耳蜗内淋巴环境阳离子成分稳态的研究进展

正常的耳蜗内淋巴环境阳离子成分稳态是听觉产生过程中的重要环节 ,内耳必须维持其内环境的相对稳定才能保持正常的生理功能。随着解剖内耳螺旋韧带技术的发现和完善 ,以及膜片钳和振动电极记录在该领域的应用 ,内耳上皮细胞已成为研究听觉机制的重要实验对象。血管纹是耳蜗内代谢活跃的组织 ,对维持耳蜗内电位及内淋巴液中高钾低钠状态有重要作用。耳蜗内淋巴具有类似细胞内液的离子成分 ,即Ｋ+浓度高 ,Ｎａ+、Ｃａ2 +浓度低 ;外淋巴液中Ｋ+浓度低 ,Ｎａ+、Ｃａ2 +浓度高。这种独特的离子环境在听觉生理机制中具有十分重要的作用。感受声音…     

耳蜗组织细胞连接蛋白-Connexin的表达及其生理与病理意义

1　耳蜗组织结构及其微环境特点1.1　耳蜗上皮结构与特殊细胞外液　哺乳类动物的耳蜗绕蜗轴呈螺旋管状 ,沿纵轴切面观 ,可将中阶蜗管侧壁分为内、外两部分。外侧壁结构主要包括血管纹与螺旋韧带 ,内侧壁则为螺旋缘 ,基底膜位于其间。这种区域的划分有助于理解耳蜗内环境的形成。从耳蜗听觉电生理及其相关离子转运活动的角度可以将耳蜗膜迷路内的上皮细胞分为 :感觉上皮———Corti器听觉毛细胞 ;离子转运上皮———维持内淋巴离子环境的上皮细胞 ,以及内、外淋巴液之间的屏障上皮细胞。三者各有其完整的形态学基础 ,共同参与维持耳蜗内环境…     

耳蜗血管纹产生耳蜗内电位的机理

耳蜗内淋巴具有独特的离子成分,以及相对于外淋巴＋80mv＋90mv的耳蜗内电位。血管纹对维持耳蜗内环境的稳定有重要作用,它在向内淋巴转运钾离子的过程中产生耳蜗内电位。本文对血管纹产生耳蜗内电位的各种相关机理的研究进展进行综述。     

耳蜗血管纹产生耳蜗内电位的机理

耳蜗内淋巴具有独特的离子成分,以及相对于外淋巴 80mv~ 90mv的耳蜗内电位。血管纹对维持耳蜗内环境的稳定有重要作用,它在向内淋巴转运钾离子的过程中产生耳蜗内电位。本文对血管纹产生耳蜗内电位的各种相关机理的研究进展进行综述。     

耳蜗内电位产生机理及钾离子动态平衡的研究进展

耳蜗作为听觉器官具有独特的离子环境。听觉感受器毛细胞的纤毛浸浴在高K^ 浓度的内淋巴液中，K^ 是主要的感受器电流。内淋巴内还有一个 80mV的耳蜗内电位(endocochlear potential，EP)。它是感受器电流最主要的驱动力。维持耳蜗电位的稳定以及内淋巴K^ 的动态平衡对维持正常听觉至关重要。研究表明血管纹在向内淋巴转运K^ 的过程中产生了EP，这一过程也是耳蜗K^ 外侧循环的一个环节，但是具体到血管纹内的哪种细胞和哪种通道产生了EP，一直存在争论。     

外淋巴腔灌注三磷酸腺苷对豚鼠耳蜗功能的影响

目的研究三磷酸腺苷(ATP)对豚鼠耳蜗功能的影响。方法外淋巴灌注ATP后，记录其对耳蜗生物电活动，包括总和电位(SP)、耳蜗微音器电位(CM)、听神经复合动作电位(CAP)、畸变产物耳声发射(DPOAE)和听性脑干反应(ABR))的影响。结果耳蜗生物电活动的变化对ATP有浓度依赖性。与人工外淋巴液相比，外淋巴灌注1mMol／L ATP引起-SP的幅度增加，CAP、DPOAE的幅度下降，ABR的阈值升高。另外，CAP和DPOAE的变化分别表现出强度和频率依赖性，当刺激声强度为20～70dB nHL时，CAP幅度显著下降；在2～8kHz中高频区DPOAE的幅度下降明显。330μMol／L ATP也引起ABR阈值升高。结论外淋巴腔灌注的ATP对耳蜗功能具有抑制性的影响。     

