Na+/H+ exchange regulates intracellular pH in a cell clone derived from bovine pigmented ciliary epithelium.
The regulation of intracellular pH (pHi) was monitored in a virus-transformed cell clone derived from bovine ciliary body exhibiting characteristics of pigmented ciliary epithelium. Data were obtained from confluent monolayers grown on plastic coverslips in nominally bicarbonate-free media using the pH-sensitive absorbance of 5- (and 6-) carboxy-4',5'-dimethylfluorescein. Under resting conditions, pHi averaged 6.98 +/- 0.01 (SEM; n = 57). When cells were acid loaded by briefly exposing them to Ringer containing NH4+ and then withdrawing the NH4+, pHi spontaneously regained its initial value. In the presence of 1 mM amiloride or in the absence of Na+, this process was blocked, indicating the involvement of an Na+/H+ exchanger in the regulation of pHi after an acid load. Removing Na+ during resting conditions decreased cytoplasmatic pH. This acidification could be slowed by amiloride, which is evidence for reversal of the Na+/H+ countertransport exchanging intracellular Na+ for extracellular protons. Application of 1 mM amiloride during steady state led to a slow acidification. Thus the Na+/H+ exchanger is operative during resting conditions extruding protons, derived from cellular metabolism, or from downhill leakage into the cell. Addition of Na+ to Na+ -depleted cells led to an alkalinization, which was sensitive to amiloride, with an IC50 of about 20 microM. This alkalinization was attributed to the Na+/H+ exchanger and exhibited saturation kinetics with increasing Na+ concentrations, with an apparent KM of 29.6 mM Na+. It is concluded that Na+/H+ exchange regulates pHi during steady state and after an acid load.
Helbig
H
H
Institut für Klinische Physiologie, Klinikum Steglitz der Freien Universität Berlin, Federal Republic of Germany.
Korbmacher
C
C
Stumpff
F
F
Coca-Prados
M
M
Wiederholt
M
M
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000502:physiology*; D002417:Cattle; D002999:Clone Cells / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers
D002352:Carrier Proteins; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride