cotransport. Changes in[Formula: see text] flux in the absence of Cl− are most likely due to Na+-[Formula: see text]cotransport-induced membrane potential changes that cannot be dissipated. Apical [Formula: see text] permeability is relatively low, but may be augmented by CO2 diffusion in conjunction with a CAIV.