P2X ANTAGONISTS INHIBIT STYRYL DYE ENTRY INTO HAIR CELLS
P2X ANTAGONISTS INHIBIT STYRYL DYE ENTRY INTO HAIR CELLS
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DOI：
10.1016/j.neuroscience.2009.02.076
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发表时间：
2009-07-21
期刊：
影响因子：
3.3
通讯作者：
Duncan, R. K.
中科院分区：
文献类型：
--
作者：
Crumling, M. A.;Tong, M.;Duncan, R. K.
苯乙烯基吡啶染料FM1-43和AM1-43是荧光分子，可以渗透内耳的感觉受体毛细胞的机械转导通道。当这些染料应用于毛细胞时，它们迅速进入细胞质，导致容易检测到的细胞内荧光，这通常被用作机械转导通道活性的分子指示。然而，这样的染料也可以渗透到ATP受体P2X(2)。因此，我们探讨了P2X受体在AM1-43负载毛细胞中的作用。鸡的内耳表达P2X受体并释放ATP，与哺乳动物的内耳相似，允许内源性的P2X受体的刺激。通过在不同实验条件下将鸡内耳感觉上皮暴露于5 MU M AM1-43，并测定组织固定后毛细胞的荧光，从药理学的角度评价了这些受体的参与。将组织预先暴露在5 mM的EGTA中15分钟，这将消除大部分机械转导通道的门控“尖端链接”，仅使荧光衰减44%。相反，P2X受体拮抗剂(pyridoxalphosphate-6-azophenyl-2‘，4’-disulfonic酸[PPADS]、苏拉明、2‘，3’-O-(2，4，6-三硝基苯基)[TNP-ATP]和d-管胡萝卜碱对染料载量的影响更大。当浓度为100mM时，PPADS、苏拉明和TNP-ATP都使毛细胞内AM1-43的荧光降低至少69%。当d-管乌拉林在阻断机械转导通道的浓度(200mM)时，d-管乌拉林处理组和对照之间的荧光差异在统计学上没有显著差异。在同样阻断P2X2受体的浓度(2 MM)下，d-管胡萝卜碱使染料载量减少了72%。从这些实验来看，AM1-43似乎可以通过内源性激活的P2X受体进入毛细胞。因此，当使用苯乙烯基吡啶染料检测毛细胞机械转导通道活动时，尤其是在缺乏明显的机械刺激的情况下，应该考虑P2X受体对染料进入的贡献。(C)2009年IBRO。爱思唯尔有限公司出版。保留所有权利。
The styryl pyridinium dyes, FM1-43 and AM1-43, are fluorescent molecules that can permeate the mechanotransduction channels of hair cells, the sensory receptors of the inner ear. When these dyes are applied to hair cells, they enter the cytoplasm rapidly, resulting in a readily detectable intracellular fluorescence that is often used as a molecular indication of mechanotransduction channel activity. However, such dyes can also permeate the ATP receptor, P2X(2). Therefore, we explored the contribution of P2X receptors to the loading of hair cells with AM1-43. The chick inner ear was found to express P2X receptors and to release ATP, similar to the inner ear of mammals, allowing for the endogenous stimulation of P2X receptors. The involvement of these receptors was evaluated pharmacologically, by exposing the sensory epithelium of the chick inner ear to 5 mu M AM1-43 under different experimental conditions and measuring the fluorescence in hair cells after fixation of the tissue. Pre-exposure of the tissue to 5 mM EGTA for 15 min, which should eliminate most of the gating "tip links" of the mechanotransduction channels, deceased fluorescence by only 44%. In contrast, P2X receptor antagonists (pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid [PPADS], suramin, 2',3'-O-(2,4,6-trinitrophenyl) ATP [TNP-ATP], and d-tubocurarine) had greater effects on dye loading. PPADS, suramin, and TNP-ATP all decreased intracellular AM1-43 fluorescence in hair cells by at least 69% when applied at a concentration of 100 mu M. The difference between d-tubocurarine-treated and control fluorescence was statistically insignificant when d-tubocurarine was applied at a concentration that blocks the mechanotransduction channel (200 mu M). At a concentration that also blocks P2X2 receptors (2 mM), d-tubocurarine decreased dye loading by 72%. From these experiments, it appears that AM1-43 can enter hair cells through endogenously activated P2X receptors. Thus, the contribution of P2X receptors to dye entry should be considered when using styryl pyridinium dyes to detect hair cell mechanotransduction channel activity, especially in the absence of explicit mechanical stimulation of stereocilia. (C) 2009 IBRO. Published by Elsevier Ltd. All rights reserved.