PH REGULATION IN TISSUE-CULTURED BOVINE LENS EPITHELIAL-CELLS

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-degrees-C with artificial aqueous humour solution (AAH) containing 16 mM HC3O- and 5% CO2, pH 7.25, pH(i) was 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'-diisothiocyanatostilbene-2,2'-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.