Visualization of flow-induced ATP release and triggering of Ca2+ waves at caveolae in vascular endothelial cells
Visualization of flow-induced ATP release and triggering of Ca2+ waves at caveolae in vascular endothelial cells
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DOI：
10.1242/jcs.087221
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发表时间：
2011-10
影响因子：
4
通讯作者：
Kimiko Yamamoto;K. Furuya;Makiko Nakamura;E. Kobatake;M. Sokabe;J. Ando
中科院分区：
文献类型：
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作者：
Kimiko Yamamoto;K. Furuya;Makiko Nakamura;E. Kobatake;M. Sokabe;J. Ando
内皮细胞 (EC) 响应剪切应力（一种由流动的血液产生的流体机械力）而释放 ATP，但是，尽管其释放在通过激活嘌呤能受体来控制多种血管功能方面发挥着至关重要的作用，但 ATP 释放的机制尚未确定。为了分析 ATP 释放的动态，我们利用细胞表面附着的萤火虫荧光素酶和 CCD 相机开发了一种新型化学发光成像方法。在剪切应力刺激下，培养的人肺动脉内皮细胞以两种不同的方式同时释放 ATP，即高度集中的局部方式和浓度较低的扩散方式。局部 ATP 释放发生在细胞膜富含小窝蛋白 1 的区域，并被 siRNA 敲低小窝蛋白 1 和甲基-β-环糊精消耗质膜胆固醇所阻断，表明小窝参与了局部 ATP 释放。 Fluo-4 的 Ca2+ 成像与 ATP 成像相结合表明，剪切应力引起细胞内 Ca2+ 浓度的增加以及随后的 Ca2+ 波，该波源自与局部 ATP 释放相同的位点。这些发现表明，小凹处的局部 ATP 释放触发了 EC 中剪切应力依赖性 Ca2+ 信号传导。
Endothelial cells (ECs) release ATP in response to shear stress, a fluid mechanical force generated by flowing blood but, although its release has a crucial role in controlling a variety of vascular functions by activating purinergic receptors, the mechanism of ATP release has never been established. To analyze the dynamics of ATP release, we developed a novel chemiluminescence imaging method by using cell-surface-attached firefly luciferase and a CCD camera. Upon stimulation of shear stress, cultured human pulmonary artery ECs simultaneously released ATP in two different manners, a highly concentrated, localized manner and a less concentrated, diffuse manner. The localized ATP release occurred at caveolin-1-rich regions of the cell membrane, and was blocked by caveolin-1 knockdown with siRNA and the depletion of plasma membrane cholesterol with methyl-β-cyclodexrin, indicating involvement of caveolae in localized ATP release. Ca2+ imaging with Fluo-4 combined with ATP imaging revealed that shear stress evoked an increase in intracellular Ca2+ concentration and the subsequent Ca2+ wave that originated from the same sites as the localized ATP release. These findings suggest that localized ATP release at caveolae triggers shear-stress-dependent Ca2+ signaling in ECs.