Visualization of flow-induced ATP release and triggering of Ca2+ waves at caveolae in vascular endothelial cells

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-beta-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.