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-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.
Laboratory of System Physiology, Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033,  Japan.
Kimiko
K
Yamamoto
Kishio
K
Furuya
Makiko
M
Nakamura
Eiry
E
Kobatake
Masahiro
M
Sokabe
Joji
J
Ando
D000255:Adenosine Triphosphate / Q000378:metabolism*; D020013:Calcium Signaling / Q000187:drug effects / Q000235:genetics; D021941:Caveolae / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D051242:Caveolin 1 / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism* / Q000473:pathology; D006439:Hemodynamics; D006801:Humans; D049409:Luciferases, Firefly / Q000378:metabolism; D008163:Luminescent Measurements / Q000295:instrumentation / Q000379:methods; D011651:Pulmonary Artery / Q000473:pathology; D034741:RNA, Small Interfering / Q000235:genetics; D013312:Stress, Physiological; D047392:beta-Cyclodextrins / Q000494:pharmacology
D051242:Caveolin 1; D034741:RNA, Small Interfering; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D000255:Adenosine Triphosphate; D002784:Cholesterol; D049409:Luciferases, Firefly