EVIDENCE FOR THE PRESENCE OF A NA-H+ EXCHANGER IN THE ENDOLYMPHATIC SAC EPITHELIUM OF GUINEA-PIGS()

The intracellular pH (pH(i)) of epithelial cells from the endolymphati c sac (ES) of the guinea-pig was measured microfluorometrically with t he pH-sensitive fluorescent dye, 2',7'-bis(carboxyethyl)-5(6)-carboxyf lu-orescein (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 extr acellular Na+ ([Na+](o)) and was inhibited by amiloride and its analog ue ethylisopropylamiloride (EIPA), suggesting that a decreased pH(i) i nduced by an acute acid load may be equilibrated by a NHE. In the stea dy-state, amiloride had no effect on pH(i), indicating that the NHE ac tivity is low at the resting pH(i). However, the intracellular acidifi cation induced by the removal of [Na+](o) was inhibited by the simulta neous 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 (beta (i)) at the corresponding pH(i), was decreased as pH(i) was increased. The concentration/response curve for the inhibition of initial J(H) b y EIPA revealed an apparent 50% inhibitory constant (K-i) of 0.85 mu M . 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+ eff lux. The results indicate that NHE in the ES epithelium belongs to an amiloride-sensitive subtype.