新生大鼠耳蜗血管纹缘细胞释放ATP的机制

目的 证实体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,并进一步探讨缘细胞释放ATP的机制.方法 分离、培养新生大鼠耳蜗血管纹缘细胞,采用生物发光法分别检测巴佛洛霉素A1、己二酸二癸酯( didecyl adipate,DDA)、细胞外K+、毒胡萝卜素、细胞外Ca2、U73122及马兜铃酸钠对细胞外液中缘细胞ATP释放的影响.结果 随着巴佛洛霉素A1浓度的增加,细胞外液中ATP的浓度明显下降;当DDA浓度增加时,细胞外液中ATP的浓度几乎呈线性增加.随着细胞外液中的K+浓度的增高,缘细胞释放的ATP浓度呈现上升趋势,当细胞外液中的K+浓度为9.15 mmol/L时,ATP的释放量达到峰值,之后随着K+浓度的继续升高ATP的释放量呈下降趋势.随着毒胡萝卜素浓度的增加,缘细胞释放的ATP浓度呈现明显下降的趋势.当细胞外Ca2+浓度为0 mmol/L时,缘细胞仍然释放ATP;Ca2+浓度增加与ATP的释放呈负相关,但当细胞外的Ca2+浓度达到1.25 mmol/L以上时,ATP的释放量维持在一个较稳定的水平.U73122的浓度在0.25～1.25 μmol/L时,其与缘细胞释放的ATP浓度呈负相关.当马兜铃酸钠的浓度为12.5 ～ 100.0 μmol/L,缘细胞ATP释放呈明显下降的趋势;当其浓度＞100.0 μmol/L时,ATP释放浓度趋于平稳.结论 体外培养的新生大鼠耳蜗血管纹缘细胞能够释放ATP,其释放量与钙泵、K+通道状态以及细胞内信号传导通路相关酶的活性有关.     

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。     

新生大鼠耳蜗血管纹缘细胞中ATP存在的证据

目的:研究体外培养的新生大鼠耳蜗血管纹缘细胞中存在ATP的证据,即细胞中是否存在ATP囊泡,体外培养液中能否检测到所释放的ATP。方法:采用出生1～3 d的Sprague-Dawley大鼠,进行体外血管纹缘细胞培养、纯化、鉴定。特异性标记ATP囊泡的喹丫因染色后在荧光显微镜下观察缘细胞中的ATP囊泡。采用生物发光法检测缘细胞细胞外液中所释放的ATP的浓度。结果:体外培养的新生大鼠耳蜗血管纹缘细胞,经流式细胞法检测上皮细胞标志性的角蛋白和波形蛋白的纯度,证实培养所获得的细胞为缘细胞。经喹丫因染色后在荧光显微镜下可见缘细胞细胞质中存在大量的绿色星点状染色。采用生物发光法检测缘细胞细胞外液中ATP的浓度,通过细胞荧光值可计算出ATP的浓度。结论:新生大鼠耳蜗血管纹缘细胞中存在ATP囊泡,并能分泌ATP。     

三磷酸腺苷在新生大鼠耳蜗血管纹缘细胞中的释放机制

目的探究新生大鼠耳蜗血管纹缘细胞中ATP释放的机制。方法原代培养新生SD大鼠血管纹缘细胞,利用钙离子荧光探针、生物发光法测定ATP浓度和β-氨基己糖苷酶释放率测定法来研究ATP和GPN与P2YR-PLC-IP3通路诱导的内质网钙库反应之间的关系,以及内质网钙库与细胞外ATP浓度和β-氨基己糖苷酶释放率的关系。结果 ATP和GPN可通过P2YR-PLC-IP3通路诱发内质网钙库释放,可被P2YR-PLC-IP3通路拮抗剂抑制。缘细胞外ATP浓度和β-氨基己糖苷酶释放率与细胞内钙离子浓度呈正相关。结论缘细胞外ATP作用于P2Y嘌呤能信号系统触发内质网钙库释放,胞内钙离子诱导溶酶体胞吐作用释放ATP至胞外,从而发挥其生物学作用。     

