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1 Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo, Japan; PRESTO, Japan Science and Technology Agency, Saitama, Japan
2 Department of Biomedical Engineering, Graduate School of Medicine, University of Tokyo, Tokyo, Japan
3 Department of Clinical Nutrition, Institution of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan
* To whom correspondence should be addressed. E-mail: joji{at}m.u-tokyo.ac.jp.
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 artery 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 the cell-surface ATP synthase and caveolin-1 are physically associated. Measurement of the extracellular metabolism of 3H-labeled 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 membrane-impermeable ATP synthase inhibitors, angiostatin and piceatannol, and by an anti-ATP synthase antibody. Depletion of plasma membrane cholesterol with methyl-
cyclodextrin (MCD) 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 MCD. Down-regulation of caveolin-1 expression by transfection of caveolin-1 siRNA also markedly suppressed ATP-releasing responses to shear stress. Neither MCD, MCD 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.
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