Proton-Coupled Organic Cation Antiporter-Mediated Uptake of Apomorphine Enantiomers in Human Brain Capillary Endothelial Cell Line hCMEC/D3
Proton-Coupled Organic Cation Antiporter-Mediated Uptake of Apomorphine Enantiomers in Human Brain Capillary Endothelial Cell Line hCMEC/D3
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DOI：
10.1248/bpb.b13-00773
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发表时间：
2014-02-01
影响因子：
2
通讯作者：
Deguchi, Yoshiharu
中科院分区：
文献类型：
--
作者：
Okura, Takashi;Higuchi, Kei;Deguchi, Yoshiharu
R（-）-阿扑吗啡是一种多巴胺激动剂，用于帕金森病患者中与左旋多巴治疗相关的运动功能障碍的抢救管理。本研究以人内皮细胞系hCMEC/D3为模型，探讨质子偶联有机阳离子逆向转运蛋白在人脑摄取R（-）-阿扑吗啡及其S-对映体中的作用。在不同条件下测定hCMEC/D3细胞摄取R（-）-或S（+）-阿扑吗啡的时间、浓度、能量和离子依赖性。抑制选定的有机阳离子也进行了检查。R（-）-和S（+）阿扑吗啡的摄取均随时间增加。R（-）-和S（+）-阿扑吗啡的初始摄取速度呈浓度依赖性，具有相似的Km和V-max值。叠氮化钠预处理可显著降低R（-）-阿扑吗啡的细胞-培养基（C/M）比，但用N-甲基葡萄糖胺或钾替代细胞外钠离子并不影响R（-）-阿扑吗啡的细胞-培养基（C/M）比。细胞内碱化显着减少的摄取，而细胞内酸化增加它，这表明摄取是由一个相反方向的质子梯度驱动。金刚烷胺、维拉帕米、吡拉明和苯海拉明（质子偶联有机阳离子逆向转运蛋白的底物或抑制剂）可显著降低C/M比，而四乙基铵（有机阳离子转运蛋白（OCT）的底物）和肉毒碱（肉毒碱/有机阳离子转运蛋白2的底物;（OCTN 2））则无影响。苯海拉明竞争性抑制R（-）-阿扑吗啡摄取。我们的结果表明，R（-）-阿扑吗啡在人血脑屏障（BBB）模型细胞中的转运类似于S（+）-阿扑吗啡的摄取。的运输是依赖于一个相反方向的质子梯度，但钠或膜电位独立。的运输特性与先前报道的质子偶联有机阳离子反向转运蛋白的参与是一致的。
R(-)-Apomorphine is a dopamine agonist used for rescue management of motor function impairment associated with levodopa therapy in Parkinson's disease patients. The aim of this study was to examine the role of proton-coupled organic cation antiporter in uptake of R(-)-apomorphine and its S-enantiomer in human brain, using human endothelial cell line hCMEC/D3 as a model. Uptake of R(-)- or S(+)-apomorphine into hCMEC/D3 cells was measured under various conditions to evaluate its time-, concentration-, energy- and ion-dependency. Inhibition by selected organic cations was also examined. Uptakes of both R(-)- and S(+)apomorphine increased with time. The initial uptake velocities of R(-)- and S(+)-apomorphine were concentration-dependent, with similar K-m and V-max values. The cell-to-medium (C/M) ratio of R(-)-apomorphine was significantly reduced by pretreatment with sodium azide, but was not affected by replacement of extracellular sodium ion with N-methylglucamine or potassium. Intracellular alkalization markedly reduced the uptake, while intracellular acidification increased it, suggesting that the uptake is driven by an oppositely directed proton gradient. The C/M ratio was significantly decreased by amantadine, verapamil, pyrilamine and diphenhydramine (substrates or inhibitors of proton-coupled organic cation antiporter), while tetraethylammonium (substrate of organic cation transporters (OCTs)) and carnitine (substrate of carnitine/organic cation transporter 2; (OCTN2)) had no effect. R(-)-Apomorphine uptake was competitively inhibited by diphenhydramine. Our results indicate that R(-)-apomorphine transport in human blood brain barrier (BBB) model cells is similar to S(+)-apomorphine uptake. The transport was dependent on an oppositely directed proton gradient, but was sodium- or membrane potential-independent. The transport characteristics were consistent with involvement of the previously reported proton-coupled organic cation antiporter.