Characterization of uptake of folates by rat and human blood-brain barrier endothelial cells
Characterization of uptake of folates by rat and human blood-brain barrier endothelial cells
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DOI：
10.1002/biof.82
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发表时间：
2010-05-01
期刊：
影响因子：
6
通讯作者：
Martel, Fatima
中科院分区：
文献类型：
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作者：
Araujo, Joao R.;Goncalves, Pedro;Martel, Fatima
本研究旨在表征大鼠（RBE4）和人（hCMEC/D3）血脑屏障（BBB）内皮细胞系对3H‐叶酸（3H‐FA）和14C‐甲基四氢叶酸（14C‐MTHF）的摄取。RBE4细胞对3h‐FA和14c‐MTHF的摄取在孵育前2分钟呈时间依赖性和线性；hCMEC/D3细胞的摄取表现出较不明显的时间依赖性和较大的实验变异性。因此，进一步的实验仅在RBE4细胞中进行。3h‐FA的摄取在酸性和碱性pH下被刺激，Na+依赖，当F−取代Cl−时被刺激，能量独立，被预甲氨曲塞抑制，被细胞松弛素D刺激，不受MTHF， DIDS， SITS，甲氨蝶呤，莫能菌素和FA的影响。发现14c - MTHF的摄取与pH‐，Na‐，Cl‐和能量无关，被预甲氨蝶呤和甲氨蝶呤抑制，被细胞松弛素D刺激，并且不受FA， DIDS， SITS，莫能菌素和MTHF的影响。RT - PCR分析显示，RBE4细胞中表达了叶酸转运蛋白（RFC），但FRα和质子偶联叶酸转运蛋白（PCFT）均未表达；hCMEC/D3细胞中表达了RFC和PCFT，但FRα未表达。总之，人和大鼠血脑屏障内皮细胞对3h - FA和14c - MTHF的顶端摄取能力都很小。因此，这些细胞系似乎不是研究血脑屏障中叶酸运输的良好模型。
This study aimed to characterize3H‐folic acid (3H‐FA) and14C‐methyltetrahydrofolic acid (14C‐MTHF) uptake by rat (RBE4) and human (hCMEC/D3) blood–brain barrier (BBB) endothelial cell lines. Uptake of3H‐FA and14C‐MTHF by RBE4 cells was time dependent and linear for the first 2 min of incubation; uptake by hCMEC/D3 cells showed a less marked time‐dependency and a greater experimental variability. So, further experiments were performed in RBE4 cells only. Uptake of3H‐FA was stimulated at acidic and alkaline pH, Na+dependent, stimulated when F−substituted for Cl−, energy independent, inhibited by premetrexed, stimulated by cytochalasin D, and unaffected by MTHF, DIDS, SITS, methotrexate, monensin, and FA. Uptake of14C‐MTHF was found to be pH‐, Na+‐, Cl−‐ and energy independent, inhibited by premetrexed and methotrexate, stimulated by cytochalasin D, and unaffected by FA, DIDS, SITS, monensin, and MTHF. RT‐PCR analysis showed mRNA expression of reduced folate transporter (RFC), but neither of FRα nor of proton‐coupled folate transporter (PCFT) in RBE4 cells, and mRNA expression of RFC and PCFT, but not of FRα, in hCMEC/D3 cells. In conclusion, both human and rat BBB endothelial cells show little capacity for3H‐FA and14C‐MTHF apical uptake. Hence, these cell lines do not appear to be a good model to study the transport of folates at the BBB.