Evidence for the presence of a Na⁺-H⁺ exchanger in the endolymphatic sac epithelium of guinea-pigs

Abstract

 The intracellular pH (pH_i) of epithelial cells from the endolymphatic sac (ES) of the guinea-pig was measured microfluorometrically with the pH-sensitive fluorescent dye, 2′,7′-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF) to examine the presence of a Na⁺-H⁺ exchanger (NHE) in the ES epithelial cells. pH_i recovery from acid loading with an NH₄ ⁺-prepulse in a nominally HCO₃-free solution was dependent on extracellular Na⁺ ([Na⁺]_o) and was inhibited by amiloride and its analogue ethylisopropylamiloride (EIPA), suggesting that a decreased pH_i induced by an acute acid load may be equilibrated by a NHE. In the steady-state, amiloride had no effect on pH_i, indicating that the NHE activity is low at the resting pH_i. However, the intracellular acidification induced by the removal of [Na⁺]_o was inhibited by the simultaneous application of amiloride. H⁺-efflux rate (J _H, mean activity of NHE), which was calculated as the product of the recovery rate (dpH_i/dt) from the acid loading and the intrinsic buffering capacity (β_i) at the corresponding pH_i, was decreased as pH_i was increased. The concentration/response curve for the inhibition of initial J _H by EIPA revealed an apparent 50% inhibitory constant (K _i ) of 0.85 µM. Kinetic analysis of initial J _H as a function of [Na⁺]_o revealed a Michaelis-Menten constant (K _m) of 24.14 mM for Na⁺-dependent H⁺ efflux. The results indicate that NHE in the ES epithelium belongs to an amiloride-sensitive subtype.