Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells.
Involvement of cell surface ATP synthase in flow-induced ATP release by vascular endothelial cells.
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DOI：
10.1152/ajpheart.01385.2006
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发表时间：
2007-09
期刊：
影响因子：
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通讯作者：
Kimiko Yamamoto;N. Shimizu;S. Obi;S. Kumagaya;Y. Taketani;A. Kamiya;J. Ando
中科院分区：
文献类型：
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作者：
Kimiko Yamamoto;N. Shimizu;S. Obi;S. Kumagaya;Y. Taketani;A. Kamiya;J. Ando
内皮细胞（EC）响应于血流产生的机械力剪切应力释放ATP，释放的ATP通过激活嘌呤受体调节EC功能。然而，剪切应力诱导ATP释放的分子机制尚未完全阐明。在这项研究中，我们已经证明，细胞表面ATP合酶参与剪切应力诱导的ATP释放。人肺动脉内皮细胞（HPAECs）的免疫荧光染色显示，细胞表面ATP合成酶分布在脂筏中，并与小窝标记蛋白caveolin-1共定位。免疫沉淀表明，细胞表面ATP合酶和小窝蛋白-1物理相关。测定细胞外[（3）H]ADP代谢证实细胞表面ATP合成酶在ATP生成中具有活性。当暴露于剪切应力时，HPAEC以剂量依赖性方式释放ATP，并且ATP释放被膜不可渗透的ATP合成酶抑制剂血管抑素和piceatannol以及抗ATP合成酶抗体显著抑制。用甲基-β-环糊精（MbetaCD）消耗质膜胆固醇破坏脂筏，并废除ATP合酶与小窝蛋白-1的共定位，这导致剪切应力诱导的ATP释放显着减少。用胆固醇预处理细胞可以防止MbetaCD的这些作用。通过转染小窝蛋白-1 siRNA下调小窝蛋白-1的表达也显著抑制了剪切应力引起的ATP释放反应。MbetaCD、MbetaCD+胆固醇和小窝蛋白-1 siRNA均对细胞表面ATP合酶的量没有任何影响。这些结果表明，ATP合酶的定位和靶向小窝/脂筏是剪切应力诱导的ATP释放的HPAECs的关键。
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.