Functional and morphological comparisons between cochlear outer hair cells and muscle tissues in the guinea-pig

Brundin , L., Wiklund , N. P., Gustafsson , L. E. & Flock , AR. 1992. Functional and morphological comparisons between cochlear outer hair cells and muscle tissues in the guinea-pig. Acta Physiol Scand 144 , 379–386. Received 24 April 1 991 , accepted 2i October 1991. ISSN 0001–6772. Departments of Physiology and Urology, and the National Institute of Environmental Medicine, Karolinska Institute, Stockholm, Sweden The effect of polylysine on the motility of outer hair cells and various muscle types was compared. Poly-L-lysine and its stereoisomer, poly-D-lysine, inhibited acoustically induced length changes of isolated outer hair cells from the guinea-pig hearing organ. The frequency specific displacements of the hearing organ in response to a tone stimulus are also inhibited to polylysine (Brundin et al. 1991). Poly-L-lysine, and its stereoisomer, irreversibly attenuated motile responses to transmural stimulation of guinea-pig ileum, vas deferens and taenia coli in a dose dependent manner, but were without significant effect on motile responses in skeletal and heart muscle. l -lysine, D-lysine, and the negatively charged polyaminoacid poly-L-aspartate, were without significant effect on outer hair cell and smooth muscle motility. The inhibitory effect of polylysine in smooth muscle is a direct effect on the muscle cell since polylysine attenuated acetylcholine- and adenosine triphosphate-induced contractions in the ileum, and ATP- or noradrenaline-induced contractions in the vas deferens. Pillar structures, believed to be of importance to excitation contraction coupling, were compared. In heart and skeletal muscle the pillars span the gap between sarcoplasmic reticulum and T-tubuli, deeply recessed into the muscle cell. In smooth muscle and outer hair cell the pillars are in closer relation to the cell exterior. The length of the pillars of the outer hair cells exceeds by two times that of smooth and skeletal muscle. The susceptibility of outer hair cells and smooth muscle tissue to the positively charged polylysine may indicate similarities in membrane or channel composition.     

Functional assembly of mammalian cochlear hair cells

Hair cells in the mammalian inner ear convert sound into electrical signals that are relayed to the nervous system by the chemical neurotransmitter glutamate. Electrical information encoding sound is then passed through the central nervous system to the higher auditory centres in the brain, where it is used to construct a temporally and spatially accurate representation of the auditory landscape. To achieve this, hair cells must encode fundamental properties of sound stimuli at extremely high rates, not only during mechano-electrical transduction, which occurs in the hair bundles at the cell apex, but also during electrochemical transduction at the specialized ribbon synapses at the cell base. How is the development of such a sophisticated cell regulated? More specifically, to what extent does physiological activity contribute to the progression of the intrinsic genetic programmes that drive cell differentiation? Hair cell differentiation takes about 3 weeks in most rodents, from terminal mitosis during embryonic development to the onset of hearing around 2 weeks after birth. Until recent years, most of the molecules involved in hair cell development and function were unknown, which was mainly due to difficulties in working with the mammalian cochlea and the very small number of hair cells, about 16,000 in humans, present in the auditory organ. Recent advances in the ability to record from the acutely isolated cochlea maintained in near-physiological conditions, combined with the use of genetically modified mouse models, has allowed the identification of several proteins and molecular mechanisms that are crucial for the maturation and function of hair cells. In this article, I highlight recent findings from my laboratory that have furthered our understanding of how developing hair cells acquire the remarkable sensitivity of adult auditory sensory receptors.     

Forces involved in length changes of cochlear outer hair cells

Motion or force generation of outer hair cells may contribute to active modulation of cochlear mechanics. In order to determine the force involved in length changes of outer hair cells, a new in vitro method was used. In the first series of experiments, apical and basolateral extracellular spaces of outer hair cells of the guinea-pig cochlea were separated. Changes of the voltage between the two extracellular spaces induced reversible, proportional changes of the cell length of 4.4 nm/ mV if the cell had a length of 80 m. In the second series of experiments, cell elongations in response to negative pressure applied to the basal end of the cells were measured and corrected for frictional effects. From these data, the compliance of the longitudinal axis of the hair cells was calculated. It was 220±130 m/N (n =25) and 240±170 m/N /(n = 24) for cells of the third and fourth cochlear turns, respectively, if the water permeability of the cell membrane was neglected. If the water permeability was taken into account, the compliance was probably around 5 km/N. Thus, a mechanism that changes the cell length by 1 m must generate a static force of at least around 200 pN in an outer hair cell of the organ of Corti. Electromotility of outer hair cells, induced by changes of the electrical potential difference across the outer hair cell, is a mechanism that generates this force.     

