Real-Time Imaging of ATP Release Induced by Mechanical Stretch in Human Airway Smooth Muscle Cells
Real-Time Imaging of ATP Release Induced by Mechanical Stretch in Human Airway Smooth Muscle Cells
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DOI：
10.1165/rcmb.2014-0008oc
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发表时间：
2014-12-01
影响因子：
6.4
通讯作者：
Hasegawa, Yoshinori
中科院分区：
文献类型：
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作者：
Takahara, Norihiro;Ito, Satoru;Hasegawa, Yoshinori
气道壁内的气道平滑肌（ASM）细胞持续暴露于机械刺激，并且响应于这些机械应力而表现出各种功能。ATP在气道中充当细胞外介质。此外，细胞外ATP被认为在哮喘和慢性阻塞性肺疾病的病理生理学中起重要作用。然而，尚不清楚ASM细胞是否是气道中ATP分泌的细胞来源。因此，我们研究了机械拉伸是否诱导ASM细胞释放ATP。使用拉伸装置将机械拉伸应用于在涂覆有I型胶原的硅酮室上培养的原代人ASM细胞。通过循环拉伸（12和20%应变），通过荧光素酶生物发光测量的细胞培养上清液中ATP的浓度显著升高。我们进一步可视化拉伸诱导的ATP释放的细胞在真实的时间使用发光成像系统，同时获得差分干涉对比度细胞图像与红外光学。在单轴拉伸1秒后，某些细胞群立即产生强烈的ATP信号并扩散到周围空间。循环牵张诱导的ATP释放显着减少钙依赖性囊泡胞吐抑制剂，1，2-双（邻氨基苯氧基）乙烷-N，N，N '，N'-四乙酸四乙酰氧基甲酯，莫能菌素，N-乙基马来酰亚胺，和巴弗洛霉素。与此相反，拉伸诱导的ATP释放不抑制半通道阻滞剂，甘珀酸，或阻断瞬时受体电位香草酸4短干扰RNA转染或钌红。这些发现揭示了ASM细胞的一个新特性：机械诱导的ATP释放可能是气道中ATP的细胞来源。
Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca2+-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.