Neural production of kynurenic acid in Caenorhabditis elegans requires the AAT-1 transporter.

Kynurenic acid (KynA) levels link peripheral metabolic status to neural functions including learning and memory. Since neural KynA levels dampen learning capacity, KynA reduction has been proposed as a therapeutic strategy for conditions of cognitive deficit such as neurodegeneration. While KynA is generated locally within the nervous system, its precursor, kynurenine (Kyn), is largely derived from peripheral resources. The mechanisms that import Kyn into the nervous system are poorly understood. Here, we provide genetic, anatomical, biochemical, and behavioral evidence showing that in C. elegans an ortholog of the human LAT1 transporter, AAT-1, imports Kyn into sites of KynA production.

L-type amino acid transporter 1 as a target for inflammatory disease and cancer immunotherapy.

Ingestion of amino acids is fundamental for cellular activity. Amino acids are important components for protein synthesis but are also crucial for intracellular metabolic reactions and signal transduction. Following activation, immune cells induce metabolic reprogramming to generate adequate energy and constitutive substances. Hence, the delivery of amino acids by transporters is necessary for the progression of metabolic rewiring. In this review, we discuss how amino acids and their transporters regulate immune cell functions, with emphasis on LAT1, a transporter of large neutral amino acids. Furthermore, we explore the possibility of targeting amino acid transporters to improve immune disorders and cancer immune therapies.

JPH203, a newly developed anti-cancer drug, shows a preincubation inhibitory effect on L-type amino acid transporter 1 function.

JPH203 is a novel anti-cancer drug targeting L-type amino acid transporter 1 (LAT1), which plays a primary role in the uptake of essential amino acids in tumor cells. Although a co-incubation inhibitory effect of JPH203 has been shown in a conventional uptake assay, its preincubation inhibitory effects have remained undetermined. Therefore, we aimed to characterize the preincubation inhibitory effects of JPH203 on LAT1 function using leucine uptake assays in LAT1-positive human colon cancer HT-29 cells. Preincubation of the cells with JPH203 (0.3 µM for 120 min) decreased the activity level to 30% of that in dimethylsulfoxide-treated cells. Similarly, in time-dependency analysis, preincubation of HT-29 cells with 10 µM JPH203 for 30, 60, and 120 min decreased the leucine uptake activity (42%, 32%, and 28% of that in control cells, respectively). Furthermore, the IC50 value of the combination of preincubation and co-incubation effects was lower than that of co-incubation inhibition alone (34.2 ± 3.6 nM vs. 99.2 ± 11.0 nM). In conclusion, we revealed that JPH203 has the capability to inhibit LAT1 function through preincubation effects. Moreover, preincubation synergistically enhances the co-incubation inhibitory effects. These findings provide a novel insight into the anti-cancer effects of JPH203 in cancer therapy.

Reciprocal regulation of amino acid import and epigenetic state through Lat1 and EZH2.

Lat1 (SLC7A5) is an amino acid transporter often required for tumor cell import of essential amino acids (AA) including Methionine (Met). Met is the obligate precursor of S-adenosylmethionine (SAM), the methyl donor utilized by all methyltransferases including the polycomb repressor complex (PRC2)-specific EZH2. Cell populations sorted for surface Lat1 exhibit activated EZH2, enrichment for Met-cycle intermediates, and aggressive tumor growth in mice. In agreement, EZH2 and Lat1 expression are co-regulated in models of cancer cell differentiation and co-expression is observed at the invasive front of human lung tumors. EZH2 knockdown or small-molecule inhibition leads to de-repression of RXRα resulting in reduced Lat1 expression. Our results describe a Lat1-EZH2 positive feedback loop illustrated by AA depletion or Lat1 knockdown resulting in SAM reduction and concomitant reduction in EZH2 activity. shRNA-mediated knockdown of Lat1 results in tumor growth inhibition and points to Lat1 as a potential therapeutic target.

