Laboratory of System Physiology, Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan.
J Cell Sci. 2011 Oct 15;124(Pt 20):3477-83. doi: 10.1242/jcs.087221.
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. Ca(2+) imaging with Fluo-4 combined with ATP imaging revealed that shear stress evoked an increase in intracellular Ca(2+) concentration and the subsequent Ca(2+) 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 Ca(2+) signaling in ECs.
内皮细胞（ECs）会在受到切应力（由血流产生的一种流体力学力）的刺激时释放 ATP，但尽管其释放通过激活嘌呤能受体在控制各种血管功能方面起着至关重要的作用，但 ATP 释放的机制尚未确定。为了分析 ATP 释放的动力学，我们通过使用细胞表面附着的萤火虫荧光素酶和 CCD 相机开发了一种新的化学发光成像方法。在切应力的刺激下，培养的人肺动脉内皮细胞以两种不同的方式同时释放 ATP，一种是高度集中、局部的方式，另一种是浓度较低、弥散的方式。局部的 ATP 释放发生在细胞膜富含 caveolin-1 的区域，并且可以通过 siRNA 敲低 caveolin-1 和甲基-β-环糊精耗尽质膜胆固醇来阻断，表明 caveolae 参与了局部 ATP 释放。与 ATP 成像相结合的 Fluo-4 钙成像显示，切应力引起细胞内 Ca2+浓度的增加和随后的 Ca2+波，其起源与局部 ATP 释放的部位相同。这些发现表明，caveolae 处的局部 ATP 释放触发了 ECs 中依赖于切应力的 Ca2+信号转导。
