Prestin, the motor protein of outer hair cells

Prestin is a gene recently cloned from mammalian cochlear outer hair cells (OHC) using a single cell type, outer minus inner hair cell, specific suppressive subtractive hybridization procedure. The localization and gene expression profile of the prestin protein fits the pattern of OHC's development of electromotility. When prestin is abundantly expressed in normally nonmotile kidney cells, nonlinear capacitance and motility that are normally only seen in OHCs can be recorded. Furthermore, both nonlinear capacitance and motility can be reduced by salicylate, a well-known inhibitor of electromotility. These data suggest that prestin is the motor protein of OHCs. Amino acid sequence and gene structure analysis indicate that prestin is the fifth member of a newly discovered anion transport family (SLC26) that includes PDS, DRA and DTDST, which are chloride-iodide transporters, Cl-/HCO3- exchangers or sulfate transporters. Prestin shares overall structure similarity with this anion transporter family. Recently, intracellular anions (chloride or bicarbonate) were found to be essential for OHC electromotility and prestin's function.     

Fast cochlear amplification with slow outer hair cells

In mammalian cochleas, outer hair cells (OHCs) produce mechanical amplification over the entire audio-frequency range (up to 100 kHz). Under the 'somatic electro-motility' theory, mechano-electrical transduction modulates the OHC transmembrane potential, driving an OHC mechanical response which generates cycle-by-cycle mechanical amplification. Yet, though the OHC motor responds up to at least 70 kHz, the OHC membrane RC time constant (in vitro upper limit approximately 1000 Hz) reduces the potential driving the motor at high frequencies. Thus, the mechanism for high-frequency amplification with slow OHCs has been a two-decade-long mystery. Previous models fit to experimental data incorporated slow OHCs but did not explain how the OHC time constant limitation is overcome. Our key contribution is showing that negative feedback due to organ-of-Corti functional anatomy with adequate OHC gain significantly extends closed-loop system bandwidth and increases resonant gain. The OHC gain-bandwidth product, not just bandwidth, determines if high-frequency amplification is possible. Due to the cochlea's collective traveling-wave architecture, a single OHC's gain need not be great. OHC piezoelectricity increases the effectiveness of negative-feedback but is not essential for amplification. Thus, emergent closed-loop network dynamics differ significantly from open-loop component dynamics, a generally important principle in complex biological systems.     

Utilizing prestin as a predictive marker for the early detection of outer hair cell damage

Purpose

To evaluate prestin as a biomarker for the identification of early ototoxicity.

Structure,pharmacology and function of GABAA receptors in cochlear outer hair cells

Summary There is evidence that the inhibitory neurotransmitter -aminobutyric acid (GABA) is released from some efferent olivocochlear nerve endings terminating at outer hair cells (OHCs). Using monoclonal antibodies against postsynaptic GABA_A receptor from bovine cerebral cortex we confirm the presence of GABA and benzodiazepine bindings sites of - and -subunits of GABA_A receptors at the basal pole of isolated OHCs. Whole-cell recording with viable OHCs revealed that the application of 10^–3–10^–8 M GABA to the cell surface was followed by a concentration-dependent hyperpolarization of the outer cell membrane. Hyperpolarization was increased in the presence of 2.5 × 10^–5 M chlorazepate, a benzodiazepine derivative. Electrophysiological effects caused by GABA alone or in combination with chlorazepate were specifically inhibited by 10^–6 M of the GABA-receptor antagonist picrotoxin. Moreover, 10^–5–10^–7 M GABA caused reversible slow elongation of the cylindrical hair cell body in OHCs examined. These neurotransmitter-induced motile responses were specifically blocked by 10^–4 M picrotoxin. The results suggest that a subpopulation of OHCs express - and -subunits of GABA_A receptors which both form a GABA/benzodiazepine-receptor complex at the basal pole of isolated OHCs. These receptors are thought to allow GABA which is released from efferent auditory nerve terminals to bind to the cell surface of OHCs, resulting in GABA_Areceptor activation. This probably gates a GABA_A-receptor-associated chloride channel in the postsynaptic OHC membrane, allowing hyperpolarization and elongation of the cell. Correspondence to: P.K. Plinkert     

Are cochlear outer hair cells the origin of otoacoustic emissions?

