Selective expression of the large neutral amino acid transporter at the blood–brain barrier

Abstract

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 blood-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 Km of tryptophan transport was 31.5 ؎ 5.5 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.biological transport ͉ endothelium ͉ gene expression A mino acid availability in brain plays an important role in the regulation of several pathways of brain amino acid metabolism, including neurotransmitter synthesis, S-adenosylmethionine production, and protein synthesis (1). The transport of essential amino acids from blood to brain intracellular space involves movement of amino acids through two biological membranes in series: the blood-brain barrier (BBB) and the plasma membrane of brain cells (neurons, glia). The brain capillary endothelial plasma membranes form the BBB in vivo. Because the surface area of the brain cell membrane is orders of magnitude greater than the surface area of the BBB (2), transport across the BBB is the rate-limiting step in amino acid movement from blood to brain intracellular spaces.The transport of large neutral amino acids across the BBB is mediated by a large neutral amino acid transporter (1), analogous to the leucine (L)-preferring system in peripheral tissues, and now designated LAT for large neutral amino acid transporter (3). However, the L-system at the BBB has a much higher affinity (lower K m ) for amino acids as compared with L-systems in peripheral tissues (1). Whereas the K m of the L-system in peripheral tissues is in the 1-10 mM range, the K m of large neutral amino acid transport by the BBB L-system is on the order of 10-100 M (4). The selective expression of a low-K m LAT at the BBB underlies the selective vulnerability of the brain to the pathologic effects of hyperaminoacidemias (1).Kanai and cowork...

IDO expression in the brain: a double-edged sword

“…The first substrate for the kynurenine pathway, Trp, is transported into the CNS by large neutral amino acid transporters [77]. However, in the absence of local inflammatory signals, the vast majority of Trp is not degradative to neurotoxic substances [78].…”

Small molecular drug transfer across the blood-brain barrier via carrier-mediated transport systems

“…4,5 LAT-1 is characterized by sodium-ion independence with Michaelis constant (Km) values varying from 10 M order for leucine to 1 mM order for glutamine, depending on the hydrophobicity and side-chain conformation of the amino acids. 6 LAT-1 is highly expressed at the BBB and requires coexpression with cell surface glycoprotein 4F2 heavy chain to be adequately expressed on the cell membrane surface. 4 -7 LAT-2 (SLC7A8), which shows differential substrate specificity from LAT-1, may also function as a transporter for amino acids and drugs at the BBB.…”

Protein Characterization of Na+-Independent System L Amino Acid Transporter 3 in Mice

“…The first identified isoform was LAT1, the transcripts of which are widely expressed in many nonepithelial cells such as those in the lung, spleen, thymus, brain, placenta, skin, liver, and testis, as well as in activated peripheral leukocytes. 20 However, despite its well-known expression in many malignant cells, 21,22 in normal tissues, the protein has been shown to be distributed only in the plasma membrane of vascular endothelial cells forming the blood-brain barrier 23,24 and of syncytiotrophoblastic cells in the placenta. 25 The second protein identified, LAT2, is highly expressed in polarized cells such as those in the small intestine and proximal tubules of the kidney, 4 -6,26 suggesting its crucial role in transepithelial amino acid transport.…”