pH Regulation in tissue-cultured bovine lens epithelial cells

Summary The intracellular pH (pH_i) of tissue-cultured bovine lens epithelial cells was measured in small groups of 6 to 10 cells using the trapped fluorescent dye 2,7-bis-(2-,carboxyethyl)-5 (and 6)carboxyfluorescein (BCECF). When perifused at 35°C with artificial aqueous humour solution (AAH) containing 16 mM HCO₃^-and 5% CO₂, pH 7.25, pH_iwas 7.19±0.02 (sem, n = 95). On removing HCO₃^-and CO₂ there was an initial transient alkalinization followed by a fall in pH to a steady value of 6.97±0.03 (sem, n = 54). Addition of 0.25 mM 4,4-diisothiocyanatostilbene2, 2-disulfonic acid (DIDS) to AAH containing HCO₃^-and CO₂ led to a rapid and pronounced fall in pH. Exposure to Na⁺-free AAH again led to a marked fall in pH_i, but in this case the addition of DIDS did not produce a further fall. Substitution of the impermeant anion gluconate for Cl^– in the presence of HCO₃^-led to a rise in pH_i, while substitution in the absence of HCO₃^-led to a fall in pH_i. The above data indicate a significant role for a sodium-dependent Cl^–-HCO₃^-exchange mechanism in the regulation of pH_i. Addition of 1 mM amiloride to control AAH in both the presence and absence of HCO₃^-led to a marked fall in pH_i, indicating that a Na⁺/H⁺ exchange mechanism also has a significant role in the regulation of pH_i. There is evidence for a lactic acid transport mechanism in bovine lens cells, as addition of lactate to the external medium produced a rapid fall in pH_i. Larger changes in pH_i were observed in control compared to HCO₃^--free AAH and in the latter case a pronounced alkalinizing overshoot was obtained on removing external lactate. Tissue-cultured bovine lens cells thus possess at least three membrane transport mechanisms that are involved in pH regulation. The buffering capacity of the lens cells was measured by perturbing pH_i with either NH₄⁺or procaine. The values obtained were similar in both cases and the intrinsic buffering capacity measured in the absence of external HCO₃^-was 5 mm/pH unit (procaine). However, in the presence of HCO₃^-and CO₂ the buffer capacity increases approximately fourfold, indicating that HCO₃^-is the principal intracellular buffer.We acknowledge financial support from the Wellcome Trust and the Humane Research Trust for this project. M.R. Williams was in receipt of a Science & Engineering Research Council studentship.