Recent investigations have shown, that cochlear outer hair cells (OHCs) influence actively the micromechanics of the cochlea, beside their capability for auditory perception. A direct evidence for these energy requiring processes has been the registration of otoacoustic emissions in the external ear canal. Motile responses of isolated OHCs following various stimuli have been suggested to be the possible source of the postulated active mechanical processes. These motile events are possibly controlled by the efferent olivocochlear innervation. By means of two monoclonal antibodies, which were directed against external and internal acetylcholine (ACh) receptor epitopes, we were able to visualize ACh-receptors on OHCs. We suspect, that ACh is released into the synaptic cleft and binds to these newly observed receptors. Thereby they may influence the biomechanics of the cochlea.     

Acute hyperpolarization and elongation of cochlear outer hair cells on superfusion with cis-platinum

The acute effects of cis-platinum on isolated cochlear outer hair cells (OHC) were investigated with whole-cell patch-clamps and measurements of cell length changes. Our findings demonstrated that cis-platinum reversibly induced a hyperpolarization and cellular elongation. These results suggest that the effects produced are the result of an interaction between cis-platinum and transduction channels in OHC. These acute effects are distinctly different from the chronic, irreversible ones that are followed by death of the OHC. The exact mechanism of these chronic effects remains unknown as yet.Presented in part at the Inner Ear Biology Workshop, August 1990, Tubingen, Germany     

Critical bands following the selective destruction of cochlear inner and outer hair cells

Critical bandwidths and absolute intensity thresholds were measured in cats before and after kanamycin treatment which induced selective inner and outer hair cell losses. Hair cell losses were measured from cochleograms constructed from surface preparations of the organ of Corti. Results suggested that, for the test frequencies and stimulus intensities employed, critical bandwidths were not affected for frequencies tonotopically located in cochlear regions where only outer hair cells were lost. Critical bands were widened or not measurable only when inner hair cell losses exceeding 40% were also associated with complete loss of outer hair cells. The experiment suggests that cochlear frequency selectivity can be mediated by inner hair cells alone.     

Motile responses of cochlear outer hair cells stimulated with an alternating electrical field

The goal of the present study was to evaluate and characterize the motile responses of guinea pig OHCs, stimulated at frequencies varying from 50 Hz to 4 kHz, using high-definition, high-speed video recording and fully automatic image analysis software. Cells stimulated in continuous, burst and sweeping modes with an external alternating electrical field showed robust fast and slow motility, which were dependent on frequency, mode and intensity of stimulation. In response to continuous stimulation, electromotile amplitude ranged from 0.3% to 3.2% of total cell length, whereas cell length usually decreased in amounts varying from 0.1% to 4.3%. Electromotile amplitude in OHCs stimulated with square wave's sweeps was near constant up to 200 Hz, progressively decreased between 200 Hz and 2 kHz, and then remained constant up to 4 kHz. In continuous and burst modes electromotility followed cycle-by-cycle the electrical stimulus, but it required 1-2 s to fully develop and reach maximal amplitude. Instead, slow cell length changes started about 0.6 s after the beginning and continuously developed up to 3 s after the end of electrical stimulation. Incubation of OHCs with 10 mM salicylate affected electromotility but not slow motility, whereas incubation with 3 mM gadolinium affected both. Thus, combination of external electrical stimulation, high-speed video recording and advanced image analysis software provides information about OHC motile responses at acoustic frequencies with an unprecedented detail, opening new areas of research in the field of OHC mechanics.     

A model of the effect of outer hair cell motility on cochlear vibrations

A model of cochlear function is presented in which deformation forces within outer hair cells are assumed to occur in synchronized response to generator potentials. Assuming a 90 degree phase lag between the generator potentials and the deformation forces, it is shown that the forces act to reduce cochlear-partition damping and thus increase frequency selectivity. A number of other experimentally observed phenomena, such as the effects of efferent-fiber stimulation and electrical polarization, can also be accounted for with this model.     