Na+-Ca2+ exchange in the isolated cochlear outer hair cells of the guinea-pig studied by fluorescence image microscopy

The outer hair cell isolated from the guinea-pig was superfused in vitro and the cytosolic calcium concentration ([Ca²⁺]_i) and sodium concentration ([Na⁺]_i) were measured using fluorescence indicators. Under the resting condition, [Ca²⁺]_i and [Na⁺]_i were 91±9 nM (n = 51) and 110±5 mM (n = 12), respectively. Removal of external Na⁺ by replacing with N-methyl-D-glucamine (NMDG⁺) increased [Ca²⁺]_i by 270±79% (n = 27) and decreased [Na⁺]_i by 23±4 mM (n = 6). Both changes in [Ca²⁺]_i and [Na⁺]_i were totally reversible on returning external Na⁺ to the initial value and were inhibited by addition of 0.1 mM La³⁺ or 100 M amiloride 5-(N,N-dimethyl) hydrochloride. Elevation of external Ca²⁺ ions to 20 mM reversibly decreased [Na⁺]_i by 8±6 mM (n = 5). Moreover, the chelation of the intracellular Ca²⁺ with 1,2-bis (2-aminophenoxy) ethane-N,N,N,N-tetraacetic acid (BAPTA) exerted an inhibitory action on the NMDG⁺-induced reduction in [Na⁺]_i. Exposure to 5 mM NaCN for 2 min significantly and reversibly increased [Ca²⁺]_i by 290±37% (n = 5), but did not affect the [Ca²⁺]_i elevation induced by the NMDG⁺ solution. The rise in [Ca²⁺]_i induced by the NMDG⁺ solution was not enhanced by ouabain pretreatment. Addition of ouabain did not alter the [Na⁺]_i. The present results are best explained by the presence of an Na⁺-Ca²⁺ exchanger in cell membrane and indicate that the activity of Na⁺/K⁺ pump is poor in outer hair cells.     

Regulation of electromotility in the cochlear outer hair cell

Mechanosensory outer hair cells play an essential role in the amplification of sound-induced vibrations within the mammalian cochlea due to their ability to contract or elongate following changes of the intracellular potential. This unique property of outer hair cells is known as electromotility. Selective efferent innervation of these cells within the organ of Corti suggests that regulation of outer hair cell electromotility may be the primary function of the efferent control in the cochlea. A number of studies demonstrate that outer hair cell electromotility is indeed modulated by the efferent neurotransmitter, acetylcholine. The effects of acetylcholine on outer hair cells include cell hyperpolarization and a decrease of the axial stiffness, both mediated by intracellular Ca²⁺. This article reviews these results and considers other potential mechanisms that may regulate electromotility, such as direct modification of the plasma membrane molecular motors, alteration of intracellular pressure, and modification of intracellular chloride concentration.     

Monoclonal antibodies specific for endoplasmic membranes of mammalian cochlear outer hair cells

Summary Monoclonal antibodies were raisedin vitro against an antigen associated with the lateral cisternal membranes of outer hair cells. Two of the antibodies were class IgM and one of these retained its specific reactivity in tissue fixed with aldehydes and embedded in the resin LR White. Immunogold labelling for electron microscopy showed that the antigen was closely associated with the membranes rather than the cytoplasmic or lumenal regions of the cisternae. The third antibody was an IgG. All three weakly labelled a protein band with an apparent molecular weight of about 60 kD on a Western blot. The antibodies did not cross-react with any other cell in the organ of Corti, including the inner hair cells. Furthermore, they showed no cross-reactivity with skeletal muscle, kidney, gut, brain, skin, blood or retina from the guinea pig. The results suggest that the lateral cisternae in outer hair cells may be functionally different from those of inner hair cells. The antibodies may provide useful markers for outer hair cells in studies of hair cell regeneration.     

