Evidence for parallel Na⁺H⁺ and Na⁺-dependent Cl^-HCO₃^- exchangers in cultured bovine lens cells

BCECF, a cell-entrapable dye with a pH-sensitive fluorescence spectrum, was used to identify transport mechanisms contributing to pH homeostasis of cultured bovine lens epithelial cells. Cells from a spontaneously established lineage were grown on glass coverslips that fit diagonally in a standard cuvette and intracellular pH (pH_i) was measured. Under perfusion with a CO₂HCO₃^--free medium (pH 7·45), pH_i was 7·19 ± 0·21 (mean ± s.d., n = 94 cell preparations). Cell acidifications (pH_i to 6·65, n = 8 induced by the 'NH₄⁺-loading' method were rapidly followed by a Na⁺-dependent, amiloride-inhibitable pH_i recovery. Introduction of a CO₂HCO₃^--rich medium (pH 7·45) resulted in a small acidification (0·18 ± 0·04 U, n = 16; P < 0·002) due to rapid CO₂ entry and an ensuing slow alkalinization to a pH_i near the control CO₂HCO₃^--free value. Subsequent removal of Cl^- resulted in a further alkalinization of 0·18 ± 0·02 U (n = 13; P < 0·001). This Cl^- effect was completely inhibited by the absence of Na⁺, but was insensitive to amiloride, suggesting the presence of a Na⁺-dependent Cl^-HCO₃^- exchanger. Consistent with this posit, the reintroduction of Na⁺ to cells perfused in the absence of the cation with a HCO₃^--containing, amiloride-complemented solution resulted in a gradual recovery from the acidic pH_i induced by the baseline conditions (n = 6). The amiloride-insensitive, Na⁺- and HCO₃^--dependent recovery was completely inhibited in cells pre-incubated with DIDS. Two independent manipulations known to depolarize cell potential failed to elicit a pH_i change indicating the absence of an electrogenic acid-base pathway, although the presence of an electroneutral Na⁺HCO₃^--cotransporter could not be excluded. It is concluded that these cells possess a highly efficient Na⁺H⁺ exchanger and a low Na⁺-dependent Cl^-HCO₃^- exchange activity since in cells pre-acidified by the 'NH₄⁺-loading' method, virtually all of the acid extruding capacity could be accounted for by the action of the former.