Regulation of cytoplasmic pH of cultured bovine corneal endothelial cells in the absence and presence of bicarbonate

Abstract

Intracellular pH (pH _i ) in confluent monolayers of cultured bovine corneal endothelial cells was determined using the pH-dependent absorbance of intracellularly trapped 5(and 6)carboxy-4′,5′-dimethylfluorescein. Steady-state pH was 7.05±0.1 in the nominal absence of bicarbonate, and 7.15±0.1 in the presence of 28mm HCO ₃ ⁻ /5% CO₂. Following an acid load imposed by a NH₄Cl prepulse, pH _i was regulated in the absence of HCO ₃ ⁻ by a Na⁺-dependent process inhibitable to a large extent by 1mm amiloride and 0.1mm dimethylamiloride. In the presence of 28mm HCO ₃ ⁻ /5% CO₂, this regulation was still dependent on Na⁺, but the inhibitory potency of amiloride was less. DIDS (1mm) partially inhibited this regulation in the presence, but not in the absence of bicarbonate. With cells pretreated with DIDS, amiloride was as effective in inhibiting recovery from acid load as in the absence of HCO ₃ ⁻ . The presence of intracellular Cl⁻ did not appreciably affect this recovery, which was still sensitive to DIDS in the absence of Cl⁻. Removal of extracellular Na⁺ led to a fall of pH _i , which was greatly attenuated in the absence of HCO ₃ ⁻ . This acidification was largely reduced by 1mm DIDS, but not by amiloride. Cl removal led to an intracellular alkalinization in the presence of HCO ₃ ⁻ . The presence of a Cl⁻/HCO ₃ ⁻ exchanger was supported by demonstrating DIDS-sensitive³⁶Cl⁻ uptake into confluent cell monolayers. Thus, bovine corneal endothelial cells express three processes involved in intracellular pH regulation: an amiloride-sensitive Na⁺/H⁻ antiport, a Na⁻−HCO ₃ ⁻ symport and a Cl⁻/HCO ₃ ⁻ exchange, the latter two being DIDS sensitive.