新生大鼠耳蜗K(o)lliker器支持细胞ATP释放的机制

目的 观察体外培养的新生大鼠耳蜗K(o)lliker器支持细胞是否存在并释放ATP,初步探讨其释放机制.方法 选取出生后ld的Sprague-Dawley大鼠,分离耳蜗膜迷路,采用机械分离与酶消化相结合的方法获得单离的K(o)lliker器支持细胞.观察膜迷路和K(o)lliker器支持细胞的喹丫因染色情况.采用生物发光法,通过影响K(o)lliker器支持细胞ATP代谢、改变细胞内外Ca2浓度、抑制细胞内磷脂酶信号通路及添加缝隙连接半通道阻断剂,观察K(o)lliker器支持细胞释放ATP浓度的变化.结果 用喹丫因染色体外培养的K(o)lliker器支持细胞,发现胞质中存在大量绿色星点状染色.采用生物发光法检测的ATP标准曲线呈明显的对数线性关系.随着巴佛洛霉素A1浓度增加,K(o)lliker 器支持细胞培养液中ATP浓度逐渐降低,而随着己二酸二癸酯浓度的增加,培养液中ATP浓度逐渐升高;在一定浓度范围内,随着细胞外Ca2浓度增加,K(o)lliker器支持细胞ATP的释放减少,而随着细胞内游离Ca2浓度增加,K(o)lliker器支持细胞释放ATP量增加;培养液中加入甘珀酸钠或乌热酸抑制半通道后可以显著的降低ATP释放.此外,抑制细胞内磷脂酶信号通路也可以减少ATP的释放.结论 体外培养的新生大鼠耳蜗K(o)lliker器支持细胞存在并释放ATP,细胞内、外液中Ca2+浓度的变化可能通过调节半通道的开放而影响其ATP的释放.     

ATP和乙酰胆碱诱发豚鼠耳蜗外毛细胞内CICR的激光共聚焦研究

目的：运用激光共聚焦研究ATP和乙酰胆碱（ACh）对豚鼠耳蜗外毛细胞内游离钙离子浓度（[Ca^2＋]i）的影响以及诱发Ca^2＋介导的Ca^2＋释放（CICR）的可能机制。方法：用酶孵育机械分离法,分离豚鼠耳蜗外毛细胞（OHC）,钙敏荧光探针Fluo-3染色后,用激光扫描共聚焦显微镜分别记录在细胞外液有钙或无钙条件下,加入ATP、ACh、斯里兰卡肉桂碱（Ryanodine）＋ATP（或ACh）和毒胡萝卜素（Thapsigargin）＋ATP（或ACh）后的OHC的[Ca^2＋],的变化。结果：在含钙的细胞外液中,ATP、Ryanodine＋ATP、Thapsigargin＋ATP、ACh、Ryanodine＋ACh和Thapsigargin＋ACh均可引起[Ca^2＋],升高,引起明显的波峰,荧光强度相对峰值分别为1．60±0．01（ATP）、1．644±0．005（Ryanodine＋ATP）、1．491±0．005（Thapsigargin＋ATP）、1．43±0．01（ACh）、1．58±0．02（Ryanodine＋ACh）、1．398±0．003（Thapsigargin＋ACh）;而在不含钙的细胞外液中,ATP和Ryanodine＋ATP仍可引起[Ca^2＋]。出现幅度较小的波峰,分别为1．341±0．006和1．386±0．008,而ACh,Ryanodine＋ACh、Thapsigargin＋ACh和Thapsigargin＋ATP未引起明显[Ca^2＋],波峰出现,其中ACh不能引起[Ca^2＋]．的升高,Ryanodine＋ACh、Thapsigargin＋ACh和Thapsigargin＋ATP分别引起[Ca^2＋]。缓慢升高。结论：在细胞外液有Ca^2＋的条件下．ATP和ACh引起OHC的[Ca^2＋]。升高,除了通过离子通道引起胞外Ca^2＋内流,还有IP。敏感钙库的释放和诱发CICR。在无Ca^2＋的条件下,ATP仍能诱发CICR,其机制可能是ATP促进IP3敏感钙库释放Ca^2＋,Ca^2＋又诱发了CICR,而ACh的反应是Ca^2＋依赖性的,在细胞外液无Ca^2＋的条件下,ACh不能引起IP。敏感钙库的释放和诱发CICR。     

