应用显微荧光测钙技术研究乙酰胆碱对豚鼠耳蜗外毛细胞内游离钙的影响

介绍了应用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（[Ca~(2 )]i）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，装载进入细胞后，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（ACh）在含钙的细胞外液中可引起［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升高的机制进行了讨论。     

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.     

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。     

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     

庆大霉素及其代谢产物对外毛细胞内游离钙浓度的影响

目的：探讨庆大霉素（GM）耳毒性与耳蜗外毛细胞（OHC）内游离钙（[Ca^2 ])之间的关系。方法：离体耳蜗OHC经钙荧光指示剂Fluo-3负载后，用激光扫描共聚焦显微镜测定高钾溶液、GM、GM代谢产物等对OHC[Ca^2 ])i的影响。结果GM使[Ca^2 ]i短暂下降，并阻滞高钾溶液引起的钙内流，GM代谢产物则使[Ca^2 ]i持续升高，对高钾引起的钙内流无明显影响，结论GM耳毒与外毛细胞[Ca^2 ]i持续升高有关。     

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。     

声波对离体耳蜗外毛细胞内游离Ca2+浓度的影响

探讨声波对离体耳蜗内外毛细内游离Ｃａ＾２＋浓度的影响。方法采用荧光染色法,用ＡＣＡＳ５７０型粘附式细胞仪对纯音刺激引起的豚鼠耳蜗ＯＨＣ的的变化进行动态观察。结果声刺激可引起明显升高,至高峰后迅速,再次刺激可引起类似反应。结论声音刺激中引起单个ＯＨＣ的生理变化。     

尼莫地平对豚鼠耳蜗外毛细胞钙电流的影响

为研究尼莫地平对豚鼠耳蜗外毛细胞钙电流的影响，应用全细胞式记录膜片钳技术，观察１８只豚鼠耳蜗分离出的２０个外毛细胞中，用全细胞方式记录到通道电流２０次。结果发现，尼莫地平可部分阻断豚鼠耳蜗外毛细胞电压依赖性钙电流，减轻毛细胞内Ｃａ＾２＋负荷，其阻断作用是可逆的，对电流的抑制率与尼莫地平的浓度有关，其半数抑制浓度为６０．１４ｎｍｏｌ／Ｌ，对电流的最大抑制率为３４．１６％。推测尼莫地平在治疗梅尼埃病等     

链霉素引起离体耳蜗外毛细胞内Ca^2＋浓度的改变

为探讨耳毒性药物与细胞内游离钙之间的关系，采用荧光素染色法，以５７０型粘附式细胞仪动态观察，发现链霉素可使豚鼠离体耳蜗外毛细胞内的游离显著增我，于６０ｓ达到最高值，持续６０ｓ后逐渐降低，再经１００ｓ降至原来水平，以后有轻度下降。该方法可显示游离钙在外毛细胞内的分布。推测外毛细胞内Ｃａ＾２＋升高可能是氨基甙类抗生素耳毒性损伤过程的一个阶段。     

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     

Glutamate induced modulation of free Ca2+ in isolated inner hair cells of the guinea pig cochlea]

OBJECTIVE: To investigate the modulation effect of glutamate (Glu) on intracellular free Ca2+ concentration([Ca2+]i) of inner hair cell (IHC). METHODS: Using the laser scanning confocal microscope (LSCM), the exogenous Glu-induced changes in [Ca2+]i of isolated 10 IHCs and 10 OHCs of guinea pig cochlea were observed with fluo-3, a fluorescent probe for [Ca2+]i. RESULTS: The IHCs were identified by their unique flask shape with a distinct neck and spherical base and a large spherical nucleus. In most cases, normal cell shapes could be maintained about two hours after isolation. The images of [Ca2+]i from LSCM were similar to those from inverted microscopy. Fluorescence of Fluo-3 distributed in the isolated IHCs with brighter staining in the nucleus. In the presence of low concentration of Glu (3.85 mumol/L), there was an increase of [Ca2+]i in IHCs, whereas no change in OHCs was found. Of the 10 IHCs, increases of [Ca2+]i were observed in 9 and no change in 1. Of the 10 OHCs, 7 showed no [Ca2+]i change and only 3 showed minor reduction of [Ca2+]i. An increase of the Glu concentration (21.88 mumol/L) induced a corresponding increase of [Ca2+]i in IHCs, but eventually resulted in a gradual decrease of [Ca2+]i with a distortion of the normal shape, which indicated that the IHCs were degenerated and swelling. CONCLUSION: These results suggested that exogenous Glu is capable of modulating [Ca2+]i of IHC and may be act on autoreceptor in a positive feedback manner. Excessive Glu induced the accumulation of IHC [Ca2+]i which finally resulted in the degeneration and edema of IHC and the reduction of IHC [Ca2+]i.     

