Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells.
Endothelial cells (ECs) release ATP in response to shear stress, a mechanical force generated by blood flow, and the ATP released modulates EC functions through activation of purinoceptors. The molecular mechanism of the shear stress-induced ATP release, however, has not been fully elucidated. In this study, we have demonstrated that cell surface ATP synthase is involved in shear stress-induced ATP release. Immunofluorescence staining of human pulmonary arterial ECs (HPAECs) showed that cell surface ATP synthase is distributed in lipid rafts and co-localized with caveolin-1, a marker protein of caveolae. Immunoprecipitation indicated that cell surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of [(3)H]ADP confirmed that cell surface ATP synthase is active in ATP generation. When exposed to shear stress, HPAECs released ATP in a dose-dependent manner, and the ATP release was markedly suppressed by the membrane-impermeable ATP synthase inhibitors angiostatin and piceatannol and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-beta-cyclodextrin (MbetaCD) disrupted lipid rafts and abolished co-localization of ATP synthase with caveolin-1, which resulted in a marked reduction in shear stress-induced ATP release. Pretreatment of the cells with cholesterol prevented these effects of MbetaCD. Downregulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MbetaCD, MbetaCD plus cholesterol, nor caveolin-1 siRNA had any effect on the amount of cell surface ATP synthase. These results suggest that the localization and targeting of ATP synthase to caveolae/lipid rafts is critical for shear stress-induced ATP release by HPAECs.
Yamamoto
Kimiko
K
Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Shimizu
Nobutaka
N
Obi
Syotaro
S
Kumagaya
Shinichiro
S
Taketani
Yutaka
Y
Kamiya
Akira
A
Ando
Joji
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D051242:Caveolin 1 / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism*; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D025261:Mitochondrial Proton-Translocating ATPases / Q000378:metabolism*; D011983:Receptors, Purinergic / Q000502:physiology; D013314:Stress, Mechanical
C506526:ATP5F1B protein, human; C495540:CAV1 protein, human; D051242:Caveolin 1; D011983:Receptors, Purinergic; D000255:Adenosine Triphosphate; D002784:Cholesterol; D025261:Mitochondrial Proton-Translocating ATPases