大鼠耳蜗中三磷酸腺苷表达及来源的初步研究

目的 研究大鼠耳蜗中三磷酸腺苷(adenosine triphosphate,ATP)的表达部位、来源及其可能的存在形式.方法 运用喹丫因染色技术,观察培养的大鼠耳蜗血管纹缘细胞、全膜迷路铺片和新鲜分离的单离外毛细胞;用免疫组化荧光染色法,观察突触素(synaptophysin,SYN)和小泡相关膜蛋白-2(vesicle-associated membrane protein-2/synaptobrevin,VAMP-2)在大鼠耳蜗中的表达.结果 培养的缘细胞胞浆中存在星点状喹丫因染色,全膜迷路铺片血管纹以外的区域和单离毛细胞中均未发现喹丫因的特异性染色.SYN和VAMP-2在大鼠耳蜗的内螺旋束、外螺旋束、Deiters细胞内侧缘和螺旋神经元(spiral ganglion neurons,SGNs)有共同表达.结论 大鼠耳蜗缘细胞胞浆中存在大量囊泡状的ATP,而毛细胞、支持细胞中的ATP可能以非囊泡的形式储存.内螺旋束、外螺旋束和SGNs中有可能存在SYN染色的ATP囊泡.     

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…     

348 Ca^2＋—ATP酶对耳蜗Ca^2＋平衡的调节作用

钙离子（C ^2＋）做为第二信使几乎参与了细胞所有的生理活动。在耳蜗水平,钙与机械－电转换、内耳声感受的频率选择性、基底膜振动非对称性等有密切关系。毛细胞及内淋巴液的Ca^2＋浓度变化与感音神经性耳聋有着密切的联系,而在Ca^2 -ATP酶（又称钙泵）在耳蜗Ca^2 稳态的调控中起了重要作用。Ca^2 －ATP酶的作用及其机理已成为感音神经性耳聋发病机制和防治研究的关键之一。     

Hensen细胞对耳蜗功能的调节

既往人们认为Hensen细胞仅仅起到支撑毛细胞、稳定基底膜结构的作用。新近研究提示,Hensen细胞可以通过释放脂滴增加网状板层和盖膜之间的空间距离,改变毛细胞纤毛与盖膜之间的剪切力,从而调制微音电位(CM);Hensen细胞中脂滴分布从底圈到顶圈逐渐增加,并通过释放脂滴调节基底膜的劲度和质量、以及基底膜不同部位的驻波共振的形成。可见Hensen细胞参与了耳蜗机械调节,增强了耳蜗的敏感性以及感受声音的频率选择性。此外,Hensen细胞还释放ANXA1蛋白参与耳蜗炎症反应、以及可能利用脂滴释放脂肪酸为毛细胞提供营养。本文为研究耳聋发生发展机制提供新线索。     

Quinacrine staining of marginal cells in the stria vascularis of the guinea-pig cochlea: a possible source of extracellular ATP?

There is accumulating evidence for a purinergic humoral system involved in the control of cochlear function. Evidence of specific P₂ purinoceptors on cochlear tissues implies a role for extracellular adenosine triphosphate (ATP) in the cochlea. To further this hypothesis a study was undertaken to determine if there was any specific source of purine compounds in cochlear tissues. Cochlear tissues (the sensory epithelium and lateral wall) from the guinea pig were incubated with the acridine derivative quinacrine dihydrochloride (5×10⁻⁶ M in phosphate-buffered saline for 30 min at room temperature) which fluoresces on binding to high concentrations of ATP. Most cochlear tissues showed a diffuse green fluorescence slightly above the background level. However, a region of the marginal cells of the stria vascularis showed a specific punctate fluorescence. Optical sectioning of these cells by confocal microscopy revealed that the fluorescent structures in these marginal cells was confined to a region up to 10 μm from their endolymphatic surface. Similar cells studied by transmission electron microscopy showed membrane-bound vesicles located in the same region of the cell. These data imply that purine compounds are localized in discrete structures, perhaps vesicles, within the marginal cells which could serve as a source of extracellular ATP in the cochlea.     