耳蜗缘细胞中三磷酸腺苷囊泡的研究现状

耳蜗外侧壁包括螺旋韧带和血管纹。国内外研究发现耳蜗血管纹缘细胞中存在大量的各种形状的囊泡结构，这些囊泡由单层或双层膜包被，并常含有绒毛状电子致密物，此囊泡可能与离子的转运、三磷酸腺苷（adenosine triphosphate，ATP）的释放密切相关。有人证实囊泡中的ATP不是来源干线粒体。有学者观察到了囊泡明显的胞吐活动，但是不能确定ATP是否通过胞吐作用分泌至内淋巴。缘细胞中的ATP囊泡是否就是溶酶体，还有待更深入的研究证实。现结合国内外文献，就耳蜗缘细胞中ATP囊泡的研究现状做一综述。     

细胞外ATP诱导的豚鼠耳蜗Ⅰ型螺旋神经节细胞的Ca~(2+)动员

该研究旨在以钙敏荧光探针Fura－2检测细胞外ATP作用下豚鼠耳蜗1型螺旋神经节细胞内游离钙（［ca’“］．）的改变．从而确定在豚鼠耳蜗毛细胞传入神经突触中ATP是否起神经介质或神经调质的作用。结果发现：细胞外ATP能诱导豚鼠耳蜗1型螺旋神经节细胞内【ca’”］；升高，并且是与剂量相关的。在没有细胞外Ca’”存在的情况下，ATP仍能诱导1型螺旋神经节细胞内［Ca’”］．升高，但升高幅度较有细胞外Ca’”时明显减小。这种现象不是对ATP的脱敏，因为ATP的作用是可重复的。细胞外液中加入Ca’一通道阻滞剂La‘－H4，结果与细胞外…     

耳蜗血管纹边缘细胞培养方法的研究现状

耳蜗内淋巴是一种特殊的细胞外液，具有高K^＋、高渗、高正电位的特点，它在听觉传导过程中发挥着重要的作用。研究证实，血管纹（stria vascularis，SV）边缘细胞（marginal cells，MCs）是内淋巴形成的必要条件，对内淋巴正电位的维持也是不可缺少的。但是，由于SV组织取材的困难性以及其结构的复杂性，边缘细胞的生理和病理生理功能仍未完全阐明。细胞培养是克服上述限制的有效方法，因而在耳蜗组织的生理、生化等研究领域得到一些学者的尝试应用。本文对SV边缘细胞培养的研究现状作一综述。     

肾上腺素β受体和胆碱能M受体对边缘细胞钾离子转运的调控

耳蜗血管纹边缘细胞向内淋巴转运钾离子对耳蜗内淋巴的生成,电化学特征的维持起到直接作用.钾离子经边缘细胞转运的过程除了受细胞内外离子浓度、膜电压的调控外还受细胞膜受体及细胞内信号转导系统调节.目前研究发现肾上腺素β受体促进边缘细胞的钾离子转运;胆碱能M受体则抑制边缘细胞的钾离子转运.肾上腺素β受体和胆碱能M受体对边缘细胞钾离子转运的调控作用具有重要的临床意义.     

The effect of spatial adaptation on auditory motion processing

Na(+) concentrations in endolymph must be controlled to maintain hair cell function since the transduction channels of hair cells are cation-permeable, but not K(+)-selective. Flooding or fluctuations of the hair cell cytosol with Na(+) would be expected to lead to cellular dysfunction, hearing loss and vertigo. This review briefly describes cellular mechanisms known to be responsible for Na(+) homeostasis in each compartment of the inner ear, including the cochlea, saccule, semicircular canals and endolymphatic sac. The influx of Na(+) into endolymph of each of the organs is likely via passive diffusion, but these pathways have not yet been identified or characterized. Na(+) absorption is controlled by gate-keeper channels in the apical (endolymphatic) membrane of the transporting cells. Highly Na(+)-selective epithelial sodium channels (ENaCs) control absorption by Reissner's membrane, saccular extramacular epithelium, semicircular canal duct epithelium and endolymphatic sac. ENaC activity is controlled by a number of signal pathways, but most notably by genomic regulation of channel numbers in the membrane via glucocorticoid signaling. Non-selective cation channels in the apical membrane of outer sulcus epithelial cells and vestibular transitional cells mediate Na(+) and parasensory K(+) absorption. The K(+)-mediated transduction current in hair cells is also accompanied by a Na(+) flux since the transduction channels are non-selective cation channels. Cation absorption by all of these cells is regulated by extracellular ATP via apical non-selective cation channels (P2X receptors). The heterogeneous population of epithelial cells in the endolymphatic sac is thought to have multiple absorptive pathways for Na(+) with regulatory pathways that include glucocorticoids and purinergic agonists.     