Alzheimer's Disease Phenotype or Inflammatory Insult Does Not Alter Function of L-Type Amino Acid Transporter 1 in Mouse Blood-Brain Barrier and Primary Astrocytes.

PURPOSE: The study aim was to evaluate the effect of Alzheimer's disease (AD) and inflammatory insult on the function of L-type amino acid transporter 1 (Lat1) at the mouse blood-brain barrier (BBB) as well as Lat1 function and expression in mouse primary astrocytes. METHODS: The Lat1 function and expression was determined in wildtype astrocytes with and without lipopolysaccharide (LPS)-induced inflammation and in LPS treated AD APP/PS1 transgenic astrocytes. The function of Lat1 at the BBB was evaluated in wildtype mice with and without LPS-induced neuroinflammation and APP/PS1 transgenic mice by in situ brain perfusion. RESULTS: There were 2.1 and 1.6 -fold decreases in Lat1 mRNA and protein expression in LPS-treated wildtype astrocytes compared to vehicle-treated astrocytes. In contrast, Lat1 mRNA and protein expression were increased by 1.7 and 1.2 -fold (not statistically significant) in the transgenic cells. A similar trend was observed in the cell uptake of [14C]-L-leucine. There were no statistically significant differences in [14C]-L-leucine BBB permeation between the groups. CONCLUSIONS: The results showed that neither LPS-induced inflammation or the presence of APP/PS1 mutations alters Lat1 function at the mouse BBB as well as Lat1 protein expression and function in mouse primary astrocytes.

Specific transport of 3-fluoro-l-α-methyl-tyrosine by LAT1 explains its specificity to malignant tumors in imaging.

3-(18)F-l-α-methyl-tyrosine ([18F]FAMT), a PET probe for tumor imaging, has advantages of high cancer-specificity and lower physiologic background. FAMT-PET has been proved useful in clinical studies for the prediction of prognosis, the assessment of therapy response and the differentiation of malignant tumors from inflammation and benign lesions. The tumor uptake of [18F]FAMT in PET is strongly correlated with the expression of L-type amino acid transporter 1 (LAT1), an isoform of system L upregulated in cancers. In this study, to assess the transporter-mediated mechanisms in FAMT uptake by tumors, we examined amino acid transporters for FAMT transport. We synthesized [14C]FAMT and measured its transport by human amino acid transporters expressed in Xenopus oocytes. The transport of FAMT was compared with that of l-methionine, a well-studied amino acid PET probe. The significance of LAT1 in FAMT uptake by tumor cells was confirmed by siRNA knockdown. Among amino acid transporters, [14C]FAMT was specifically transported by LAT1, whereas l-[14C]methionine was taken up by most of the transporters. Km of LAT1-mediated [14C]FAMT transport was 72.7 µM, similar to that for endogenous substrates. Knockdown of LAT1 resulted in the marked reduction of [14C]FAMT transport in HeLa S3 cells, confirming the contribution of LAT1 in FAMT uptake by tumor cells. FAMT is highly specific to cancer-type amino acid transporter LAT1, which explains the cancer-specific accumulation of [18F]FAMT in PET. This, vice versa, further supports the cancer-specific expression of LAT1. This study has established FAMT as a LAT1-specific molecular probe to monitor the expression of a potential tumor biomarker LAT1.

Spermine modulates the expression of two probable polyamine transporter genes and determines growth responses to cadaverine in Arabidopsis.

KEY MESSAGE: Two genes, LAT1 and OCT1 , are likely to be involved in polyamine transport in Arabidopsis. Endogenous spermine levels modulate their expression and determine the sensitivity to cadaverine. Arabidopsis spermine (Spm) synthase (SPMS) gene-deficient mutant was previously shown to be rather resistant to the diamine cadaverine (Cad). Furthermore, a mutant deficient in polyamine oxidase 4 gene, accumulating about twofold more of Spm than wild type plants, showed increased sensitivity to Cad. It suggests that endogenous Spm content determines growth responses to Cad in Arabidopsis thaliana. Here, we showed that Arabidopsis seedlings pretreated with Spm absorbs more Cad and has shorter root growth, and that the transgenic Arabidopsis plants overexpressing the SPMS gene are hypersensitive to Cad, further supporting the above idea. The transgenic Arabidopsis overexpressing L-Amino acid Transporter 1 (LAT1) absorbed more Cad and showed increased Cad sensitivity, suggesting that LAT1 functions as a Cad importer. Recently, other research group reported that Organic Cation Transporter 1 (OCT1) is a causal gene which determines the Cad sensitivity of various Arabidopsis accessions. Furthermore, their results suggested that OCT1 is involved in Cad efflux. Thus we monitored the expression of OCT1 and LAT1 during the above experiments. Based on the results, we proposed a model in which the level of Spm content modulates the expression of OCT1 and LAT1, and determines Cad sensitivity of Arabidopsis.

Aspartate-modified doxorubicin on its N-terminal increases drug accumulation in LAT1-overexpressing tumors.

L-type amino acid transporter 1 (LAT1), overexpressed on the membrane of various tumor cells, is a potential target for tumor-targeting therapy. This study aimed to develop a LAT1-mediated chemotherapeutic agent. We screened doxorubicin modified by seven different large neutral amino acids. The aspartate-modified doxorubicin (Asp-DOX) showed the highest affinity (Km = 41.423 µmol/L) to LAT1. Aspartate was attached to the N-terminal of DOX by the amide bond with a free carboxyl and a free amino group on the α-carbon atom of the Asp residue. The product Asp-DOX was characterized by HPLC/MS. In vitro, Asp-DOX exerted stronger inhibition on the cancer cells overexpressing LAT1 and the uptake of Asp-DOX was approximately 3.5-fold higher than that of DOX in HepG2 cells. Pharmacokinetic data also showed that Asp-DOX was expressed over a longer circulation time (t1/2 = 49.14 min) in the blood compared to DOX alone (t1/2 = 15.12 min). In HepG2 and HCT116 tumor-bearing mice, Asp-DOX achieved 3.1-fold and 6.4-fold accumulation of drugs in tumor tissue, respectively, than those of the unmodified DOX. More importantly, treatment of tumor-bearing mice with Asp-DOX showed a significantly stronger inhibition of tumor growth than mice treated with free DOX in HepG2 tumor models. Furthermore, after Asp modification, Asp-DOX avoided MDR mediated by P-glycoprotein. These results suggested that the Asp-DOX modified drug may provide a new treatment strategy for tumors that overexpress LAT1 and MDR1.

LAT1 is a central transporter of essential amino acids in human umbilical vein endothelial cells.

Endothelial cell proliferation supporting angiogenesis requires sufficient nutrient supply because of facilitated intracellular metabolism. However, little is known about the mechanism for the promotion of nutrient incorporation in proliferating endothelial cells. Here we show that L-type amino acid transporter 1 (LAT1) is a major transporter of essential amino acids in human umbilical vein endothelial cells (HUVECs). Growing HUVECs express a certain level of LAT1. A LAT1-specific inhibitor suppressed leucine uptake, cell proliferation, and tube formation of HUVECs. Therefore, LAT1 acts to support effective uptake of amino acids, which is critical for the optimal function of HUVECs for angiogenesis.

Cancer-associated fibroblasts-derived exosomal METTL3 promotes the proliferation, invasion, stemness and glutaminolysis in non-small cell lung cancer cells by eliciting SLC7A5 m6A modification.

