Kageyama T, Nakamura M, Matsuo A, Yamasaki Y, Takakura Y, Hashida M, Kanai Y, Naito M, Tsuruo T, Minato N, Shimohama S
Department of Neurology, Graduate School of Medicine, Kyoto University, 54 Shogoin-Kawaharacho, Sakyo-ku, 606-8507, Kyoto, Japan.
Brain Res. 2000 Oct 6;879(1-2):115-21. doi: 10.1016/s0006-8993(00)02758-x.
L-DOPA is transported across the blood-brain barrier (BBB) by an amino acid transporter, system L. Recently, it has been demonstrated that system L consists of two subunits, 4F2hc and either LAT1 or LAT2. 4F2hc/LAT1 and 4F2hc/LAT2 show different transport characteristics, while their distribution in the brain has not been determined. To clarify whether 4F2hc/LAT1 participates in L-DOPA transport across the BBB, we first examined the expression of 4F2hc/LAT1 in the mouse brain capillary endothelial cell line, MBEC4, as an in vitro BBB model. Northern hybridization and immunoblotting revealed that both 4F2hc and LAT1 are expressed and form a heterodimer in MBEC4 cells. To confirm whether 4F2hc/LAT1 acts as system L to transport L-DOPA, we characterized L-DOPA uptake into the cells. The uptake process was time-dependent, temperature-sensitive, and Na(+)-independent. Neutral amino acids with bulky side chains and a bicyclic amino acid, 2-aminobicyclo-[2, 2,1]-heptane-2-carboxylic acid (BCH), inhibited L-DOPA uptake into MBEC4 cells to a great extent, while an acidic amino acid, basic amino acids, and glycine had no effect. Other neutral amino acids, such as alanine, asparagine, glutamine, serine, and threonine inhibited L-DOPA uptake by 40-70% at most. These characteristics are more compatible with those of 4F2hc/LAT1, rather than those of 4F2hc/LAT2. Finally, immunohistochemistry with anti-LAT1 antibody demonstrated that LAT1 is predominantly expressed in the microvessels of the central nervous system. This is the first report showing that the 4F2hc/LAT1 complex participates in L-DOPA transport across the BBB.
左旋多巴通过氨基酸转运体L系统穿过血脑屏障（BBB）。最近，已证明L系统由两个亚基4F2hc和LAT1或LAT2组成。4F2hc/LAT1和4F2hc/LAT2表现出不同的转运特性，但其在脑中的分布尚未确定。为了阐明4F2hc/LAT1是否参与左旋多巴穿过血脑屏障的转运，我们首先在小鼠脑微血管内皮细胞系MBEC4（一种体外血脑屏障模型）中检测了4F2hc/LAT1的表达。Northern杂交和免疫印迹显示4F2hc和LAT1均有表达并在MBEC4细胞中形成异二聚体。为了证实4F2hc/LAT1是否作为L系统转运左旋多巴，我们对细胞摄取左旋多巴的特性进行了研究。摄取过程具有时间依赖性、温度敏感性且不依赖于Na(+)。带有大侧链的中性氨基酸和双环氨基酸2-氨基双环-[2, 2,1]-庚烷-2-羧酸（BCH）在很大程度上抑制了MBEC4细胞对左旋多巴的摄取，而酸性氨基酸、碱性氨基酸和甘氨酸则无影响。其他中性氨基酸，如丙氨酸、天冬酰胺、谷氨酰胺、丝氨酸和苏氨酸最多抑制40 - 70%的左旋多巴摄取。这些特性与4F2hc/LAT1的特性更相符，而非4F2hc/LAT2的特性。最后，用抗LAT1抗体进行免疫组织化学显示LAT1主要在中枢神经系统的微血管中表达。这是首次报道表明4F2hc/LAT1复合物参与左旋多巴穿过血脑屏障的转运。
