Reevaluation of Cl-/HCO3- exchange in cultured bovine corneal endothelial cells

PURPOSE. To determine the apical versus basolateral polarity of the putativ e anion exchanger in cultured bovine corneal endothelial cells (BCECs) and to examine the influence of Cl--dependent membrane potential (E-m) changes on HCO3- transport. METHODS. BCECs grown on permeable supports were used for independent perfus ion of epical and basolateral surfaces. Intracellular pH (pH(i)) was measur ed using the fluorescent dye BCECF. Relative changes in E-m were measured u sing the fluorescent dye bis-oxonol. Western blot analysis was used to dete ct immunoreactivity against the anion exchanger (AE1 or AE2). RESULTS. Cl- removal from apical and basolateral surfaces produced cellular alkalinization (apical side, 0.07 pH units; basolateral side, 0.06 pH unit s; both sides, 0.20 pH units). Application of 100 mu M H-2-4,4'-diisothiocy anatodihydrostilbene-acid (DIDS), an anion exchange inhibitor, on the apica l side produced an alkalinization (0.02 pH units) followed by acidification (-0.05 pH units), whereas basolateral H2DIDS caused a substantial acidific ation (-0.16 pH units). In the absence of Na+, Cl- removal from the apical side caused a transient alkalinization (0.03 pH units) followed by a return to baseline; Cl- removal from the basolateral side caused a small (-0.03) acidification. In Na+-free Ringer, apical H2DIDS produced a transient alkal inization (0.02 pH units), whereas basolateral exposure had no effect. 5-Ni tro-2(3-phenylpropylamino)benzoic acid (NPPB), N-phenylanthranilic acid (DP C), and niflumic acid (50-200 mu M), known Cl- channel blockers, produced c ellular acidification in control Ringer. Niflumic acid hyperpolarized E-m a nd inhibited depolarization after Cl- removal. Western blot analysis failed to detect AE2 expression in cultured BCECs. However, fresh BCECs produced a trace response. CONCLUSIONS. Physiological activity of an apical anion exchanger is weak in cultured BCECs. Cultured BCECs have significant Cl- conductance. Thus, cel lular alkalinization after Cl- removal is caused primarily by depolarizatio n of E-m, which drives HCO3- influx through the basolateral electrogenic Na +:nHCO(3)(-) cotransporter. In contrast with cultured BCECs, AE2 may be pre sent in fresh cells.