豚鼠壶腹嵴毛细胞内钙离子活动及ATP 的影响

目的　探讨离体前庭毛细胞（ｖｅｓｔｉｂｕｌａｒ ｈａｉｒｃｅｌｌｓ,ＶＨＣ）去极化时细胞内钙离子活动特征及内耳活性物质ＡＴＰ对离体ＶＨＣ内钙离子浓度的影响及其机制。方法　采用酶孵育后机械分离法分离豚鼠壶腹嵴ＶＨＣ,６ μｍｏｌ／Ｌ钙荧光探针Ｆｌｕｏ３ 染色,利用激光扫描共聚焦显微镜（ｌａｓｅｒ ｓｃａｎｎｉｎｇｃｏｎｆｏｃａｌ ｍｉｃｒｏｓｃｏｐｙ,ＬＳＣＭ） 记录静息状态下、加入高Ｋ＋ 液及不同浓度ＡＴＰ时代表ＶＨＣ内［Ｃａ２＋ ］ｉ 的荧光强度相对值的变化。结果　细胞外有Ｃａ２＋ 存在时,７５ ｍｍｏｌ／ＬＫ＋ 可引起所观察５ 个ＶＨＣ内［Ｃａ２＋］ｉ均升高,细胞体和皮板区的［Ｃａ２＋］ｉ 变化一致;在细胞外无Ｃａ２＋ 的情况下,所观察的５ 个ＶＨＣ 中,高Ｋ＋ 并不能引起细胞内［Ｃａ２ ＋］ｉ 升高。在细胞外存在Ｃａ２ ＋ 时,不同浓度（１ ．０ ｍｍｏｌ／Ｌ,０．１ ｍｍｏｌ／Ｌ）ＡＴＰ可引起绝大多数（１８／１９）ＶＨＣ 内［Ｃａ２＋ ］ｉ 迅速升高,达高峰后下降,恢复至静息时的水平;细胞外无Ｃａ２ ＋ 时,ＡＴＰ仅引起极少数（１／１０）ＶＨＣ内［Ｃａ２＋］ｉ 升高;而对照组加入与ＡＴＰ等体积的Ｈａｎｋ 液,不引起ＶＨＣ 内［Ｃａ     

听神经瘤患者离体前庭毛细胞的分离及胞内钙离子活动

目的 进一步了解离体人前庭毛细胞的胞内钙离子（Ｃａ＾２＋）活动。方法 从３例经迷路听神经瘤切除术患者中采取半规管壶腹中的前庭毛细胞，用倒置显微镜观察分离出的离体前庭毛细胞形态，用Ｃａ＾２＋敏感的荧光染料Ｆｕｒａ－２和数字影像显微镜监测细胞内Ｃａ＾２＋浓度的变化。结果 分离的前庭毛细胞主要为Ｉ型毛细胞，可在体外存活２．５ｈ。用１５０ｍｍｏｌ／Ｌ Ｋ＾＋液灌流单离的前庭毛细胞引起胞内Ｃａ＾２＋的显著升     

温度变化对豚鼠离体前庭毛细胞内钙离子浓度的影响

目的 为探讨温度改变诱发前庭眼震的机理。方法 酶孵育后机械分离成功豚鼠前庭毛细胞（ＶＨＣ）３４个，Ｆｌｕｏ－３荧光染色后，改变其外环境的温度，用激光扫描共聚焦显微镜（ＬＳＣＭ）记录静息状态下及温度改变时ＶＨＣ内钙离子浓度（［Ｃａ＾２＋］ｉ）的变化。结果 将分离的ＶＨＣ分为两型，ＬＳＣＭ下钙离子分布图像与相差显微镜下所见细胞外形相似，其钙离子分布在核区略高。１８／２２的Ｉ型与７／１２的Ⅱ型ＶＨＣ其细     