电离辐射对大鼠耳蜗外毛细胞Prestin蛋白表达影响的实验研究

目的探讨电离辐射下大鼠耳蜗外毛细胞Prestin蛋白表达的改变及意义。方法建立大鼠耳放射损伤模型,检测其听力复合动作电位（CAP）阈值及畸变产物耳声发射（DPOAEs）幅值,验证其电离辐射晚期感音神经性耳聋（SNHL）的发生。提取大鼠耳蜗组织mRNA及蛋白,荧光实时定量PCR检测耳蜗Prestin蛋白mRNA水平的表达,以及Western杂交检测Prestin蛋白水平的表达。结果成功建立了电离辐射晚期SNHL大鼠模型,其耳蜗Prestin蛋白无论是mRNA表达水平还是蛋白表达水平均较未照射组明显降低。结论放疗晚期感音神经性耳聋的发生机制可能与电离辐射导致内耳外毛细胞Prestin蛋白的表达异常有关。     

Relationship between stiffness,internal cell pressure and shape of outer hair cells isolated from the guinea-pig hearing organ

The mechanical properties of outer hair cells are of importance for normal hearing, and it has been shown that damage of the cells can lead to a reduction in the hearing sensitivity. In this study, we measured the stiffness of isolated outer hair cells in hyper- and hypotonic conditions, and examined the change in stiffness in relation to the corresponding changes in internal cell pressure and cell shape. The results showed that the axial stiffness of isolated outer hair cells (30–90 μm in length, 8–12 μm in diameter), ranging from 0.13–5.39 mN m^?1, was inversely related to cell length. Exposure to hyper- and hypotonic external media with a small percentage change in osmolality caused a similar magnitude of change in cell length and cell diameter, but an average 60% change in cell stiffness. Therefore, a moderate osmotic change in the external medium can lead to a significant alteration in cell stiffness. The findings thus indicate an important contribution of internal cell pressure to cell stiffness.     

The potential and electric field in the cochlear outer hair cell membrane

Outer hair cell electromechanics, critically important to mammalian active hearing, is driven by the cell membrane potential. The membrane protein prestin is a crucial component of the active outer hair cell’s motor. The focus of the paper is the analysis of the local membrane potential and electric field resulting from the interaction of electric charges involved. Here the relevant charges are the ions inside and outside the cell, lipid bilayer charges, and prestin-associated charges (mobile—transferred by the protein under the action of the applied field, and stationary—relatively unmoved by the field). The electric potentials across and along the membrane are computed for the case of an applied DC-field. The local amplitudes and phases of the potential under different frequencies are analyzed for the case of a DC + AC-field. We found that the effect of the system of charges alters the electric potential and internal field, which deviate significantly from their traditional linear and constant distributions. Under DC + AC conditions, the strong frequency dependence of the prestin mobile charge has a relatively small effect on the amplitude and phase of the resulting potential. The obtained results can help in a better understanding and experimental verification of the mechanism of prestin performance.     

Block by amiloride and its derivatives of mechano-electrical transduction in outer hair cells of mouse cochlear cultures.

1. The effects of amiloride and amiloride derivatives on mechano-electrical transducer currents in outer hair cells of the cultured neonatal mouse cochlea were examined under whole-cell voltage clamp. 2. At -84 mV transducer currents were reversibly blocked by the extracellular application of the pyrazinecarboxamides amiloride, benzamil, dimethylamiloride, hexamethyleneiminoamiloride, phenamil and methoxynitroiodobenzamil with half-blocking concentrations of 53, 5.5, 40, 4.3, 12 and 1.8 microM, respectively. Hill coefficients were determined for all but the last of these compounds and were 1.7, 1.6, 1.0, 2.2 and 1.6, respectively, suggesting that two drug molecules co-operatively block the transducer channel. 3. Both the structure-activity sequence for amiloride and its derivatives and the mechanism of the block of the transducer channel appear to be different from those reported for the high-affinity amiloride-sensitive epithelial Na+ channels but similar to those of stretch-activated channels in Xenopus oocytes. 4. The block by all pyrazinecarboxamides was voltage dependent with positive membrane potentials releasing the block. The form of the voltage dependence is consistent with a voltage-independent binding of the drug to a site that is accessible at hyperpolarized but not at depolarized potentials, suggesting that the transducer channel undergoes a voltage-dependent conformational change. The channel was not blocked by 1 mM amiloride from the intracellular side at either negative or positive membrane potentials. 5. The kinetics of the block were studied using force steps or voltage jumps. The results suggest that the drug binding site is only accessible when the transducer channel is open (open-channel block) and that the channel cannot close when the drug molecules are bound. 6. The time dependence and voltage dependence of the block together reveal that the transducer channel has at least two open conformational states, the transition between which is voltage dependent.     