乙酰胆碱和三磷酸腺苷介导的豚鼠外毛细胞和Deiters细胞的离子电流

OBJECTIVE: To determine ACh- and ATP-induced currents in isolated outer hair cells of guinea pig cochlea, respectively. METHOD: The whole-cell patch-clamp technique was used to investigate potential and concentration dependence of ACh- and ATP-induced ion currents. RESULTS: ACh(100 mumol/L) induced an early inward current followed by a late outward current when the cells were voltage-clamped at -70 mV. Only outward current occurred when potential was over -60 mV and its reversal potential was (-80 +/- 5.61) mV(n = 6). When depolarization protocols were applied, amplitude of inward currents activated by ATP(100 mumol/L) decreased and reversed at (3.2 +/- 0.23) mV(n = 8). Ion currents activated by ATP (100 mumol/L) were also recorded from isolated Deiters' cells. CONCLUSION: ACh-induced outward current was carried by K+ and ATP-induced inward current was through non-selective cations channel. Effects of ACh and ATP on OHCs were voltage dependent.     

谷氨酸调节耳蜗内毛细胞游离钙的实验观察

目的探索谷氨酸(glutamate,Glu)对离体耳蜗内毛细胞(innerhaircells,IHC)内钙信号的调控作用及其生理病理意义。方法在激光共聚焦显微镜下用钙敏荧光探针Fluo-3作为指示剂,观察外源性谷氨酸对分离的10个豚鼠耳蜗IHC胞内游离钙离子浓度(［Ca2+］i)的影响。结果分离好的正常IHC呈烧瓶形状,有明显的颈部,皮板上可观察到静纤毛,大球形的细胞体中间可见圆形的细胞核。形态完好的IHC大约存活2h,Fluo-3钙敏荧光探针染色后IHC胞体、胞核及表皮板有明显的染色梯度,表明游离Ca2+浓度从细胞核向细胞质逐渐减少。终浓度为3.85μmol/L的Glu对游离IHC内［Ca2+］i有增高趋势,而对游离外毛细胞(outerhaircells,OHC)内［Ca2+］i浓度无影响。观察10个IHC,发现9个［Ca2+］i浓度增加,1个无变化;观察10个OHC,发现7个［Ca2+］i无变化,3个略有下降。当Glu浓度增高后,IHC内［Ca2+］i先是迅速升高,继而逐渐下降。IHC外形由烧瓶状逐渐变成球形,提示IHC水肿变性。结论Glu可选择性调控IHC内［Ca2+］i,而对OHC内［Ca2+］i无影响,而过量的Glu刺激,可造成IHC［Ca2+］i的堆积,从而IHC水肿变性。     

水杨酸钠对豚鼠耳蜗单离外毛细胞游离钙浓度影响及其调控机制研究

目的　观察水杨酸钠对豚鼠耳蜗外毛细胞 (outerhaircells,OHC)内游离钙浓度 [Ca2 ]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。 10 μmol/L的Fluo 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[Ca2 ]i的变化。结果　OHC在标准细胞外液灌流过程中 [Ca2 ]i保持稳定。 1mmol/L水杨酸钠灌流致 8/ 10个细胞的 [Ca2 ]i明显升高。无钙细胞外液中灌流亦有 10 / 10个细胞 [Ca2 ]i明显的升高。经 3mmol/L利多卡因作用1h后 ,水杨酸钠灌流不能使细胞 [Ca2 ]i升高 (n =10 )。盐酸氟桂嗪可以明显阻断水杨酸钠导致的OHC[Ca2 ]i升高效应 (3/ 12细胞的 [Ca2 ]i升高 ) ,而尼莫地平无此效应 (7/ 7细胞的 [Ca2 ]i升高 )。结论　水杨酸钠可以导致OHC[Ca2 ]i明显增加 ,OHC[Ca2 ]i升高来源于细胞内钙库的动员和释放。使细胞内 [Ca2 ]i升高可能是水杨酸钠的耳毒性机制之一。     

豚鼠耳蜗外毛细胞的变性机制

目的:探讨外毛细胞维持形态及变性过程发生的可能机制。方法:倒置相差显微镜下,对于急性酶分离所得的外毛细胞进行6 h的连续性观察。结果:半数以上活性良好的单离外毛细胞可保持活性平均约6 h,变性过程中均出现自表皮板至基底部的纵行褶线。含钙和不含钙的细胞维持活性时间相同。静纤毛缺失可以同样维持较长时间的活性。结论:钙离子浓度和静纤毛损伤不是外毛细胞变性的决定性因素,而环细胞表面的某种弹性机制可能是维持形态保持活性的根本原因。