温度变化对豚鼠离体前庭毛细胞内钙离子浓度的影响

目的为探讨温度改变诱发前庭眼震的机理。方法酶孵育后机械分离成功豚鼠前庭毛细胞（ＶＨＣ）３４个，Ｆｌｕｏ－３荧光染色后，改变其外环境的温度，用激光扫描共聚焦显微镜（ＬＳＣＭ）记录静息状态下及温度改变时ＶＨＣ内钙离子浓度（［Ｃａ２＋］ｉ）的变化。结果将分离的ＶＨＣ分为两型，ＬＳＣＭ下钙离子分布图像与相差显微镜下所见细胞外形相似，其钙离子分布在核区略高。１８／２２的Ⅰ型与７／１２的Ⅱ型ＶＨＣ其细胞内［Ｃａ２＋］ｉ随温度的升高而增加，随温度的降低而减少，呈规律性变化，但Ｉ型ＶＨＣ的变化幅度较大；对照试验观察到变温仅对少数（２／９）耳蜗外毛细胞（ＯＨＣ）内［Ｃａ２＋］ｉ有影响。本研究结果显示变温对ＶＨＣ有直接刺激作用，表现为ＶＨＣ内［Ｃａ２＋］ｉ变化。结论对冷热诱发眼震的解释，除Ｂａｒａｎｙ的热对流学说外，尚存在冷热对ＶＨＣ的直接作用。     

三种神经活性物质对离体豚鼠耳蜗外毛细胞内游离钙浓度的影响

目的研究神经活性物质对耳蜗外毛细胞内游离钙离子浓度（［Ｃａ２＋］ｉ）的影响。方法应用显微荧光测钙技术检测豚鼠耳蜗离体外毛细胞［Ｃａ２＋］ｉ在加入乙酰胆碱、ＡＴＰ、碳酰胆碱之后的变化。结果在含钙的细胞外液中，乙酰胆碱、ＡＴＰ和碳酰胆碱均可引起［Ｃａ２＋］ｉ升高，幅度分别为（０．７４±０．１２９）μｍｏｌ／Ｌ（乙酰胆碱），（０．６５±０．１１）μｍｏｌ／Ｌ（ＡＴＰ），（１．１６±０．２７）μｍｏｌ／Ｌ（碳酰胆碱）；而细胞外液中无钙时，ＡＴＰ仅可引起缓慢而小幅度（０．１８±０．０５）μｍｏｌ／Ｌ的［Ｃａ２＋］ｉ升高。结论同属胆碱能毒蕈碱受体激动剂的乙酰胆碱和碳酰胆碱可作用于受体，打开离子通道，使细胞外Ｃａ２＋进入细胞内，导致外毛细胞［Ｃａ２＋］ｉ升高；细胞外液有Ｃａ２＋时，ＡＴＰ引致的［Ｃａ２＋］ｉ升高，可能是因为引发了内向的Ｃａ２＋流，细胞外液无Ｃａ２＋时，［Ｃａ２＋］ｉ升高则可能源于细胞内钙库的释放。     

听神经瘤患者离体前庭毛细胞的分离及胞内钙离子活动

目的进一步了解离体人前庭毛细胞的胞内钙离子（Ｃａ２＋）活动。方法从３例经迷路听神经瘤切除术患者中采取半规管壶腹中的前庭毛细胞，用倒置显微镜观察分离出的离体前庭毛细胞形态，用Ｃａ２＋敏感的荧光染料Ｆｕｒａ２和数字影像显微镜监测细胞内Ｃａ２＋浓度的变化。结果分离的前庭毛细胞主要为Ⅰ型毛细胞，可在体外存活２．５ｈ。用１５０ｍｍｏｌ／ＬＫ＋液灌流单离的前庭毛细胞引起胞内Ｃａ２＋的显著升高，荧光比率从０．５４升至１．１６；这种胞内Ｃａ２＋的升高在胞外液中Ｃａ２＋缺如的情况下消失。０．２５μｍｏｌ／ＬＩｏｎｏｍｙｃｉｎ可以引起前庭毛细胞内Ｃａ２＋的不可逆性升高，荧光比率保持在０．７３。结论人前庭毛细胞膜上存在电压敏感的Ｃａ２＋通道。     

豚鼠耳蜗外毛细胞的共聚焦图像

建立了用激光扫描共聚焦显微镜豚鼠耳蜗外毛细胞生理变化及细胞形态的方法，以荧光染料Ｆｌｕｏ－３作胞内Ｃａ＾２＋指示剂，用共聚焦显微观察乙酰胆碱（Ａｃｈ），ＡＴＰ、高钾引起的外毛细胞（ＯＨＣ）胞内Ｃａ＾２＋浓度的变化，三种物质均引起Ｃａ＾２＋升高，但升高的幅度，进相及所在细胞部位不同，高钾还引起ＯＨＣ短缩，偏折，利用共聚焦显微镜“光学切片”的特性，对Ｆｌｕｏｒｅｓｃｅｉｎ荧光素染色的活的ＯＨＣ形态及磺     

