Na(+)-dependent HCO3- transport and Na+/H+ exchange regulate pHi in human ciliary muscle cells.
We investigated intracellular pH (pHi) 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 pHi 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 pHi 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 pHi recovery after an intracellular acid load was completely dependent on extracellular sodium. In HEPES-buffered solution the pHi recovery was almost completely mediated by Na+/H+ exchange, since it was blocked by amiloride (1 mmol/liter). In contrast, a marked amiloride-insensitive pHi 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 pHi recovery after NH4Cl prepulse revealed Vmax = 0.57 pH units/min, Km = 39.7 mmol/liter extracellular sodium for the amiloride-sensitive component and Vmax = 0.19 pH units/min, Km = 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 pHi regulation of cultured human ciliary muscle cells.
Stahl
F
F
Institut für Klinische Physiologie, Universitätsklinikum Steglitz, Freie Universität Berlin, Germany.
Lepple-Wienhues
A
A
Koch
M
M
Wiederholt
M
M
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D000584:Amiloride / Q000494:pharmacology; D001639:Bicarbonates / Q000378:metabolism*; D001692:Biological Transport; D002245:Carbon Dioxide / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D002924:Ciliary Body / Q000378:metabolism*; D005110:Extracellular Space / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007231:Infant, Newborn; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism*; D009130:Muscle, Smooth / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D017693:Sodium Bicarbonate; D017923:Sodium-Hydrogen Exchangers
D001639:Bicarbonates; D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D002245:Carbon Dioxide; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D000584:Amiloride; D017693:Sodium Bicarbonate; D012964:Sodium; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid