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. 1999 Oct 12;96(21):12079-84.
doi: 10.1073/pnas.96.21.12079.

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

R J Boado  1 , J Y Li, M Nagaya, C Zhang, W M Pardridge
Affiliations

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

R J Boado et al. Proc Natl Acad Sci U S A. .

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 K(m) of tryptophan transport was 31.5 +/- 5.5 microM. 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.

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Figures

Figure 1
Figure 1
(A) Rat LAT1 cDNA was generated by reverse transcription–PCR amplification of rat C6 poly(A)+ RNA. Duplicate aliquots of PCR products were analyzed by gel electrophoresis on 0.8% agarose, and the ethidium bromide staining of the gel showed a major ≈290-nt band corresponding to the amplified rat-LAT1 cDNA. The sizes (kb) of the DNA standards are shown on the left. The gel was scanned and the image was inverted. (B) Micrograph of freshly isolated bovine brain capillaries, which are free of adjoining brain tissue; erythrocytes are seen trapped in the capillary lumen. (×100.) Poly(A)+ RNA was isolated from the brain capillary preparation. (C) The brain capillary mRNA was size-fractionated in a 5–20% sucrose gradient, and 0.5-μg aliquots of the first 7 gradient fractions were subjected to agarose gel electrophoresis followed by ethidium bromide staining; RNA standards are shown on the left.
Figure 2
Figure 2
Nucleotide and deduced amino acid sequence of the bovine LAT. Sequences with high conservation in the UTRs among species are underlined. In the 5′-UTR, nucleotides 28–67 of bovine LAT are 70% and 68% similar to human and rat LAT1, respectively. In the 3′-UTR, the underlined region is 88% similar to the rat LAT1. A putative binding site for the adenosine-uridine binding protein (AUUUA) is also underlined.
Figure 3
Figure 3
Expression of bovine LAT in Xenopus laevis oocytes. Oocytes were injected with 50 nl water or cRNA solution containing 80 ng of bovine LAT and/or 10 ng of rat 4F2hc and incubated for 4 days at 18°C. (A) The volume of distribution (VD) of [3H]tryptophan in oocytes is increased after the coinjection of 4F2hc mRNA and bovine LAT mRNA. (B) Inhibition of bovine LAT-mediated [3H]tryptophan uptake by amino acids added in 500 μM concentrations. BCH, 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid; MeAIB, N-methylaminoisobutyric acid. (C) Kinetic analysis of the LAT-mediated uptake of [3H]tryptophan. Oocytes were injected with 4F2hc cRNA alone (♦, ▴ in Inset) or in combination with bovine LAT (■). Competition studies were performed by varying the concentration of [3H]tryptophan (14–140 nM) or by increasing the levels of unlabeled tryptophan (0.3–500 μM). The Inset represents the beginning of the curve shown in the main figure, and it is used to demonstrate that there are no changes in Km. Each bar or point represents mean ± SE, n = 5.
Figure 4
Figure 4
Northern blot analyses of poly(A)+ RNA using 32P-labeled bovine LAT clone IV-5, 4F2hc, or actin. Northern blotting was performed with 2 μg (A and B) or 10 μg (C) of poly(A)+ RNA. Membranes were exposed to Kodak BioMax film and intensifying screens as follows: A, LAT, 2 hr at 22°C; 4F2hc and actin, 20 hr at −70°C; B, LAT, 7 days at −70°C; 4F2hc and actin, 20 hr at −70°C; and C, LAT, 7 days at −70°C. The mRNA was derived from rat brain, lung, spleen, testis, or heart; C6 rat glioma cells; or bovine brain capillaries (BBB).
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