Selective expression of the large neutral amino acid transporter at the blood-brain barrier
Selective expression of the large neutral amino acid transporter at the blood-brain barrier
复制标题
DOI：
10.1073/pnas.96.21.12079
复制
发表时间：
1999-10-12
影响因子：
11.1
通讯作者：
Pardridge, WM
中科院分区：
文献类型：
--
作者：
Boado, RJ;Li, JY;Pardridge, WM
脑毛细血管内皮细胞上的大分子中性氨基酸转运体（LAT）的活性调节脑内氨基酸的供应，而LAT在体内形成了巢脑屏障（BBB）。从2.0 kg新鲜牛脑中分离牛BBB poly（A）（+）RNA，并在蔗糖密度梯度上进行大小分级，制备pSPORT载体中大小分级的牛BBB cDNA文库。从该文库中分离出编码牛BBB LAT的全长cDNA，预测的氨基酸序列与LAT 1同种型具有89-92%的同一性。牛BBB LAT 1 mRNA产生10倍的增强色氨酸转运到青蛙卵母细胞共注射牛BBB LAT 1 mRNA和mRNA的4F 2 hc，它编码的异源二聚体的重链。色氨酸转运到注射mRNA的卵母细胞中是不依赖于钠的，并且被其他大的中性氨基酸特异性抑制，色氨酸转运的Km为31.5 +/- 5.5 μ M。牛血脑屏障LAT 1 cDNA的北方印迹显示，与C6大鼠胶质瘤细胞或大鼠脑相比，在分离的牛脑毛细血管中的LAT 1 mRNA高100倍，并且在大鼠肝、心、肺或肾中未检测到LAT 1 mRNA。这些研究表明，与其他组织相比，LAT 1转录物选择性地在BBB表达，并且在BBB的LAT 1 mRNA的丰度比诸如4F 2 hc抗原、肌动蛋白或Glut 1葡萄糖转运蛋白的转录物的丰度高许多倍。
Amino acid supply in brain is regulated by the activity of the large neutral amino acid transporter (LAT) at the brain capillary endothelial cell, which forms the brood-brain barrier (BBB) in vivo. Bovine BBB poly(A)(+) RNA was isolated from 2.0 kg of fresh bovine brain and size fractionated on a sucrose density gradient, and a size-fractionated bovine BBB cDNA library in the pSPORT Vector was prepared. The full-length cDNA encoding the bovine BBB LAT was isolated from this library, and the predicted amino acid sequence was 89-92% identical to the LAT1 isoform. The bovine BBB LAT1 mRNA produced a 10-fold enhancement in tryptophan transport into frog oocytes coinjected with bovine BBB LAT1 mRNA and the mRNA for 4F2hc, which encodes the heavy chain of the heterodimer. Tryptophan transport into the mRNA-injected oocytes was sodium independent and was specifically inhibited by other large neutral amino acids, and the K-m of tryptophan transport was 31.5 +/- 5.5 mu M. Northern blotting with the bovine BBB LAT1 cDNA showed that the LAT1 mRNA is 100-fold higher in isolated bovine brain capillaries compared with C6 rat glioma cells or rat brain, and the LAT1 mRNA was not detected in rat liver, heart, lung, or kidney. These studies show that the LAT1 transcript is selectively expressed at the BBB compared with other tissues, and the abundance of the LAT1 mRNA at the BBB is manyfold higher than that of transcripts such as the 4F2hc antigen, actin, or the Glut1 glucose transporter.