Regulation of intracellular pH in cultured bovine retinal pigment epithelial cells

Regulation of intracellular pH (pHi) in bovine retinal pigment epithelium (RPE) was investigated in cell culture. pHi was measured using the pH-sensitive absorbance of intracellularly trapped 5 (and 6)-carboxy-dimethyl-fluorescein (CDMF). (1) Regulation of pHi after induction of an acid load by removal of NH4Cl could be blocked either totally by removal of extracellular sodium, or subtotally (about 90%) by application of amiloride (1 mmol/l). Additional flux measurements revealed a dose-dependent, amiloride-sensitive 22Na+-uptake into Na+-loaded cells. Both results suggest the presence of a Na+/H+ antiport. (2) When alkalinization of the cells was induced by preincubation with 50 mmol/l acetate in HCO3(-)-Ringer's and subsequent removal of the weak acid, the following regulation was dependent on the presence of extracellular chloride. This process could be blocked with DIDS (1 mmol/l), suggesting the presence of a Cl-/HCO3- exchange mechanism. (3) We found no evidence for a Na+/HCO3(-)-cotransport, which had been postulated to be present in RPE by others. We conclude that two processes are involved in regulation of pHi in RPE: A Na+/H+ antiport responsible for recovery of pHi from acid load, and a DIDS-sensitive Cl-/HCO3- exchange mechanism responsible for recovery of pHi after alkalinization.