Cancer-associated fibroblasts (CAFs) can promote the crosstalk between cancer cells and tumor microenvironment by exosomes. METTL3-mediated N6-methyladenine (m6A) modification has been proved to promote the progression of non-small cell lung cancer (NSCLC). Here, we focused on the impacts of CAFs-derived exosomes and METTL3-mediated m6A modification on NSCLC progression. Functional analyses were conducted using Cell Counting Kit-8, EdU, colony formation, sphere formation and transwell assays, respectively. Glutamine metabolism was evaluated by detecting glutamate consumption, and the production of intercellular glutamate and α-ketoglutarate (α-KG). qRT-PCR and western blotting analyses were utilized to measure the levels of genes and proteins. Exosomes were isolated by kits. The methylated RNA immunoprecipitation assay detected the m6A modification profile of Amino acid transporter LAT1 (SLC7A5) mRNA. The NSCLC mouse model was established to conduct in vivo experiments. We found that CAFs promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells. METTL3 was enriched in CAFs and was packaged into exosomes. After knockdown of METTL3 in CAF exosomes, it was found the oncogenic effects of CAFs on NSCLC cells were suppressed. CAFs elevated m6A levels in NSCLC cells. Mechanistically, exosomal METTL3-induced m6A modification in SLC7A5 mRNA and stabilized its expression in NSCLC cells. Moreover, SLC7A5 overexpression abolished the inhibitory effects of exosomal METTL3-decreased CAFs on NSCLC cells. In addition, METTL3 inhibition in CAF exosomes impeded NSCLC growth in vivo. In all, CAFs-derived exosomal METTL3 promoted the proliferation, invasion, stemness and glutaminolysis in NSCLC cells by inducing SLC7A5 m6A modification.

Oncogenicity of L-type amino-acid transporter 1 (LAT1) revealed by targeted gene disruption in chicken DT40 cells: LAT1 is a promising molecular target for human cancer therapy.

L-type amino-acid transporter 1 (LAT1) is the first identified light chain of CD98 molecule, disulfide-linked to a heavy chain of CD98. Following cDNA cloning of chicken full-length LAT1, we have constructed targeting vectors for the disruption of chicken LAT1 gene from genomic DNA of chicken LAT1 consisting of 5.4kb. We established five homozygous LAT1-disrupted (LAT1(-/-)) cell clones, derived from a heterozygous LAT1(+/-) clone of DT40 chicken B cell line. Reactivity of anti-chicken CD98hc monoclonal antibody (mAb) with LAT1(-/-) DT40 cells was markedly decreased compared with that of wild-type DT40 cells. All LAT1(-/-) cells were deficient in L-type amino-acid transporting activity, although alternative-splice variant but not full-length mRNA of LAT1 was detected in these cells. LAT1(-/-) DT40 clones showed outstandingly slow growth in liquid culture and decreased colony-formation capacity in soft agar compared with wild-type DT40 cells. Cell-cycle analyses indicated that LAT1(-/-) DT40 clones have prolonged cell-cycle phases compared with wild-type or LAT1(+/-) DT40 cells. Knockdown of human LAT1 by small interfering RNAs resulted in marked in vitro cell-growth inhibition of human cancer cells, and in vivo tumor growth of HeLa cells in athymic mice was significantly inhibited by anti-human LAT1 mAb. All these results indicate essential roles of LAT1 in the cell proliferation and occurrence of malignant phenotypes and that LAT1 is a promising candidate as a molecular target of human cancer therapy.

[Drug Discovery Targeting an Amino Acid Transporter for Diagnosis and Therapy].

Nutrients are essential for all living organisms. Because growing cancer cells have strong metabolic demands, nutrient transporters are constitutively increased to facilitate the nutrient uptake. Among these nutrient transporters, L-type amino acid transporter 1 (LAT1), which transports large neutral amino acids including essential amino acids, is critical for cancer growth. Therefore, LAT1 has been considered as an attractive target for diagnosis and therapy of cancers. We have developed several lines of compounds for cancer diagnosis and therapy. To diagnose cancer by using positron emission tomography (PET) probes, we have created amino acid derivatives which are selectively transported by LAT1 and accumulated in cancer cells. In addition to amino acid derivatives as the LAT1 inhibitors, we also have made non-amino acid small compounds as anti-cancer drugs which inhibit LAT1 function and suppress tumor growth. The LAT1 targeting anti-cancer drug showed low toxicity but strong effects on various types of cancer cells in animal models. The novel PET probe is approved for clinical research and the new anti-cancer drug has been under clinical trial. Small compounds targeting the amino acid transporter bring us new tools for cancer diagnosis and therapy.