水杨酸钠豚鼠耳蜗单离外毛细胸游离钙浓度影？…

目的 观察水杨酸钠对豚鼠耳蜗外毛细胞（ｏｕｔｅｒ ｈａｉｒ ｃｅｌｌｓ，ＯＨＣ）内游离钙浓度「Ｃａ＾２＋」ｉ的影响，并对钙通道调控药物的拮抗作用进行观察，以深入探讨水杨酸钠耳毒性的分子生物学机制。方法 豚鼠全身麻醉后断头活杀，酶－机械法分离获得耳蜗单离ＯＨＣ。１０ｕｍｏｌ／Ｌ的Ｅｌｕｏ－３／ＡＭ负载３０ｍｉｎ后用ＡＣＡＳＵｌｔｉｍａ型激光扫描共聚焦显微镜观察不同药物灌流下ＯＨＣ「Ｃａ＾２＋」ｉ的变     

豚鼠膜迷路积水模型耳蜗Ca^2＋ATP酶的变化

目的 了解Ｃａ＾２＋－ＡＴＰ酶在耳蜗活动位点及膜迷路积水后耳蜗Ｃａ＾２＋－ＡＴＰ酶的变化。方法 选成年健康、Ｐｒｅｙｅｒ反射正常豚鼠１４只，左耳装圆窗电极一阻塞肉淋巴，经声反应阈（ＣＡＰ阀值）证明膜迷路积水形成；右侧为对照耳。用枸橼酸铅细胞组化法测定Ｃａ＾２＋－ＡＴＰ酶，在透射电镜下Ｃａ＾２＋－ＡＴＰ酶以磷酸铅黑色颗粒显示。结果 对照耳Ｃａ＾２＋－ＡＴＰ酶活动部位在蜗管前庭膜内淋巴侧、内外毛细胞皮     

应用显微荧光测钙技术研究乙酰胆碱对豚鼠耳蜗对外毛细胞内 …

介绍了应用最显微荧光测钙技术研究豚鼠耳蜗外毛细胞（ＯＨＣ）内游离钙浓度的变化的方法。该技术以钙敏感染料Ｆｕｒａ－２作为胞内Ｃａ＾２＋的指示剂，装协进入细胞后，与胞内游离Ｃａ＾２＋结合，在特定波长光的激发下可发出荧光，由此可推算「Ｃａ＾２＋」ｉ的变化。结果发现乙酰胆碱在含钙的细胞上液中可引起「Ｃａ＾２＋」ｉ升高。对显微荧光测钙技术在研究耳蜗毛细胞中的应用以及ＡＣｈ引起「Ｃａ＾２＋」ｉ升高的机制进行讨     

Extracellular adenosine 5'-ATP-induced calcium signaling in isolated vestibular ganglion cells of the guinea pig

Extracellular adenosine 5'-triphosphate (ATP)-induced intracellular calcium concentration ([Ca2+]i) changes in acutely isolated vestibular ganglion cells (VGCs) of the guinea pig were investigated using the Ca2+ -sensitive dye Fura-2. Extracellular ATP induced an increase in [Ca2+]i in VGCs in a dose-dependent manner. ATP induced an increase in [Ca2+]i even in the absence of extracellular Ca2+ (1 mM Ethylene Glycol-bis (beta-aminoethyl Ether) N,N,N',N'-Tetraacetic Acid (EGTA)), thus suggesting that ATP induces Ca2+ release from the intracellular stores. The P2-receptor antagonists suramin and reactive blue 2 inhibited the ATP-induced [Ca2+]i increase in a dose-dependent manner. The P1-receptor agonist adenosine did not induce any changes in [Ca2+]i. These results suggest that VGCs may possess a P2-purinergic receptor but not a P1-purinergic receptor. La3+, a receptor-mediated calcium channel blocker, inhibited the ATP-induced [Ca2+]i increase but, in contrast, nifedipine, a L-type calcium channel blocker, did not. These results suggest that ATP induces both a Ca2+ -release from the intracellular stores and a Ca2+ influx from the extracellular space through La3+ -sensitive and nifedipine-insensitive Ca2+ channels in VGCs. Our results also suggest that extracellular ATP may act as a neurotransmitter or neuromodulator of the vestibular peripheral system in the guinea pig.     

