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

Abstract

The intracellular pH (pHi) 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. pHi 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 pHi induced by an acute acid load may be equilibrated by a NHE. In the steady-state, amiloride had no effect on pHi, indicating that the NHE activity is low at the resting pHi. However, the intracellular acidification induced by the removal of [Na+]o was inhibited by the simultaneous application of amiloride. H+-efflux rate (JH, mean activity of NHE), which was calculated as the product of the recovery rate (dpHi/dt) from the acid loading and the intrinsic buffering capacity (betai) at the corresponding pHi, was decreased as pHi was increased. The concentration/response curve for the inhibition of initial JH by EIPA revealed an apparent 50% inhibitory constant (Ki ) of 0.85 microM. Kinetic analysis of initial JH as a function of [Na+]o revealed a Michaelis-Menten constant (Km) of 24.14 mM for Na+-dependent H+ efflux. The results indicate that NHE in the ES epithelium belongs to an amiloride-sensitive subtype.