化学修饰电极对雏鸡耳蜗神经元一氧化氮的定性检测

目的比较雏鸡和哺乳动物耳蜗生理功能的异同。过去已有哺乳动物耳蜗及前庭神经元中一氧化氮合酶（ＮＯＳ）活动证据的报道，其所释放的一氧化氮（ＮＯ）在听觉和平衡活动中可能发挥重要作用。方法用经化学修饰的碳纤维微电极，定性检测雏鸡离体耳蜗神经元在不同激动剂的诱发下所释放的ＮＯ。根据电极所检测的电流变化的幅度和持续时间判断ＮＯ的释放。结果由激动剂乙酰胆碱和Ｌ精氨酸所诱发的ＮＯ释放反应幅度相似，但乙酰胆碱的高电流持续时间较短，而ＡＴＰ所致者幅度大，持续时间长。预加ＮＧ硝基Ｌ精氨酸，再加上述激动剂，电流无变化。结论雏鸡耳蜗神经元细胞中存在ＮＯＳ。     

化学修饰电极对雏鸡耳蜗神经元一氧化氮的定性检测

目的 比较雏鸡和哺乳动物耳蜗生理功能的异同。过去已有哺乳动物耳蜗及前庭神经元中一氧化氮合酶（ＮＯＳ）活动证据的报道，其所释放的一氧化氮（ＮＯ）在听觉和平衡活动中可能发挥重要作用。方法 用经修饰的碳和有、定性检测鸡离体耳神经元在不同激动剂的诱发下所释放的ＮＯ。根据电极所检测的电流变化的幅度和持续时间判断ＮＯ的释放。结果 由激动剂乙酰胆碱和Ｌ－精氨酸所诱发的ＮＯ释放反应幅度相似，但乙酰胆碱的高电流持续     

用电化学方法鉴别雏鸡耳蜗神经元中一氧化氮合酶的类型

目的：鉴别雏鸡耳蜗神经元中一氧化氮合酶的类型。方法：用ＣｕＰｔＣｌ６化学修饰电极,检测了离体的雏鸡耳蜗神经元在含Ｃａ＾２＋的谷氨酸、缓激肽,不含Ｃａ＾２＋的主Ｌ－精氨酸、三磷酸腺苷、乙酰胆碱、缓激肽等溶液刺激下一氧化氮的释放反应,结果：有Ｃａ＾２＋情况下谷氨酸可以诱发一氧化氮释放,其余均无释放反应,结论,雏鸡耳蜗元中的一氧化氮合酶为神经型。     

噪声刺激对耳蜗一氧化氮合酶的影响

目的：探讨一氧化氮（ＮＯ）在噪声性聋发病中的作用。方法：用中高频连续稳态噪声制作噪声性聋的动物模型,用ＮＡＤＰＨ－黄递酶组织化学、原位杂效和Ｎｏｒｔｈｅｒｎ印迹法,观察噪声刺激对耳蜗一氧化氮合酶（ＮＯＳ）表达的影响。结果：组织化学法显示ＮＯＳ主要分布于内外毛细胞、螺旋神经节细胞和血管纹边缘细胞;原位杂效法发现ＮＯＳｍＲＮＡ在内外毛细胞、螺旋神经节细胞胞浆内均可见阳性染色,但血管纹边缘细胞无阳性染色     

Vesicular storage of adenosine triphosphate in the guinea-pig cochlear lateral wall and concentrations of ATP in the endolymph during sound exposure and hypoxia