L-type amino acid transporter 1 is associated with chemoresistance in breast cancer via the promotion of amino acid metabolism.

18F-FDG PET/CT has been used as an indicator of chemotherapy effects, but cancer cells can remain even when no FDG uptake is detected, indicating the importance of exploring other metabolomic pathways. Therefore, we explored the amino acid metabolism, including L-type amino acid transporter-1 (LAT1), in breast cancer tissues and clarified the role of LAT1 in therapeutic resistance and clinical outcomes of patients. We evaluated LAT1 expression before and after neoadjuvant chemotherapy and examined the correlation of glucose uptake using FDG-PET with the pathological response of patients. It revealed that LAT1 levels correlated with proliferation after chemotherapy, and amino acid and glucose metabolism were closely correlated. In addition, LAT1 was considered to be involved in treatment resistance and sensitivity only in luminal type breast cancer. Results of in vitro analyses revealed that LAT1 promoted amino acid uptake, which contributed to energy production by supplying amino acids to the TCA cycle. However, in MCF-7 cells treated with chemotherapeutic agents, oncometabolites and branched-chain amino acids also played a pivotal role in energy production and drug resistance, despite decreased glucose metabolism. In conclusion, LAT1 was involved in drug resistance and could be a novel therapeutic target against chemotherapy resistance in luminal type breast cancer.

L-type amino acid transporter 1 inhibitors inhibit tumor cell growth.

Most tumor cell membranes overexpress L-type amino acid transporter 1, while normal cell membranes contain l-type amino acid transporter 2; both are Na(+)-independent amino acid transporters. Therefore, compounds that selectively inhibit L-type amino acid transporter 1 offer researchers with a novel cancer molecular target. Synthetic chemistry efforts and in vitro screening have produced a variety of novel compounds possessing high in vitrol-type amino acid transporter 1 selectivity; KYT-0353 was one such compound. The present studies illustrate that KYT-0353 inhibited (14)C-leucine uptake and cell growth in human colon cancer-derived HT-29 cells; IC(50)s were 0.06 microm and 4.1 microm, respectively. KYT-0353 also inhibited (14)C-leucine uptake in mouse renal proximal tubule cells expressing l-type amino acid transporter 1, and inhibited cell growth; IC(50)s were 0.14 microm and 16.4 microm, respectively. Compared to control animals, intravenously administered KYT-0353 (12.5 mg/kg and 25.0 mg/kg) showed statistically significant growth inhibition against HT-29 tumors transplanted to nude mice with maximal inhibition ratios of 65.9% and 77.2%, respectively. Body weight increase with time--a safety indicator--was slightly depressed at 12.5 mg/kg and 25.0 mg/kg with maximal ratios of 3.7% (day 2) and 6.3% (day 11), respectively. Thus, KYT-0353 showed significant growth inhibitory effects on HT-29 cells both in vitro and in vivo, whereas it only caused a slight body weight depression. Therefore, KYT-0353 appears to have potential as a novel anti-tumor agent, presumably via selective in vivol-type amino acid transporter 1 inhibition.

Slc7a5 alters Kvß-mediated regulation of Kv1.2.

