Contribution of Pannexin 1 and Connexin 43 Hemichannels to Extracellular Calcium-Dependent Transport Dynamics in Human Blood-Brain Barrier Endothelial Cells
Contribution of Pannexin 1 and Connexin 43 Hemichannels to Extracellular Calcium-Dependent Transport Dynamics in Human Blood-Brain Barrier Endothelial Cells
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DOI：
10.1124/jpet.114.220210
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发表时间：
2015-04-01
影响因子：
3.5
通讯作者：
Terasaki, Tetsuya
中科院分区：
文献类型：
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作者：
Kaneko, Yosuke;Tachikawa, Masanori;Terasaki, Tetsuya
血脑屏障（BBB）转运功能失调被认为会加剧急性缺血性中风的神经元损伤。本研究的目的是阐明人 BBB 内皮细胞系 hCMEC/D3 中潘连接蛋白 (Px) 和/或连接蛋白 (Cx) 半通道介导的有机阴离子和阳离子转运的特征，并鉴定在细胞外 Ca2+ 缺乏（模拟急性缺血性中风的情况）的情况下 hCMEC/D3 细胞中半通道开放的抑制剂。在缺乏细胞外 Ca2+ 的情况下，细胞表现出有机离子荧光染料的摄取和流出运输增加。经典的半通道抑制剂显着抑制增强的摄取和流出。定量靶向绝对蛋白质组学证实了 hCMEC/D3 细胞质膜中 Px1 和 Cx43 蛋白的表达。用小干扰 RNA 敲低 Px1 和 Cx43 显着抑制有机阴离子和阳离子荧光染料的增强摄取和流出。临床上使用的西尼地平和黄体酮在动物缺血模型中具有神经保护作用，被认为是半通道开放的抑制剂。这些发现表明，在缺乏细胞外 Ca2+ 的情况下，人类 BBB 运输动力学的改变至少部分归因于 Px1 和 Cx43 半通道的开放。因此，我们推测Px1和Cx43可能是改善急性缺血期间BBB转运失调的潜在药物靶点。
Dysregulation of blood-brain barrier (BBB) transport function is thought to exacerbate neuronal damage in acute ischemic stroke. The purpose of this study was to clarify the characteristics of pannexin (Px) and/or connexin (Cx) hemichannel(s)-mediated transport of organic anions and cations in human BBB endothelial cell line hCMEC/D3 and to identify inhibitors of hemichannel opening in hCMEC/D3 cells in the absence of extracellular Ca2+, a condition mimicking acute ischemic stroke. In the absence of extracellular Ca2+, the cells showed increased uptake and efflux transport of organic ionic fluorescent dyes. Classic hemichannel inhibitors markedly inhibited the enhanced uptake and efflux. Quantitative targeted absolute proteomics confirmed Px1 and Cx43 protein expression in plasma membrane of hCMEC/D3 cells. Knockdown of Px1 and Cx43 with the small interfering RNAs significantly inhibited the enhanced uptake and efflux of organic anionic and cationic fluorescent dyes. Clinically used cilnidipine and progesterone, which have neuroprotective effects in animal ischemia models, were identified as inhibitors of hemichannel opening. These findings suggest that altered transport dynamics at the human BBB in the absence of extracellular Ca2+ is at least partly attributable to opening of Px1 and Cx43 hemichannels. Therefore, we speculate that Px1 and Cx43 may be potential drug targets to ameliorate BBB transport dysregulation during acute ischemia.