NA+-DEPENDENT HCO-3 TRANSPORT AND NA+/H+ EXCHANGE REGULATE PH(I) IN HUMAN CILIARY MUSCLE-CELLS

We investigated intracellular pH (pH(i)) regulation in cultured human ciliary muscle cells by means of the pH-sensitive absorbance of 5(and 6)-carboxy-4',5'-dimethylfluorescein (CDMF). The steady-state pH(i) was 7.09 +/- 0.04 (n = 12) in CO2/HCO3--buffered and 6.86 +/- 0.03 (n = 12) in HEPES-buffered solution. Removal of extracellular sodium for 6 min acidified the cells by 1.11 +/- 0.06 pH units (n = 12) in the presence of CO2/HCO3- and by 0.91 +/- 0.05 pH units (n = 8) in its absence. Readdition of external sodium resulted in a rapid pH(i) recovery, which was almost completely amiloride-sensitive in the absence of CO2/HCO3- but only slightly influenced by amiloride in its presence. Application of DIDS under steady-state conditions significantly acidified the ciliary muscle cells by 0.25 +/- 0.02 (n = 4) in 6 min, while amiloride had no effect. The pH(i) recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pH(i) recovery was almost completely mediated by Na+/H+ exchange. since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amiloride-insensitive pH(i) recovery was observed in CO2/HCO3--buffered solution which was mediated by chloride-independent and chloride-dependent Na+ HCO3- cotransport. This recovery, inhibited by DIDS (0.2 mmol/liter), was also observed if the cells were preincubated in chloride-free solution for 4 hr. Analysis of the sodium dependence of the pH(i) recovery after NH4Cl prepulse revealed V(max) = 0.57 pH units/min, K(m) = 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and V(max) = 0.19 pH units/min, K(m) = 14.3 mmol/liter extracellular sodium for the amiloride-insensitive component. We conclude that Na+/H+ exchange and chloride-independent and chloride-dependent Na+-HCO3- cotransport are involved in the pH(i) regulation of cultured human ciliary muscle cells.