The voltage-gated potassium channel Kv1.2 plays a pivotal role in neuronal excitability and is regulated by a variety of known and unknown extrinsic factors. The canonical accessory subunit of Kv1.2, Kvß, promotes N-type inactivation and cell surface expression of the channel. We recently reported that a neutral amino acid transporter, Slc7a5, alters the function and expression of Kv1.2. In the current study, we investigated the effects of Slc7a5 on Kv1.2 in the presence of Kvß1.2 subunits. We observed that Slc7a5-induced suppression of Kv1.2 current and protein expression was attenuated with cotransfection of Kvß1.2. However, gating effects mediated by Slc7a5, including disinhibition and a hyperpolarizing shift in channel activation, were observed together with Kvß-mediated inactivation, indicating convergent regulation of Kv1.2 by both regulatory proteins. Slc7a5 influenced several properties of Kvß-induced inactivation of Kv1.2, including accelerated inactivation, a hyperpolarizing shift and greater extent of steady-state inactivation, and delayed recovery from inactivation. These modified inactivation properties were also apparent in altered deactivation of the Kv1.2/Kvß/Slc7a5 channel complex. Taken together, these findings illustrate a functional interaction arising from simultaneous regulation of Kv1.2 by Kvß and Slc7a5, leading to powerful effects on Kv1.2 expression, gating, and overall channel function.

Regulation of Melanogenesis by the Amino Acid Transporter SLC7A5.

Integration of chromatin immunoprecipitation-sequencing and microarray data enabled us to identify previously unreported MITF-target genes, among which the amino acid transporter SLC7A5 is also included. We reported that small interfering RNA-mediated SLC7A5 knockdown decreased pigmentation in B16F10 cells but neither affected morphology nor dendricity. Treatment with the SLC7A5 inhibitors 2-amino-2-norbornanecarboxylic acid (BCH) or JPH203 also decreased melanin synthesis in B16F10 cells. Our findings indicated that BCH was as potent as reference depigmenting agent, kojic acid, but acted through a different pathway not affecting tyrosinase activity. BCH also decreased pigmentation in human MNT1 melanoma cells or normal human melanocytes. Finally, we tested BCH on a more physiological model, using reconstructed human epidermis and confirmed a strong inhibition of pigmentation, revealing the clinical potential of SLC7A5 inhibition and positioning BCH as a depigmenting agent suitable for cosmetic or dermatological intervention in hyperpigmentation diseases.

[Transfer Mechanisms of Compounds between Mother and Fetus/Infant Aimed for Optimized Medication during Pregnancy and Breastfeeding].

Potential risks to the fetus or infant should be considered prior to medication during pregnancy and lactation. It is essential to evaluate the exposure levels of drugs and their related factors in addition to toxicological effects. Epilepsy is one of the most common neurological complications in pregnancy; some women continue to use antiepileptic drugs (AEDs) to control seizures. Benzodiazepines (BZDs) are widely prescribed for several women who experience symptoms such as anxiety and insomnia during the postpartum period. In this review, we describe the 1) transport mechanisms of AEDs across the placenta and the effects of these drugs on placental transporters, and 2) the transfer of BZDs into breast milk. Our findings indicated that carrier systems were involved in the uptake of gabapentin (GBP) and lamotrigine (LTG) in placental trophoblast cell lines. SLC7A5 was the main contributor to GBP transport in placental cells. LTG was transported by a carrier that was sensitive to chloroquine, imipramine, quinidine, and verapamil. Short-term exposure to 16 AEDs had no effect on folic acid uptake in placental cells. However, long-term exposure to valproic acid (VPA) affected the expression of folate carriers (FOLR1, SLC46A1). Furthermore, VPA administration changed the expression levels of various transporters in rat placenta, suggesting that sensitivity to VPA differed across gestational stages. Lastly, we developed a method for quantifying eight BZDs in human breast milk and plasma using LC/MS/MS, and successfully applied it to quantify alprazolam in breast milk and plasma donated by a lactating woman.

Inhibition of system L (LAT1/CD98hc) reduces the growth of cultured human breast cancer cells.