小鼠耳蜗内外毛细胞胞吞功能的实验研究

目的研究小鼠内外毛细胞胞吞功能的异同,探讨毛细胞胞吞功能与动物听功能之间的关系。方法选择出生后1月龄的正常C57BL/6J小鼠耳蜗基底膜在体外培养,以染料FM1-43为胞吞示踪剂,应用活细胞成像技术观察耳蜗内外毛细胞胞吞现象。结果内毛细胞的胞吞活动主要集中在细胞底部及核下区,而外毛细胞的胞吞活动则主要集中在核上区和外侧壁区。而且,在不同的观察时间点上,内毛细胞对FM1-43的摄入量均显著高于外毛细胞(P<0.05)。结论内毛细胞的胞吞活动集中出现在细胞底部及核下区,说明这种胞吞活动与内毛细胞带状突触的功能密切相关;外毛细胞的胞吞活动主要出现在核上区及细胞外侧壁,表明这种活动更多参与了外毛细胞纤毛及离子通道。内毛细胞比外毛细胞具有更强大的胞吞功能,表明内毛细胞在听功能的发育和维持中发挥着更为关键的作用。     

Visualization and functional testing of acetylcholine receptor-like molecules in cochlear outer hair cells

The efferent nerve endings at outer hair cells (OHCs) have been suggested to regulate active mechanical processes in the cochlea. The discovery of acetylcholine (ACh)-producing and -degrading enzymes in these synapses gave rise to the speculation that ACh might be one of the efferent transmitters. However, there has as yet been no identification and characterization of any corresponding receptor in OHCs which is required for further clarification of this question. In the present paper existence, location and first characterization of acetylcholine receptors (AChRs) in OHCs are reported. Using two anti-AChR monoclonal antibodies, AChR epitopes were found forming a cup at the basal end of the OHCs opposite to the efferent nerve endings. Furthermore, the studied molecules could be shown to extend through the cell membrane. In addition, the denervated OHC AChR-epitopes seem to move by lateral diffusion. Application of Carbachol and ACh to the basal pole of OHCs induced a weak, reversible cell contraction. Pharmacological controls revealed, that hte motile responses were mediated by the AChRs.     

Brief report: the cochlear microphonic as an indication of outer hair cell function

The extra-cellular cochlear microphonic is believed to be generated predominantly by outer hair cells and therefore it would seem reasonable to assume that the presence of a cochlear microphonic excludes outer hair cell dysfunction. Indeed, a diagnosis of auditory neuropathy might be, and has been, made on the basis of a cochlear microphonic present with an abnormal auditory brainstem response. Animal studies, however, have shown that the cochlear microphonic recorded from the round window is dominated by cellular generators located in the base of the cochlea. Primarily on this basis, it is argued that the presence of a cochlear microphonic does not exclude outer hair cell pathology and so outer hair cell integrity should not necessarily be inferred from the presence of the cochlear microphonic alone. In contrast, the absence of an otoacoustic emission in such cases is consistent with outer hair cell dysfunction.     

Effect of aminoglycoside otic drops on isolated cochlear outer hair cells with and without liver extract activation

OBJECTIVE: To assess the ototoxicity of commercially available Gentacidin and TobraDex ear drops with and without liver extract activation using isolated cochlear outer hair cells (OHCs). MATERIAL AND METHODS: OHCs from adult chinchilla cochleae were exposed to standard bathing solution (SBS), liver extract alone and Gentacidin and TobraDex ear drops with and without liver extract. All experiments were performed at an osmolality of 305 +/-5 mOsm, at room temperature and for up to 60 min. OHC images were recorded using an inverted microscope and analyzed electronically. Time to cell death and changes in cell length were measured. RESULTS: The time to cell death and the percent change in cell length were significantly shorter in the Gentacidin+liver extract group than in the Gentacidin alone group (p < 0.05). The TobraDex+liver extract group showed a significantly decreased time to cell death compared to the SBS control group (p < 0.05). There were no significant differences in cell length or time to cell death between the TobraDex+liver extract group and the TobraDex alone group (p > 0.05). CONCLUSION: This study suggests that the cytotoxicity of aminoglycoside ear drops to isolated OHCs in vitro requires     

Construction of an expression system for the motor protein prestin in Chinese hamster ovary cells

The electromotility of outer hair cells (OHCs) is believed to be a major factor in cochlear amplification that enables the high sensitivity of hearing in mammals. This motility is thought to be based on voltage-dependent conformational changes of a motor protein embedded in the lateral wall of the OHC. In 2000, this motor protein was identified and termed prestin. To obtain knowledge on the function of prestin, research at the molecular level is necessary. For this purpose, a method of obtaining a large amount of prestin is required. In this study, an attempt was therefore made to construct an expression system for prestin. Prestin cDNA was introduced into Escherichia coli (E. coli), insect cells and Chinese hamster ovary (CHO) cells, and the expression of prestin was examined by Western blotting. As CHO cells expressed prestin well, we generated prestin-expressing cell lines using CHO cells by limiting dilution cloning. The stable expression and the activity of prestin in generated cell lines were then confirmed. Finally, to obtain prestin from these cell lines efficiently, culture conditions of the cells were examined, and it was clarified that cells should be cultured in serum-free medium and harvested around 48 h after passage.     