ATP and Nitric Oxide Modulate Intracellular Calcium in Isolated Pillar Cells of the Guinea Pig Cochlea

Supporting cells in the mammalian cochlea have recently received attention as potential targets of neurotransmitters, neuromodulators, and neurohumoral agents. Calcium homeostasis in Deiters' and Hensen's cells, for example, is regulated by ATP and nitric oxide. We studied the intracellular calcium concentration [Ca2+]i in isolated pillar cells of the guinea pig cochlea in response to extracellular ATP and nitric oxide using the fluorescent indicator fluo-3. [Ca2+]i increased rapidly and significantly throughout the pillar cell in response to a bolus of ATP or 2-methylthio ATP while a, b-methylene ATP was ineffective. The response to ATP was inhibited by suramin and Cibacron Blue but not by pyridoxal phosphate 6-azopheny1-2',4'-disulfonic acid. This pharmacological profile is consistent with a [Ca2+]i increase largely mediated by P2Y receptors. In Ca2+-free medium supplemented with EGTA, the response to extracellular ATP was reduced by 33%, suggesting a contribution of calcium influx to the overall effect. The ATP-induced increase of [Ca2+]i was attenuated by NO donors (sodium nitroprusside or diethylamine NONOate), and this attenuation was reversed by KT5823, an antagonist to protein kinase G. The results indicate the involvement of purinergic mechanisms and the nitric oxide/cyclic GMP/protein kinase G pathway in the regulation of [Ca2+]i in cochlear pillar cells.     

巯基氧化剂硫汞撒对豚鼠耳蜗外毛细胞胞内钙离子流的影响

目的 观察外毛细胞内源性外钙离子的释放。方法 分离豚鼠耳蜗外毛细胞,用硫汞撒（ｔｈｉｍｅｒｏｓａｌ）为激动剂,用钙离子敏感染料ｆｕｒａ－２和数字荧光显微镜观察离体外毛细胞内钙离子浓度的波动。结果 硫汞撒可诱发离体耳蜗外毛细胞内钙离子的明显增加,其增加程度在一定范围内与硫汞撒的浓度呈正比。在清除了细胞外液中钙离子后,硫汞撒仍可诱发离体耳蜗外毛细胞胞内钙离子浓度的升高。在此钙离子浓度升高的基础上向胞外     

三种神经活性物质对离体豚鼠耳蜗外毛细胞内游离钙浓度的 …

OBJECTIVE: To measure the effects of neuro-active substances on intracellular free Ca2+ concentration ([Ca2+]i) in isolated outer hair cells(OHCs) of the guinea pig cochlea. METHODS: The fura-2 microfluorimetry was used to measure changes of [Ca2+]i in OHCs of the guinea pig cochlea after application of acetylcholine, ATP and carbacholine. RESULTS: Acetylcholine, ATP and carbacholine increased [Ca2+]i (acetylcholine: 0.74 +/- 0.12 mumol/L, ATP: 0.65 +/- 0.11 mumol/L, carbacholine: 1.16 +/- 0.27 mumol/L) in OHCs in the presence of extracellular Ca2+. In the absence of extracellular Ca2+, however, only ATP induced a gradual and small [Ca2+]i elevation, whereas other substances did not. CONCLUSION: Acetylcholine and carbacholine, the cholinergic mascarinic agonists, increased [Ca2+]i in OHCs by acting at receptor-induced ion channel resulting in Ca2+ efflux. ATP-induced elevation of [Ca2+]i without Ca2+ in extracellular medium is due to a release of Ca2+ from an intracellular reservoir.     

Effect of neuroregulators on the intracellular calcium level in the outer hair cell isolated from the guinea pig.