It has been suggested that system L (LAT1/CD98hc) is up-regulated in cancer cells, including breast tumour cells, and is therefore a promising molecular target to inhibit or limit tumour cell growth. In view of this, we have examined the effect of BCH and other inhibitors of system L on the growth of MCF-7, ZR-75-1 and MDA-MB-231 cells. Treating cells with BCH markedly inhibited the metabolism of WST-1 in a dose-dependent fashion. Similarly, melphalan and D-leucine inhibited the growth of cultured breast cancer cells whereas MeAIB, an inhibitor of system A, was without effect. The effects of BCH and melphalan on cell growth were non-additive suggesting that both compounds were acting at a single locus. The results indicate that system L is required to maintain MCF-7, ZR-75-1 and MDA-MB-231 cell growth and support the notion that LAT1/CD98hc may be a suitable target to inhibit breast cancer progression.

High throughput silencing identifies novel genes in endometrioid endometrial cancer.

OBJECTIVE: To validate the gene expression profile obtained from the previous microarray analysis and to further study the biological functions of these genes in endometrial cancer. From our previous study, we identified 621 differentially expressed genes in laser-captured microdissected endometrioid endometrial cancer as compared to normal endometrial cells. Among these genes, 146 were significantly up-regulated in endometrial cancer. MATERIALS AND METHODS: A total of 20 genes were selected from the list of up-regulated genes for the validation assay. The qPCR confirmed that 19 out of the 20 genes were up-regulated in endometrial cancer compared with normal endometrium. RNA interference (RNAi) was used to knockdown the expression of the upregulated genes in ECC-1 and HEC-1A endometrial cancer cell lines and its effect on proliferation, migration and invasion were examined. RESULTS: Knockdown of MIF, SOD2, HIF1A and SLC7A5 by RNAi significantly decreased the proliferation of ECC-1 cells (p < 0.05). Our results also showed that the knockdown of MIF, SOD2 and SLC7A5 by RNAi significantly decreased the proliferation and migration abilities of HEC-1A cells (p < 0.05). Moreover, the knockdown of SLC38A1 and HIF1A by RNAi resulted in a significant decrease in the proliferation of HEC1A cells (p < 0.05). CONCLUSION: We have identified the biological roles of SLC38A1, MIF, SOD2, HIF1A and SLC7A5 in endometrial cancer, which opens up the possibility of using the RNAi silencing approach to design therapeutic strategies for treatment of endometrial cancer.

L-type amino acid transporter 1-mediated L-leucine transport at the inner blood-retinal barrier.

PURPOSE: L-type amino acid transporters (LATs) prefer branched-chain and aromatic amino acids, including neurotransmitter precursors. The objective of this study was to clarify the expression and function of LAT at the inner blood-retinal barrier (BRB). METHODS: [3H]L-Leucine transport at the inner BRB was characterized by using in vivo integration plot analysis and a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2). The expression of the LAT1 was demonstrated by quantitative real-time RT-PCR, immunoblot, and immunohistochemical analyses. RESULTS: The apparent influx permeability clearance of [3H]L-leucine in the rat retina was found to be 203 microL/(min.g retina), supporting a carrier-mediated influx transport of L-leucine at the BRB. [3H]L-Leucine uptake by TR-iBRB2 cells was an Na+-independent and concentration-dependent process with a Km of 14.1 microM. This process was more potently cis inhibited by substrates of LAT1, D-leucine, D-phenylalanine, and D-methionine, than those of LAT2, L-alanine, and L-glutamine. [3H]L-Leucine efflux from TR-iBRB2 cells was trans-stimulated by substrates of LAT1. The expression of LAT1 mRNA was 100- and 15-fold greater than that of LAT2 in TR-iBRB2 and magnetically isolated rat retinal vascular endothelial cells, respectively. The expression of LAT1 protein was observed in TR-iBRB2 and primary cultured human retinal endothelial cells and immunostaining of LAT1 was observed along the rat retinal capillaries. CONCLUSIONS: LAT1 is expressed at the inner BRB and mediates blood-to-retina L-leucine transport. This transport system plays a key role in maintaining large neutral amino acids as well as neurotransmitters in the neural retina.