Structural evidence for ion transport and tectorial membrane maintenance in the gerbil limbus

Cells medial to the tunnel of Corti were examined to assess fine structural features relevant to their proposed role in cochlear K(+) homeostasis. A dense network of canaliculi referred to as canalicular reticulum (CR) resided in the foot body of inner pillar cells, where it bordered and could resorb ions released from inner radial and spiral nerves. Lateral interdental cells (IDCs) formed columns which connected the inner sulcus epithelium with the base of the tectorial membrane's (TM) middle zone. A spout-like neck in cells at the top of lateral IDC columns housed a dense concentration of CR which resembled that characteristic of ion transporting epithelia and appeared to be located here for transporting ions and fluid toward the TM. Clustered IDCs in the center of the limbus connected underlying limbal stroma with the TM's limbal zone and appeared capable of transporting ions from stroma to TM. Abundant CR in limbal stellate fibrocytes evidenced their capacity to transport ions and fluid, presumably from inner sulcus epithelium toward central IDCs. The most medial IDCs possibly function as the terminus of an ion cycling path from scala vestibuli to endolymph. Light fibrocytes situated between supralimbal fibrocytes and medial IDCs appeared to serve as a link in this pathway. The limbal zone of the TM overlying central IDCs consisted of three distinct regions which offered a structural basis for transformation of an amorphous matrix supplied by central IDCs into the protofibrils of the membrane's middle zone.     

K+ and Na+ absorption by outer sulcus epithelial cells.

Transduction of sound into nerve impulses by hair cells depends on modulation of a current carried primarily by K+ into the cell across apical transduction channels that are permeable to cations. The cochlear function thus depends on active secretion of K+ accompanied by absorption of Na+ by epithelial cells enclosing the cochlear duct. The para-sensory cells which participate in the absorption of Na+ (down to the uniquely low level of 1 mM) were previously unidentified and the existence of a para-sensory pathway which actively absorbs K+ was previously unknown. A relative short circuit current (Isc,probe, measured as the extracellular current density with a vibrating electrode) was directed into the apical side of the outer sulcus epithelium, decreased by ouabain (1 mM), an inhibitor of Na+, K(+)-ATPase, and found to depend on bath Na+ and K+ but on neither Ca2+ nor Cl-. Isc,probe was shown to be an active current by its sensitivity to ouabain. On-cell patch clamp recordings of the apical membrane of outer sulcus cells displayed a channel activity, which carried inward currents under conditions identical to those used to measure Isc,probe. Both Isc,probe and non-selective cation channels (27.4+/-0.6 ps, n = 22) in excised outside-out patches from the apical membrane were inhibited by Gd3+ (1 mM). Ics,prob was also inhibited by 5 mM lidocaine, 1 mM quinine and 500 microM amiloride but not by 10 microM amiloride. These results demonstrate that outer sulcus epithelial cells contribute to the homeostasis of endolymph by actively absorbing Na+ and K+. An entry pathway in the apical membrane was shown to be through non-selective cation channels that were sensitive to Gd3+.     

Purinergic signaling in the inner ear

Epithelial cells of the inner ear coordinate their ion transport activity through a number of mechanisms. One important mechanism is the autocrine and paracrine signaling among neighboring cells in the ear via nucleotides, such as adenosine, ATP and UTP. This review summarizes observations on the release, detection and degradation of nucleotides by epithelial cells of the inner ear. Purinergic signaling is thought to be important for endolymph ion homeostasis and for protection from acoustic over-stimulation.     

Ablation of inner hair cells by carboplatin alters cells in the medial K(+) flow route and disrupts tectorial membrane.

The thesis that K(+) effluxing from inner hair cells (IHCs) cycles medially back to endolymph through inner sulcus and interdental cells (IDCs) was tested by comparing control chinchilla cochleas with those in which IHCs were selectively destroyed by carboplatin. By light microscopy inner sulcus cells appeared tall and nearly empty in control ears, but 4 months after the carboplatin treatment many showed vacuolization and shrinkage. Inner pillar cells also consistently developed abnormal vacuoles after carboplatin treatment. Control cochleas exhibited lateral columns and central clusters of IDCs which at their apex possessed expanded presumably hydrated phalanges. Four months after carboplatin, the IDC epithelium enclosed empty looking spaces and the apical phalangeal compartment collapsed into a thin, apparently dehydrated layer. This alteration was accompanied by changes in the tectorial membrane (TM) whereby the membrane's limbal zone thickened progressively to form a tall hollow mound in advanced lesions. The clear spaces in the epithelium and collapse of the phalanges are thought to reflect diminished flow of ions and fluid through IDCs. The accumulation of limbal TM supports the premise that IDCs secrete macromolecules for TM turnover as well as ions and fluid for promoting lateral migration of its precursor constituents. Occurring after ablation of IHCs by carboplatin, the changes in inner pillar, inner sulcus and IDCs and limbal TM can be viewed as a secondary effect of the interrupted ion efflux from IHCs and as further evidence that this effluent follows a medial route.     