A calcium-activated nonselective cationic channel in the basolateral membrane of outer hair cells of the guinea-pig cochlea

The patch-clamp technique was used to investigate ion channels in the basolateral perilymph-facing membrane of freshly isolated outer hair cells (OHCs) from the guinea-pig cochlea. These sensory cells probably determine, via their motile activity, the fine tuning of sound frequencies and the high sensitivity of the inner ear. A Ca²⁺-activated nonselective cationic channel was found in excised inside-out membrane patches. The current/voltage relationship was linear with a unit conductance of 26.3±0.3 pS (n=15) under symmetrical inger conditions. The channel excluded anions (P _Na/P _Cl=18 whereP _Na/P _Cl denotes the relative permeability of Na to Cl); it was equally permeant to the Na⁺ and K⁺ ions and exhibited a low permeability toN-methyl-D-glucamine and Ba²⁺ or Ca²⁺. Channel opening required a free Ca²⁺ concentration of about 10^–6 mol/l on the internal side of the membrane and the open probability (P _o) was maximal at 10^–3 mol/l (P _o=0.72±0.06,n=12). Adenosine 5mono-, tri- and di-phosphate reducedP _o to 29±14 (n=5), 42±10 (n=8) and 51±12 (n=5) % of controlP _o, respectively, when they were added at a concentration of 10^–3 mol/l to the internal side. The channel was partially blocked by flufenamic acid (10^–4 mol/l) and 3,5-dichlorodiphenylamine-2-carboxylic acid (DCDPC, 10^–5 mol/l). This type of channel, together with Ca²⁺-activated K⁺ channels, might participate in the control of membrane potential and modulate the motility of OHCs.     

耳毒性药物对小鼠耳蜗外毛细胞prestin表达的影响

目的探讨氨基糖甙类药物庆大霉素对小鼠外毛细胞马达蛋白prestin的影响以及和听力损害之间的关系。方法选择鼠龄为5周的C57BL/6J小鼠,每天腹腔注射庆大霉素1次,药物浓度为100mg/kg,连续给药14d。实验期间,分别在第4d,7d和第14d时检测受试小鼠的ABR(Click)以观察耳毒性药物对小鼠听功能的影响,并以未进行腹腔注射给药的小鼠(0d)作为对照组。采用免疫组织化学结合激光共聚焦显微镜方法,观察小鼠耳蜗基底膜铺片标本上prestin表达变化,并采用比较荧光强度的方法定量分析prestin在各组之间表达量的差异。结果听功能检测显示,实验组小鼠听力在第4d时开始下降,到第7d时下降达到峰值(P<0.01),14d时较第7d有所降低但无显著差异(P>0.05),但仍高于对照组(P<0.05)。prestin在外毛细胞表达量在第4d开始上调,表现为圆环状的绿色荧光变得模糊,第7d时圆环的形状已经变得不规则并且多带有比较长的尾迹,而prestin表达量达到了最高水平(P<0.01),第14d时表达量和第7d相比有所降低(P<0.05),但是仍然高于对照组(P<0.05)。结论氨基糖甙类耳毒性药物持续暴露导致prestin过表达,与小鼠的听力损害过程存在一致性,提示prestin过表达可能参与致聋过程。     

Axial and transverse stiffness measures of cochlear outer hair cells suggest a common mechanical basis

 The function of the hearing organ is based on mechanical processes occurring at the cellular level. The mechanical properties of guinea-pig isolated sensory cells were investigated using two different techniques. The stiffness of the outer hair cells along the longitudinal axis was measured by compressing the cell body using stiffness-calibrated quartz fibres. For cells with a mean length of 69 μm, the mean axial compression stiffness was 1.1±0.8 mN/m (±SD). There was an inverse relation between stiffness and cell length. The stiffness of the cell membrane perpendicular to the longitudinal axis of the sensory cell was measured by indenting the cell membrane with a known force. The mean lateral indentation stiffness was 3.3±1.5 mN/m (±SD) for cells with a mean length of 64 μm. Longer cells were less stiff than short cells. Modelling the hair cell as a shell with bending resistance, finite element calculations demonstrated that the axial compression stiffness correlated well with the lateral indentation stiffness, and that a simple isotropic model is sufficient to explain the experimental observations despite the different stress strain states produced by the two techniques. The results imply that the two different stiffness properties may originate from the same cytoskeletal structures. It is suggested that the mechanical properties of the outer hair cells are designed to influence the sound-induced motion of the reticular lamina. In such a system, stiffness changes of the outer hair cell bodies could actively control the efficiency of the mechanical coupling between the basilar membrane and the important mechanoelectrical transduction sites at the surface of the hearing organ. Received: 12 June 1997/Received after revision: 13 November 1997/Accepted: 19 January 1998