Membrane Transport Mechanism of Aluminum and its Effect on the Inositol Phospholipid Metabolism in the Neuronal System

虽然已知铝（Al）在大脑中的积聚可引起神经退行性疾病，并主要受血脑屏障（BBB）的调节，但Al在脑组织中的积聚机制尚不清楚。在本研究中，我们利用大鼠永生化脑内皮细胞系、RBEC1细胞和人神经母细胞瘤SH-SY5Y细胞，重点研究了谷氨酸转运蛋白家族参与了脑血脑屏障中柠檬酸铝的运输，柠檬酸铝是脑中主要的铝种类。柠檬酸铝的吸收表现出温度和浓度依赖性，并且不需要向内定向的Na^+梯度作为驱动力，排除了Na^+依赖性谷氨酸转运体参与其运输的可能性。通过RT-PCR检测Na^+非依赖性谷氨酸转运体系统Xc^-的表达，在RBEC1和SH-SY5Y细胞中均发现了xCT和4F2hc的mrna作为其组成部分。在RBEC1和SH-SY5Y细胞中，l -谷氨酸辅助l -胱氨酸显著抑制柠檬酸铝的摄取，并将其加载到细胞中，刺激其摄取，而l -天冬氨酸在完整细胞中不是该系统的配体，没有任何影响。这些结果表明，柠檬酸铝通过Xc^-系统进入RBEC1和SH-SY5Y细胞，该系统可能在血脑屏障和神经元的柠檬酸铝运输中起重要作用。我们进一步研究了柠檬酸铝处理对SH-SY5Y细胞转运蛋白表达和氧化应激易感性的影响。用枸橼酸铝处理细胞2周，而不是1天，xCT和4F2hc （xCT的一个伴侣子）mRNA的表达减少，免疫细胞化学证实了枸橼酸铝处理对xCT蛋白的影响。虽然细胞活力和谷胱甘肽含量没有受到葡萄糖剥夺的影响，但柠檬酸铝处理的细胞中硝基酪氨酸的形成和死亡细胞的数量增加。这些发现表明，柠檬酸铝是Xc^-系统的底物，长期用柠檬酸铝治疗会导致xCT的下调，增加细胞对氧化应激的脆弱性，而不会直接影响细胞活力或GSH含量。少

Although accumulation of aluminum (Al) in the brain is known to cause neurodegenerative disorders and to be regulated mainly by the blood-brain barrier (BBB), the mechanism for Al accumulation in brain tissue has not been clarified yet. In this study, we investigated what kind of transporter is involved in the transport of Al citrate, which is the major species of Al in the brain, at the BBB using a rat immortalized brain endothelial cell line, RBEC1 cells and human neuroblastoma SH-SY5Y cells, focusing on the glutamate transporter family. The uptake of Al citrate showed temperature- and concentration-dependency, and did not require an inwardly directed Na^+-gradient as a driving force, ruling out the involvement of Na^+-dependent glutamate transporters in its transport. Examination of the expression of a Na^+-independent glutamate transporter, system Xc^-, by RT-PCR, mRNAs for xCT and 4F2hc, as its components, were found in both RBEC1 and SH-SY5Y cells. L-Glutamate aid L-cystine, repr … More esentative ligands for system Xc^-, significantly inhibited the uptake of Al citrate, and loading of them into the cells resulted in stimulation of its uptake in RBEC1 and SH-SY5Y cells, while L-aspartate, which is not a ligand for the system in intact cells, did not have any effect. These results demonstrated that Al citrate is taken up into RBEC1 and SH-SY5Y cells via system Xc^-, and that this system might play an important role in Al citrate transport at the BBB and neurons. We further examined the effect of Al citrate treatment on expression of the transporter and on the susceptibility to oxidative stress of SH-SY5Y cells. When the cells were treated with Al citrate, but not citrate, for 2 weeks, but not a day, the expression of mRNA for xCT and 4F2hc, a chaperon of xCT was decreased, and the effect of the Al citrate treatment on xCT protein was confirmed immunocytochemically. Although the cell viability and glutathione content of the cells were not altered by the treatment, the formation of nitrotyrosine and the number of dead cells among the Al citrate-treated cells increased on exposure to glucose deprivation. These findings demonstrate that Al citrate is a substrate for system Xc^-, and that chronic treatment with Al citrate causes downregulation of xCT and increases the vulnerability of the cells to oxidative stress without a direct effect on the viability or GSH content. Less