In vitro morphological study of cochlear epithelium.

1. The mammalian cochlear epithelium was successfully kept alive in long-term tissue cultures. The Na and K concentration of the culture medium was similar to that of perilymph. 2. The most vulnerable cells in the culture epithelium were the outer and inner hair cells followed by the inner zones cells and the interdental cells of the spiral limbus. The difference in disintegration time between the outer and inner hair cells was about 48 hrs under the same culture conditions. The same phenomenon was noted between the rows of outer hair cells. 3. When the outer hair cells were not supported by Deiters' cells or when the cochlear epithelium was separated between Deiters' cells and Hensens cells, the outer hair cells degenerated quickly. 4. The interdental cells appeared to be relatively highly differentiated and showed much activity. No evidence of secretory function of interdental cells could be obtained. 5. The inner sulcus cells, Hensens cells, Claudius' cells and Reissners membrane cells were flattened and arranged like covering epithelium.     

Cytochemical localization of Ca++-ATPase activity in the lateral cochlear wall of the guinea pig

Summary Ca⁺⁺-ATPase activity was examined cytochemically in the lateral cochlear wall of the guinea pig. The reaction products showing Ca⁺⁺-ATPase activity were found along the folded plasma membrane of the strial marginal cells. In contrast, little or no reaction was seen on the apical surfaces of these cells. There were also marked reaction products on the microvilli and the endolymphatic cell surface of Reissner's membrane, and the apical and lateral plasma membranes of the spiral prominence and the external sulcus cells. These reactions completely disappeared when Ca⁺⁺ or ATP was removed from the incubation medium. Our results strongly suggest that Ca⁺⁺-ATPase plays an important role in Ca⁺⁺ transport system for the regulation of Ca⁺⁺ concentration in the cochlear endolymph.     

Distribution and significance of endothelin 1 in guinea pig cochlear lateral wall

Endothelin 1 is a vasoconstrictive peptide with many biological functions. To investigate the distribution of endothelin 1 in guinea pig cochlear lateral wall and the significance of endothelin 1 in maintaining cochlear homeostasis, the immunohistochemistry avidin biotin complex method was applied by using rabbit anti-endothelin 1 polyclonal antibody as primary antibody. Endothelin-1-like activities were detected in the marginal cells, spiral prominence epithelial cells, outer sulcus cells, stria vascularis capillaries, basal cells and spiral ligament fibrocytes. These results suggest that endothelin 1 may play an important role in maintaining cochlear homeostasis.     

Changes in ultrastructural characteristics of endolymphatic sac ribosome-rich cells of the rat during development

It has recently been demonstrated that endolymphatic sac (ES) ribosome-rich (dark) cells respond to induced endolymph changes and are thus likely to be involved in endolymph homeostasis. Therefore, we studied the ultrastructural characteristics of rat ES ribosome-rich cells during development in order to determine the cellular distribution of organelles involved in protein metabolism, secretion and absorption, indicative for their contribution to endolymph homeostasis. During embryonal stages ribosome-rich cells contain a limited number and variety of organelles and are predominantly involved in the production of components for cell growth and differentiation. In the young adult stage (P60) three different states of ribosome-rich cells may be distinguished. State A resembles a cell with only limited metabolic activities whereas state B is characterized by numerous different intracellular organelles and is considered to be involved in production and secretion as well as absorption and degradation of complex proteins. A third cellular state, state C, is filled with phagolysosomes and contains very few other organelles. This is considered to be a final (pre)apoptotic state. Autoradiography data suggest that ES ribosome-rich cells are capable of synthesis and secretion of tyrosine-containing proteins and may thus be involved in regulation of the osmolarity of endolymph based on the capacity to bind cations as well as water molecules. In addition, ES ribosome-rich cells appear to synthesize and secrete fucosylated glycoproteins into the endolymph. In conclusion, the present data suggest that ES ribosome-rich cells are actively involved in endolymph homeostasis through secretion and absorption of complex proteins and it is hypothesized that they are able to adapt their function or activities in response to changes in endolymph composition.