Regulation of intracellular sodium and pH by the electrogenic H⁺ pump in frog skin

Abstract

We have investigated the role of the electrogenic hydrogen ion pump in the regulation of intracellular sodium ion activity (a ⁱ_Na ) and intracellular pH (pH_i) in frog skin epithelial cells using double-barreled ion sensitive microelectrodes. WhenRana esculenta skin is mounted in an Ussing chamber and bathed in 1 mM Na₂SO₄ buffered to pH 7.34 with imidazole on the apical side and in normal Ringer on the serosal side, the apical addition of the carbonic anhydrase inhibitor, ethoxzolamide (10⁻⁴M) blocks net H⁺ ion excretion and Na absorption, producing a depolarization of 25–30 mV of the apical membrane, potential (Ψmc). We demonstrate the these changes are accompanied by a fall ina ⁱ_Na from 6.2±0.5 mmol/l to 3.4±0.6 mmol/l and an increase in pH_i from 7.20±0.03 to 7.38±0.08 (n=12 skins). Voltage clamping Ψmc to its control value in the presence of ethoxzolamide restoreda ⁱ_Na but the pH_i remained alkaline. Furthermore, the fall ina ⁱ_Na produced by ethoxzolamide could be mimicked by voltage clamping Ψmc towards the value of the Nernst potential for Na at the apical membrane. These results indicate that the maintenance of the cellular Na⁺ transport pool is dependent on a favourable electrical driving force and counter-current generated by an electrogenic H⁺ pump at the apical membrane.

Addition of amiloride (10⁻⁵ mol/l) or substitution of external Na⁺ by Mg²⁺ or K⁺ caused a hyperpolarization of Ψmc and a fall ina ⁱ_Na . These effects were accompanied by an inhibition of H⁺ excretion and a fall in pH_i of 0.14 ±0.08 units (n=6 skins). However, when the effect, of Na⁺ transport inhibition on Ψmc was prevented by imposing a voltage clamp no effects of amiloride on H⁺ excretion or pH_i were observed. Moreover, the effect of amiloride on pH_i could be reproduced in control skins by voltage clamping Ψmc to −100 mV. The metabolic inhibitors vanadate (1 mmol/l) and di-cyclo hexyl carbodiimide (5×10⁻⁵ mol/l) inhibited H⁺ excretion and decreased pH_i from 7.28±0.08 to 7.02±0.06 and from 7.30±0.06 to 7.12±0.05 (n=6 skins), respectively.

These results indicate that an apical membrane H⁺ ATPase plays a role in regulating pH_i and the mechanism is sensitive to membrane potential.