硝普钠对豚鼠耳蜗外毛细胞全细胞钙电流作用的实验研究

目的　探讨一氧化氮供体———硝普钠 (sodiumnitroprusside ,SNP)对豚鼠单离耳蜗外毛细胞钙电流的影响及作用机制。方法　利用急性分离的豚鼠耳蜗外毛细胞 ,在全细胞膜片钳电压钳记录技术下 ,通过分离离子电流成分的方法 ,记录豚鼠耳蜗外毛细胞全细胞钙电流。结果　SNP对内向钙电流有抑制作用 ,在钳制电位为 -60mV ,刺激电压为 + 10mV的条件下 ,10mmol/L的SNP能抑制 (61 12± 1 99) %的内向钙电流 ( x±s ,n =5)。从 5～ 8个细胞得到的量效关系曲线中 ,其半数作用浓度为 1 9mmol/L ,最大抑制浓度为 10 0mmol/L ,斜率为 0 98。作用的机制为SNP可选择性地阻断外毛细胞膜上的L 型钙通道 (n =6)。结论　SNP作为一氧化氮的一种供体 ,能影响豚鼠耳蜗外毛细胞的生理功能 ,是通过阻断外毛细胞L 型钙通道电流的内流来实现的     

Effects of common topical otic preparations on the morphology of isolated cochlear outer hair cells

Otic drops are commonly used not only for otitis externa but also for otorrhea in the presence of tympanostomy tube or tympanic membrane perforation. Many studies have demonstrated the ototoxicity of common otic preparations such as Cortisporin otic drops. Recent studies have suggested the use of fluoroquinolone antibiotic drops as an alternative owing to their excellent antimicrobial coverage and no ototoxic effect. The purpose of this study was to assess the relative ototoxicity of four common otic preparations by direct exposure to isolated cochlear outer hair cells (OHCs). OHCs from adult chinchilla cochlea were exposed to standard bathing solution (control), Cortisporin, Cipro HC, Ciloxan, and Floxin. The cells were observed using an inverted microscope, and the images recorded in digital still-frame and video, and analyzed on the Image Pro-Plus 3.0 program. As measured by time to cell death and change in morphology of OHCs, Cortisporin was most toxic to OHCs. Among the fluoroquinolone drops, Floxin was more toxic than Ciloxan or Cipro HC.     

Nitrosoglutathione suppresses cochlear potentials and DPOAEs but not outer hair cell currents or voltage-dependent capacitance

Biochemical and pharmacological evidence support a role for nitric oxide (NO) and glutathione (GSH) in the cochlea. GSH combines with NO in tissue to form nitrosoglutathione (GSNO) that can act as a storage form for GSH and NO. Therefore, we tested GSNO on sound-evoked responses of the cochlea (cochlear microphonic, CM; summating potential, SP; compound action potential, CAP; cubic distortion product otoacoustic emission, DPOAE), on the endocochlear potential (EP), on isolated outer hair cell (OHC) currents and voltage-dependent capacitance, and on Deiters' cell currents. In vivo application of GSNO in increasing concentrations reversibly reduced low-intensity sound-evoked CAP, SP and DPOAEs starting at about 1 mM (CAP) and 3.3 mM (SP, DPOAE). However, even at 10 mM, GSNO had little effect on the EP. In vitro, salicylate (10 mM) but not GSNO (3 and 10 mM) suppressed the early capacitative transients of OHCs. GSNO (3 and 10 mM) had no effect on the whole cell currents of OHCs or Deiters' cells. Results show that GSNO suppresses cochlear function. This suppression may be due to an effect of GSNO on the cochlear amplifier. The actions of GSNO were different from those of other NO donors; therefore, the effects of GSNO may not be mediated by NO. The mechanisms underlying GSNO effects seem to be different from those of salicylate.