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Department of Otolaryngology, Tohoku University School of Medicine, Sendai, Japan.

1. The intracellular pH (pHi) regulation mechanisms of the outer hair cell (OHC) isolated from the guinea-pig were studied using fluorescence ratio imaging microscopy. 2. The OHC pHi in the resting condition was 7.26 +/- 0.08 (mean +/- S.D., n = 49) when the standard solution buffered with HEPES-Tris was superfused. 3. Exposure to 25 mM-NH4+ in the absence of HCO3- caused biphasic changes in pHi; a transient increase (7.89 +/- 0.14, n = 22) followed by a slow decrease (7.57 +/- 0.12; mean +/- S.D.). Removal of external NH4+ by introducing the N-methyl-D-glucamine (NMDG+) solution in the absence of HCO3- markedly acidified the pHi to 6.38 +/- 0.12 with little pHi recovery. Subsequent application of the standard Na+ solution restored the pHi to the initial value. The recovery was inhibited by 0.5 mM-amiloride but not by 0.3 mM-DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid). 4. In the presence of HCO3-, removal of both external NH4+ and Na+ promptly caused an intracellular acidification followed by a pHi recovery. The pHi recovery from an acid load was inhibited by 0.3 mM-DIDS or 10 microM-NPPB (5-nitro-2-(3-phenylpropyl-amino)-benzoate). However, the pHi in the steady state in the presence or absence of HCO3- was not altered by addition of 0.5 mM-amiloride or NMDG+ solution. 5. The intracellular buffering power obtained from the NH4+ exposure and withdrawal was -15.1 +/- 8.7 mM (pH unit)-1 (n = 6) and -14.3 +/- 5.8 mM (pH unit)-1, respectively. 6. Replacement of external Cl- with gluconate in the HCO3- solution increased the pHi from 7.22 +/- 0.12 to 7.51 +/- 0.20 (n = 6), which was inhibited by 0.3 mM-DIDS. Moreover, addition of DIDS to the HCO3- solution increased the pHi by 0.13 +/- 0.08 (n = 8). 7. When the external standard solution buffered with HEPES-Tris was replaced with the HCO3- solution, the basal pHi (7.27 +/- 0.10) was promptly acidified to 6.87 +/- 0.10 then relaxed slowly to 7.00 +/- 0.15 (n = 16). 8. The pHi showed an initial alkalinization and a subsequent slow acidification after the HCO3(-)-free standard solution replaced the HCO3- solution. The slow acidification was inhibited by low external Cl- concentration or by addition of 0.3 mM-DIDS.(ABSTRACT TRUNCATED AT 400 WORDS)

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