Evidence for a Na⁺/H⁺ exchanger at the basolateral membranes of the isolated frog skin epithelium: Effect of amiloride analogues

Abstract

We have investigated the possible existence of a Na⁺/H⁺ ion exchanger in the frog skin epithelium by using isotopic methods and two amiloride analogues: 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) and phenamil. We found phenamil to be a specific blocker of sodium entry to its cellular transport compartment since it inhibited both the transepithelial Na⁺ influxes (J ₁₃) with aK _I of 4·10⁻⁷ mol/l and the Na⁺ pool (control: 77±4 neq·h⁻¹·cm⁻²; phenamil: 21±1 neq·h⁻¹·cm⁻²). On the contrary EIPA (10⁻⁵ mol/l) had no effect onJ ₁₃ nor on the apical Na⁺ conductance. Acidification of the epithelium by passing from a normal Ringer (25 mmol/l HCO ⁻₃ , 5% CO₂, pH 7.34) to a HCO ⁻₃ -free Ringer (5% CO₂, pH 6.20) while blocking the Na⁺ conductance with phenamil, produced a large stimulation of Na⁺ influxes exclusively across the basolateral membranes (J ₃₂), after return to a normal Ringer (J ₃₂=706±76 and 1635±199 neq·h⁻¹·cm⁻² in control and acid-loaded epithelia respectively). The stimulation ofJ ₃₂ was initiated when the epithelia were acid-loaded with Ringer of pH lower than 6.90 and was blocked by amiloride (K _I=7·10⁻⁶ mol/l) and EIPA (K _I=5·10⁻⁷ mol/l) whereas phenamil had no effect. In na⁺-loaded epithelia (ouabain treated) the Na⁺ efflux across the basolateral membranes was stimulated by an inwardly directed proton gradient and was blocked by EIPA (10⁻⁵ mol/l) or amiloride (10⁻⁴ mol/l), a result suggesting reversibility of the mechanism. We conclude that a Na⁺ permeability mediated by a Na⁺/H⁺ ion exchanger exists in the basolateral membranes, which is stimulated by intracellular acidification and is sensitive to amiloride or EIPA. This exchanger is proposed to be involved in intracellular pH regulation.