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

虽然铝在脑内的蓄积可导致神经退行性疾病，并且主要受血脑屏障(BBB)的调节，但其在脑组织中蓄积的机制尚不清楚。在这项研究中，我们利用永生化的大鼠脑内皮细胞系、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体家族，研究了什么样的转运体参与了脑中主要铝物种柠檬酸铝在血脑屏障的转运。柠檬酸铝的摄取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，排除了依赖Na~+的谷氨酸转运体参与其运输。RT-PCR检测到一个Na~+非依赖性谷氨酸转运蛋白系统XC^-的表达，在RBEC1和SH-SY5Y细胞中都发现了XCT和4F2hc的mRNAs。L-谷氨酸援助L-胱氨酸，REPR…较多的XC^-系统配体显著抑制柠檬酸铝的摄取，细胞内负载这些配体可刺激细胞摄取柠檬酸铝，而不是完整细胞中该系统的配体L-天冬氨酸对柠檬酸铝摄取无影响。这些结果表明，柠檬酸铝是通过XC^-系统被吸收到RBEC1和SH-SY5Y细胞中的，该系统可能在血脑屏障和神经元的柠檬酸铝转运中起重要作用。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敏感性的影响。当用柠檬酸铝处理细胞2周而不是处理1天时，XCT和XCT的伴侣4F2hc的mRNA表达减少，免疫细胞化学证实了柠檬酸铝对XCT蛋白的影响。虽然细胞活力和谷胱甘肽含量没有因缺糖而改变，但柠檬酸铝处理的细胞中硝基酪氨酸的形成和死亡细胞数在缺糖条件下增加。这些发现表明，柠檬酸铝是XC^-系统的底物，而长期使用柠檬酸铝处理会导致XCT表达下调，并增加细胞对氧化应激的脆弱性，而不会直接影响细胞的活力或GSH含量。较少

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