Intracellular pH regulation in isolated cochlear outer hair cells of the guinea‐pig.

Abstract

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)