Functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood-brain barrier model.
Functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood-brain barrier model.
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DOI：
10.1186/2045-8118-10-8
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发表时间：
2013-01-26
影响因子：
7.3
通讯作者：
Deguchi Y
中科院分区：
文献类型：
--
作者：
Shimomura K;Okura T;Kato S;Couraud PO;Schermann JM;Terasaki T;Deguchi Y
了解人血脑屏障（BBB）的分子基础和转运功能不仅对了解人类大脑生理学非常重要，而且对开发新的中枢神经系统（CNS）作用药物也很重要。然而，由于人脑材料难以获得，利用人脑毛细血管内皮细胞的研究很少。本研究的目的是阐明质子偶联有机阳离子（H+/OC）逆向转运蛋白在人脑毛细血管内皮细胞系hCMEC/D3中的功能表达，该细胞系最近被开发为体外人血脑屏障模型。苯海拉明，[3 H]吡拉明和羟考酮被用作阳离子药物，证明是H+/OC逆向转运蛋白底物。在几种条件下进行了hCMEC/D3细胞的体外摄取实验。苯海拉明和[3 H]吡拉明均以时间和浓度依赖性方式转运至hCMEC/D3细胞，Km值分别为59 μM和19 μM。每一种都以竞争的方式抑制另一种的吸收，这表明它们的运输中涉及一种共同的机制。金刚烷胺和奎尼丁显著抑制苯海拉明的摄取，但不抑制四乙基铵和1-甲基-4-苯基吡啶（众所周知的有机阳离子转运蛋白的底物）。代谢抑制剂抑制摄取，但对细胞外钠和膜电位不敏感。此外，通过细胞外碱化和细胞内酸化增加摄取。这些转运特性与先前在大鼠血脑屏障中表征的H+/OC反向转运蛋白的转运特性完全一致。本研究结果提示H+/OC逆向转运蛋白在hCMEC/D3细胞中有功能性表达。
Knowledge of the molecular basis and transport function of the human blood–brain barrier (BBB) is important for not only understanding human cerebral physiology, but also development of new central nervous system (CNS)-acting drugs. However, few studies have been done using human brain capillary endothelial cells, because human brain materials are difficult to obtain. The purpose of this study is to clarify the functional expression of a proton-coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, which has been recently developed as an in vitro human BBB model. Diphenhydramine, [3H]pyrilamine and oxycodone were used as cationic drugs that proved to be H+/OC antiporter substrates. The in vitro uptake experiments by hCMEC/D3 cells were carried out under several conditions. Diphenhydramine and [3H]pyrilamine were both transported into hCMEC/D3 cells in a time- and concentration-dependent manner with Km values of 59 μM and 19 μM, respectively. Each inhibited uptake of the other in a competitive manner, suggesting that a common mechanism is involved in their transport. The diphenhydramine uptake was significantly inhibited by amantadine and quinidine, but not tetraethylammonium and 1-methyl-4-phenylpyridinium (substrates for well-known organic cation transporters). The uptake was inhibited by metabolic inhibitors, but was insensitive to extracellular sodium and membrane potential. Further, the uptake was increased by extracellular alkalization and intracellular acidification. These transport properties are completely consistent with those of previously characterized H+/OC antiporter in rat BBB. The present results suggest that H+/OC antiporter is functionally expressed in hCMEC/D3 cells.