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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: To investigate the effects of ATP and acetylcholine (ACh) on intracellular free calcium concentration ([Ca2 +] i) in cochlear outer hair cells of guinea pigs and the possible mechanism of Ca2 + -mediated Ca2 + release (CICR) by laser scanning confocal microscope. Methods: The isolated outer hair cells (OHC) of guinea pig cochlea were separated by enzyme-catalyzed mechanical separation. Fluorescence-sensitive fluorescent probe Fluo-3 was stained with laser scanning confocal microscope and recorded in extracellular solution with or without calcium. Changes in [Ca2 +] i of OHC after addition of ATP, ACh, Sri Lanka Ryanodine + ATP (or ACh) and Thapsigargin + ATP (or ACh). Results: ATP, Ryanodine + ATP, Thapsigargin + ATP, ACh, Ryanodine + ACh and Thapsigargin + ACh all induced the increase of [Ca2 +] i in Ca2 + -containing extracellular fluid, causing obvious peaks and relative fluorescence intensity peaks respectively 1.64 ± 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 the non-calcium-containing extracellular fluid, ATP and Ryanodine + ATP still caused the smaller peaks of [Ca2 +] i, which were 1.341 ± 0.006 and 1.386 ± 0.008 respectively, while ACh, Ryan-odine + ACh, Thapsigargin + ACh and Thapsigargin + ATP did not cause obvious peak of [Ca2 +] i, but ACh did not induce the increase of [Ca2 +] i. Ryanodine + ACh, Thapsigargin + ACh and Thapsigargin + ATP caused a slowly increase of [Ca2 +] i, respectively. CONCLUSION: ATP and ACh cause the increase of [Ca2 +] i in OHC under the condition of Ca2 + in extracellular fluid. In addition to the influx of extracellular Ca2 + through ion channels, release of IP3 sensitive calcium pool and induction of CICR are also possible. In the absence of Ca2 +, ATP still induced CICR. The mechanism may be that ATP promotes the release of Ca2 + from IP3-sensitive Ca2 +, and Ca2 + induces CICR. However, the response of ACh is Ca2 + -dependent. , ACh did not induce the release of IP3-sensitive calcium pool and induce CICR.