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

ATP and ACh induced CICR in outer hair cells of the guinea pig cochlea:study of confocal microscopy

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([Ca2+]i)的影响以及诱发Ca2+介导的Ca2+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[Ca2+]i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[Ca2+]i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[Ca2+]i出现幅度较小的波峰,分别为1.341±0.006和1.386±0.008,而ACh、Ryan-odine+ACh、Thapsigargin+ACh和Thapsigargin+ATP未引起明显[Ca2+]i波峰出现,其中ACh不能引起[Ca2+]i的升高,Ryanodine+ACh、Thapsigargin+ACh和Thapsigargin+ATP分别引起[Ca2+]i缓慢升高。结论:在细胞外液有Ca2+的条件下,ATP和ACh引起OHC的[Ca2+]i升高,除了通过离子通道引起胞外Ca2+内流,还有IP3敏感钙库的释放和诱发CICR。在无Ca2+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Ca2+,Ca2+又诱发了CICR,而ACh的反应是Ca2+依赖性的,在细胞外液无Ca2+的条件下,ACh不能引起IP3敏感钙库的释放和诱发CICR。

Objective：Effects of ATP and acetylcholine （ACh） on intracellular Ca^2＋ concentrations （[Ca^2＋]i） and possible mechanism of Ca^2＋induced Ca^2＋ release（CICR） of the isolated outer hair cells（OHCs） in the guinea pig cochlea were studied with confocal microscopy. Method：OHCs were isolated from guinea pig cochlea by enzymatic and mechanical methods. The effects of ATP, ACh,Ryanodine＋ATP （or ACh） and Thapsigargin＋ATP （or ACh） in the presence or absence of extracellular Ca^2＋ on [Ca^2＋ ]i in OHCs were examined by confocal microscopy. Result： In the presence of ATP, Ryanodine＋ ATP, Thapsigargin＋ ATP, ACb, Ryanodine ＋ ACh and Thapsigargin＋ACh increased [Ca^2＋ ]i and evoked an evident wave, respectively, the relative magnitude of fluorescence were 1. 604±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） in OHCs in the presence of extracellular Ca^2＋ respectively. In the absence of extracellular Ca^2＋ , ATP and Ryanodine＋ ATP induced a gradual and small [Ca^2＋ ]i wave, the relative magnitude of fluorescence were 1. 341 ± 0. 006 and 1. 386 ± 0. 008, however, ACh,Ryanodine＋ACh,Thapsigargin＋ACh and Thapsigargin＋ATP can not induce wave but a gradual [Ca^2＋ ]i elevation. ACh can not increase [Ca^2＋ ]i. Conclusion： In the presence of extracellular Ca^2＋ , ATP and ACh increased [Ca^2＋ ]i in OHCs not only by Ca^2＋ influx through ion channel on cell membrance but also a release of Ca^2＋ from IP3-sensitive calcium reservoir and CICR. In the absence of extracellular Ca^2＋ , ATP activated IPa sensitive calcium reservoir and Ca^2＋ release through IPa sensitive calcium reservoir, in turn CICR was induced. ACh can not activate IPa sensitive calcium reservoir and CICR in the absence of extracellular Ca^2＋ , therefore, the effect of ACh was dependent of extracellular Ca^2＋.