BICARBONATE TRANSPORT UNDER NOMINALLY BICARBONATE-FREE CONDITIONS IN BOVINE CORNEAL ENDOTHELIUM

Fluid transport by the corneal endothelium is supported in air equilibrated, nominally HCO3— free Ringer's, yet it is inhibited by carbonic anhydrase inhibitors indicating dependence on the presence of CO2. It is possible that the CO2/HCO3— contained in air and/or the CO2 derived from oxidative metabolism is sufficient to support HCO3— transport. This study investigates whether endothelial HCO3— transport mechanisms, Na+: HCO3—cotransport and Cl—/HCO3— exchange, are active in the nominal absence of HCO3—. In cultured bovine corneal endothelial cells the rate of intracellular pH (pHi) recovery following acid loading in air-equilibrated, nominally HCO3—-free solutions was slowed 75% by 0.5 mM amiloride (inhibitor of Na+/H+ exchange), yet 60% of the acid load was still pumped out. This residual acid efflux (or base influx) was Na+-dependent, slowed 60 ± 15% by equilibration of solutions with 100% N2 (to minimize CO2 availability) and totally blocked by pretreatment with 100 μM DIDS, an inhibitor of Na+: HCO3— cotransport. Resting cells were acidified 0.19 ± 0.01 pH units by addition of 0.5 mM H2 DIDS when perfused with air-equilibrated solutions, but only 0.03 ± 0.02 when perfused with 100% N2 equilibrated solutions. Application of 500 μM acetazolamide (a carbonic anhydrase inhibitor) also acidified resting cells, 0.11 ± 0.01 pH units, but the acidification was eliminated when cells were perfused with 100% N2-equilibrated solutions, indicating the presence of carbonic anhydrase activity under nominally HCO3—-free conditions. Cl—/HCO3— exchange activity was eliminated by 100% N2 perfusion and slowed 29% by acetazolamide. Removal of exogenous CO2 blocked acetazolamide induced acidifications while treatment with 10 μM oligomycin (or oligomycin plus 10 mM NaN3) did not inhibit acetazolamide induced acidifications, indicating no contribution from metabolic CO2. Thus, it is concluded that under the conditions of these experiments: (1) both Na+: HCO3— cotransport and Cl—/HCO3— exchange are active under nominally HCO3—-free conditions, which may explain observations of fluid transport under these conditions and (2) only exogenous CO2 contributes toward HCO3—transport under nominally HCO3—-free conditions. © 1994 Academic Press, Inc.