The cytosolic calcium concentration [( Ca2+]i) of the isolated outer hair cell of the guinea pig was measured using fluorescence imaging microscopy and the effects of efferent neuroregulators such as acetylcholine, ATP, GABA, substance P, enkephalin, calcitonin gene-related peptide, serotonin, dopamine, norepinephrine, and glutamate were investigated. Among the drugs tested only ATP induced an elevation of the [Ca2+]i of the outer hair cell. In the resting condition, [Ca2+]i averaged 104.5 +/- 31.1 nM (n = 27), while 100 microM ATP significantly increased [Ca2+]i to 146.3 +/- 43.5 nM (n = 19). Superfusion with Ca2(+)-free solution (pCa = 7.5) abolished the increase in [Ca2+]i induced by ATP, suggesting that ATP causes an entry of external Ca2+. The relevance of [Ca2+]i to the inhibitory actions of efferent neuroregulators is discussed.     

The effects of streptomycin on the calcium influx in SGCs during depolarization]

OBJECTIVE: To examine the effect of streptomycin on the calcium influx in spiral ganglion cells (SGCs) during depolarization and the differences among different concentrations of streptomycin and SGCs sources with the aim of exploring the mechanism of acute streptomycin ototoxicity. METHODS: The SGCs of guinea pig cochlea were isolated using an enzyme-machine method and loaded with 10 mumol/L Fluo-3/AM for 30 min at 37 degrees C. Dissociated SGCs loaded with Fluo-3 were examined with a confocal microscope (ACAS Ultima, USA) using a 20 x objective lens and linear scan mean. The fluorescent images collected every 5-sec for a total of 300 sec were stored in a computer. The fluorescent intensity of the images was analyzed by a software cooperated with the confocal microscope, and a curve of fluorescent intensity changes against time was obtained. RESULTS: The [Ca2+]i of SGCs was steady under the perfusion with standard extracellular solution. The [Ca2+]i of SGCs increased in 12/14 cells under the perfusion with 150 mmol/L high potassium solution, but increased in only 1/10 cells induced by the high potassium calcium free solution. After treating with 1 mmol/L, 0.1 mmol/L, 0.01 mmol/L streptomycin, the [Ca2+]i of the SGCs perfused with the potassium solution was increased in 0/9, 6/14, 7/13 cells, respectively. After the SGCs were treatment with 0.02 mmol/L streptomycin, the [Ca2+]i of the SGCs perfused with the high potassium solution was increased in 5/10 SGCs from the apical turn and in 0/15 SGCs from the basal turn. CONCLUSION: Perfusion with high potassium media can result in obviously increased [Ca2+]i of the SGCs, and the increase of the [Ca2+]i in the SGCs may originate from the extracellular calcium influx. The calcium influx can be blocked by streptomycin and such effects depend on streptomycin concentrations and SGCs sources.     

Isolation of and calcium mobilization in vestibular hair cells of the guinea pig

Vestibular hair cells were isolated from the macula utriculi, macula sacculi and crista ampullaris of the guinea pig, using enzymatic and mechanical techniques. The cells could be classified into two types: flask-shaped ones with a neck and rod- or round-shaped ones without a neck. The flask-shaped cells were thought to be type I cells, the rod-shaped cells type II cells, while the round-shaped cells could have been type I or type II cells. The intracellular free calcium ion concentrations [( Ca2+]i) of these cells were determined using the Ca2(+)-sensitive dye Fura-2 and digital imaging miroscopy. In the resting state, [Ca2+]i in the nucleus and cuticular plate was variable in both types of cells. The 150 mM KCl stimulation, which might induce a depolarization, resulted in a temporary increase in [Ca2+]i in both types of cells. In the presence of 1 micron ionomycin, there was an irreversible increase in [Ca2+]i in both types of cells. These observations aid in elucidating vestibular function.     

Isolation of and calcium kinetics in cochlear inner hair cells of the guinea pig.

Single inner hair cells of the guinea pig cochlea were isolated using enzymatic and mechanical techniques. The intracellular free calcium ion concentrations [( Ca2+]i) of the isolated inner hair cells were determined using the Ca2+ sensitive dye fura-2 and digital imaging microscopy. In the presence of 1 micron ionomycin, a Ca2+ ionophore, there was an irreversible increase in [Ca2+]i. The 150 mM KCl stimulation, which induces a depolarization, resulted in a temporary increase in [Ca2+]i. This increase in [Ca2+]i was not observed under conditions of depolarization, in Ca(2+)-free medium. These observations are interpreted to mean that the [Ca2+]i during membrane depolarization mainly originates from an influx of extracellular Ca2+ into the cytoplasm.