Evidence for the presence of a Na+-H+ exchanger in the endolymphatic sac epithelium of guinea-pigs.
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.
Wu
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D
Department of Otolaryngology, Kagawa Medical University, Kagawa 761-0793, Japan.
Mori
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N
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002021:Buffers; D004712:Endolymphatic Sac / Q000737:chemistry*; D004848:Epithelium / Q000737:chemistry; D005260:Female; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008297:Male; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D012964:Sodium / Q000494:pharmacology; D017923:Sodium-Hydrogen Exchangers / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism
D002021:Buffers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D000644:Quaternary Ammonium Compounds; D017923:Sodium-Hydrogen Exchangers; D000584:Amiloride; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D012964:Sodium; C039614:ethylisopropylamiloride