pH regulation of the globular substance in the otoconial membrane of the guinea-pig inner ear.

Physiological and pharmacological characteristics of the globular substance, a precursor of otoconia, are not well understood. In the present study, we investigated the variations and regulation of internal pH (pHi) of the globular substance of the guinea-pig inner ear. The otoconial membrane was dissected out from the utricular macula and loaded with the pH-sensitive fluorophore, carboxyseminaphthorhodafluor-1. Dynamic changes of fluorescence were directly observed under a confocal laser scanning microscope, and pHi was calculated from dual emission ratio. In the NaCl standard solution buffered with 5 mM HEPES/Tris at pH 7.4, the pHi of the globular substance varied from 6.26 to 8.55 with an average of 7.21 (n = 270). Exposure to 25 mM NH+4 induced a rapid increase of the pHi followed by a slow relaxation. The, wash-out of NH+4 caused a prompt and pronounced acidification followed by a gradual pHi recovery to the initial level. This gradual pHi recovery was significantly inhibited by the absence of external Na+, indicating the presence of an external Na(+)-dependent H+ extrusion mechanism. This pHi recovery was also inhibited by 1 mM amiloride and 10 microM 3-amino-N-(aminoiminomethyl)-6-[ethyl(2-propyl)amino]pyrazine-2-ca rboxamide. These results suggest the presence of an Na(+)-H+ exchanger in the globular substance of the guinea pig. However, HCO3(-)-transporting mechanisms were not determined. The working hypothesis for the otoconial formation is discussed.