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

Abstract

SUMMARY1. 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+O008 (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 pH1; 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 HC03-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 HC03-, 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,sM-NPPB (5-nitro-2-(3-phenylpropyl-amino)-benzoate). However, the pHi in the steady state in the presence or absence of HC03-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 + 0X08(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). 11. The above-mentioned findings indicate that the Na+-H+ and Cl--HCO3-exchangers and HC03-conductive pathway in the cell membrane play a key role in the pHi regulation in OHCs. Also participation of the carbonic anhydrase in the pH1 regulation by facilitating CO2 transfer across the membrane was suggested.

Vestibular Hair Bundles Control pH with (Na+, K+)/H+Exchangers NHE6 and NHE9

“…If 2 mM K ϩ equilibrates H ϩ across the bundle membrane and there are no other H ϩ transporters, then the intracellular concentration of K ϩ must be close to 2 mM, not the expected ϳ120 mM. In that case, leaky hair cells might have equilibrated their Na ϩ and K ϩ with the bathing saline; consistent with that suggestion, dissociated frog hair cells in vitro have high levels of intracellular Na ϩ (data not shown), as also shown in cochlear hair cells (Ikeda et al, 1992;Shi et al, 2005). Alternatively, K ϩ may be permissive for K ϩ /H ϩ exchange rather than the driving force; for example, H ϩ transport by NHE9 may be augmented by an electrogenic transporter that also requires K ϩ .…”

“…؉ extrusion by hair bundles The resting pH of hair bundles was similar to that measured in outer hair cells (pH 7.26) (Ikeda et al, 1992) and in other cells (Roos and Boron, 1981) Hair bundles also restore pH to the resting level after acid loading with 25 mM H ϩ equivalents. Nevertheless, bundles likely tightly control their pH during physiological stimuli that trigger elevated hair-bundle Ca 2ϩ pumping.…”

“…Although HCO 3 Ϫ transport could play a role in hair-bundle pH homeostasis as it does in the soma (Ikeda et al, 1992), rapid pH recovery in bundles did not depend on HCO 3 Ϫ . Not only did our extracellular solutions not include HCO 3 Ϫ , but the HCO 3 Ϫ / Cl Ϫ antiporter inhibitor 4,4-diisothiocyanatostilbene-2,2Ј-disulfonic acid (Romero et al, 2004) had no effect on bundle pH recovery (data not shown).…”

“…Nevertheless, somas of outer hair cells do supplement NHE-mediated pH control with HCO 3 Ϫ mechanisms (Ikeda et al, 1992); it is possible that hair bundles do as well.…”

“…Although bundles presumably have endogenous H ϩ buffers to blunt pH changes on a short timescale, sustained maintenance of neutral pH requires H ϩ removal from the bundle. In somas of hair cells (Ikeda et al, 1992) and in many other cell types (Brett et al, 2005a), Na ϩ /H ϩ exchangers (NHEs) transport H ϩ across the plasma membrane, but Na ϩ -dependent exchangers cannot operate in hair bundles because of the lack of extracellular Na ϩ in the endolymph. Although H ϩ transport could be achieved by diffusion to the soma, this mechanism would generate transepithelial H ϩ flow, requiring H ϩ pumping to maintain pH in the endolymph, the K ϩ -rich extracellular solution bathing the bundles.…”