Previous studies have revealed putative vesicular stores of adenosine triphosphate (ATP) in the marginal cells of the cochlear stria vascularis which may serve as a source of ATP for purinergic signalling. This study aimed to provide further evidence of ATP storage in the cochlea and to see whether ATP levels in the endolymph are affected by noise and hypoxia. Tissues from the lateral wall and organ of Corti of the guinea-pig cochlea were fractionated to obtain vesicular (VF) and mitochondrial (MF) fractions. Free and total ATP were then measured by the luciferase-luciferin reaction from which membrane-bound vesicular ATP was calculated. In the lateral wall, the VF contained 2.02+/-0.04 nmol ATP/mg protein (n = 5), significantly greater (p < 0.001; paired Student's t-test) than the concentration of ATP in the MF (0.36+/-0.05). In the organ of Corti, the VF contained 0.69+/-0.08 nmol ATP/mg protein (n = 4), significantly smaller than the amount in the VF of the lateral wall tissues (p < 0.001; non-paired Student's t-test). Small amounts of fumarase. an enzyme of the mitochondrial matrix, in the VF, excluded the possibility of mitochondrial ATP contamination. To investigate the effect of hypoxia and noise on the ATP concentrations in the endolymph, fluid samples were collected from the first (basal) cochlear turn of anaesthetized guinea-pigs. As a result of hypoxia (15 min, 13% F1O2), ATP concentrations (nM, mean +/- SEM) increased from 6.2+/-2.3 to 9.3+/-4.5 (n = 4), but the difference was not statistically significant. As a result of noise (15 min, 10 kHz, 110 dB SPL. broad band), the ATP levels increased significantly from 7.4+/-1.2 to 16.0+/-1.8 (p = 0.01; Student's t-test: n = 4). This study has demonstrated the presence of a vesicular store of ATP in the stria vascularis of the cochlea and described an increase in the ATP levels in the endolymph during noise exposure. The findings suggest that ATP is actively secreted from the vesicular store under conditions of metabolic stress. The presence of ATP under basal conditions supports a role for ATP in the sound transduction process during normal function.     

Permanent threshold shift caused by acute cochlear mitochondrial dysfunction is primarily mediated by degeneration of the lateral wall of the cochlea

Mitochondrial dysfunction in the cochlea is thought to be an important cause of sensorineural hearing loss. Recently, we have established a novel rat model with acute hearing impairment caused by exposure to the mitochondrial toxin 3-nitropropionic acid (3-NP) to analyze the mechanism of cochlear mitochondrial dysfunction. Both permanent and temporary threshold shifts were observed in this model depending on the amount of 3-NP used to induce hearing impairment. In this study, we demonstrate cochlear morphological changes in the permanent threshold shift model. Marked degeneration was detected in type 2 fibrocytes in the spiral prominence, type 4 fibrocytes in the spiral ligament, marginal cells and intermediate cells in the stria vascularis 3 h after 3-NP administration; these changes were progressive for at least 14 days. Less prominent degeneration was detected in type 1 and type 3 fibrocytes in the spiral ligament. These results indicate that permanent threshold shift caused by acute cochlear mitochondrial dysfunction is primarily mediated by cellular degeneration in the lateral wall of the cochlea, and suggest that therapy of cochlear hearing loss due to acute energy failure may be achieved through protection and regeneration of the cochlear lateral wall.     

Comparison of the quantity of cochlear melanin in young and old C57BL/6 mice

OBJECTIVE: To elucidate the functional relationship between cochlear melanin and aging. DESIGN: Melanin has been described in the cochlear labyrinth and has been suggested to protect the cochlea from various types of trauma. The quantity of melanin has been shown to change with aging in several organs; however, to our knowledge, aging changes in the cochlea have not been documented. Therefore, we chemically quantified cochlear eumelanin and pheomelanin contents and compared these in young and old C57BL/6 mice using high-performance liquid chromatography. Because melanin deposits in the cochlea present most extensively in the stria vascularis, we morphologically examined the stria using transmission electron microscopy. SUBJECTS: Cochleae from an inbred strain of C57BL/6 male and female mice; 6 at the age of 10 weeks and 5 at the age of 100 weeks were studied. RESULTS: The quantities of cochlear eumelanin and pheomelanin were 421 and 480 ng per cochlea in young mice, and 2060 and 765 ng per cochlea in old mice, respectively. Under transmission electron microscopy, the number of pigmented granules seemed to be greater in older mice compared with younger mice, especially in marginal cells. CONCLUSION: To our knowledge, our findings are the first quantitative evidence to show an age-related overexpression of cochlear melanin and an alteration in the proportion of eumelanin and pheomelanin with aging, suggesting a possible otoprotective function of eumelanin against age-related cochlear deterioration.