Antagonists of the system L neutral amino acid transporter (LAT) promote endothelial adhesivity of human red blood cells.

The system L neutral amino acid transporter (LAT; LAT1, LAT2, LAT3, or LAT4) has multiple functions in human biology, including the cellular import of S-nitrosothiols (SNOs), biologically active derivatives of nitric oxide (NO). SNO formation by haemoglobin within red blood cells (RBC) has been studied, but the conduit whereby a SNO leaves the RBC remains unidentified. Here we hypothesised that SNO export by RBCs may also depend on LAT activity, and investigated the role of RBC LAT in modulating SNO-sensitive RBC-endothelial cell (EC) adhesion. We used multiple pharmacologic inhibitors of LAT in vitro and in vivo to test the role of LAT in SNO export from RBCs and in thereby modulating RBC-EC adhesion. Inhibition of human RBC LAT by type-1-specific or nonspecific LAT antagonists increased RBC-endothelial adhesivity in vitro, and LAT inhibitors tended to increase post-transfusion RBC sequestration in the lung and decreased oxygenation in vivo. A LAT1-specific inhibitor attenuated SNO export from RBCs, and we demonstrated LAT1 in RBC membranes and LAT1 mRNA in reticulocytes. The proadhesive effects of inhibiting LAT1 could be overcome by supplemental L-CSNO (S-nitroso-L-cysteine), but not D-CSNO or L-Cys, and suggest a basal anti-adhesive role for stereospecific intercellular SNO transport. This study reveals for the first time a novel role of LAT1 in the export of SNOs from RBCs to prevent their adhesion to ECs. The findings have implications for the mechanisms of intercellular SNO signalling, and for thrombosis, sickle cell disease, and post-storage RBC transfusion, when RBC adhesivity is increased.

Efficacy of system l amino acid transporter 1 inhibition as a therapeutic target in esophageal squamous cell carcinoma.

System l amino acid transporter 1 (LAT1) is highly expressed in various types of human cancer, and contributes to cancer growth and survival. Recently, we have shown that LAT1 expression is closely related to the growth and aggressiveness of esophageal cancer, and is an independent marker of poor prognosis. However, it remains unclear whether LAT1 inhibition could suppress esophageal cancer growth. In this study, we investigated the tumor-suppressive effects of the inhibition of LAT1. Both LAT1 and CD98, which covalently associates to LAT1 on the membrane, were expressed in human esophageal cancer cell lines KYSE30 and KYSE150. Quantitative PCR analysis showed that the expression of LAT1 was much higher than other subtypes of LAT. A selective inhibitor of LAT, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), suppressed cellular uptake of l-14 C-leucine and cell proliferation in a dose-dependent manner. It also suppressed phosphorylation of mammalian target of rapamycin, 4E-BP1, and p70S6K protein, and induced cell cycle arrest at G1 phase. These results suggest that suppression of both mammalian target of rapamycin signaling and cell cycle progression is involved in BCH-induced growth inhibition. In tumor-bearing mice, daily treatment with BCH significantly delayed tumor growth and decreased glucose metabolism, indicating that LAT1 inhibition potentially suppresses esophageal cancer growth in vivo. Thus, our results suggest that LAT1 inhibition could be a promising molecular target for the esophageal cancer therapy.

Regulation of the plasma amino acid profile by leucine via the system L amino acid transporter.

Plasma concentrations of amino acids reflect the intracellular amino acid pool in mammals. However, the regulatory mechanism requires clarification. In this study, we examined the effect of leucine administration on plasma amino acid profiles in mice with and without the treatment of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) or rapamycin as an inhibitor of system L or mammalian target of rapamycin complex 1, respectively. The elevation of plasma leucine concentration after leucine administration was associated with a significant decrease in the plasma concentrations of isoleucine, valine, methionine, phenylalanine, and tyrosine; BCH treatment almost completely blocked the leucine-induced decrease in plasma amino acid concentrations. Rapamycin treatment had much less effects on the actions of leucine than BCH treatment. These results suggest that leucine regulates the plasma concentrations of branched-chain amino acids, methionine, phenylalanine, and tyrosine, and that system L amino acid transporters are involved in the leucine action.

JPH203, an L-type amino acid transporter 1-selective compound, induces apoptosis of YD-38 human oral cancer cells.

Compared to most normal cells that express L-type amino acid transporter 2, L-type amino acid transporter 1 is highly expressed in cancer cells and presumed to support their elevated growth and proliferation. This study examined JPH203, a potent and selective L-type amino acid transporter 1 inhibitor, and its ability to suppress YD-38 human oral cancer cell growth. The YD-38 cells express L-type amino acid transporter 1 with its associating protein 4F2 heavy chain, but not L-type amino acid transporter 2. JPH203 and BCH, a non-selective L-type amino acid transporter inhibitor, completely inhibited l-leucine uptake in YD-38 cells. As expected, the intrinsic affinity of JPH203 to inhibit l-leucine uptake was far more efficient than BCH. Likewise, JPH203 and BCH inhibited YD-38 cell growth, with JPH203 being superior to BCH. JPH203 up-regulated the population of apoptotic YD-38 cells through the activation of apoptotic factors, including caspases and PARP. These results suggest that the inhibition of L-type amino acid transporter 1 activity via JPH203, which may act as a potential novel anti-oral-cancer agent, leads to apoptosis by inducing the intracellular depletion of the neutral amino acids essential for cancer cell growth in YD-38 human oral cancer cells.

Radiosynthesis and biological evaluation of L- and D-S-(3-[18F]fluoropropyl)homocysteine for tumor imaging using positron emission tomography.

Interest in radiolabeled amino acids for metabolic imaging of cancer and limitations with [(11)C]methionine has prompted the development of a new (18)F-labeled methionine derivative S-(3-[(18)F]fluoropropyl)homocysteine ([(18)F]FPHCys). The L and D enantiomers of [(18)F]FPHCys were prepared from their respective protected S-(3-tosyloxypropyl)homocysteine precursors 1 by [(18)F]fluoride substitution using K(2.2.2) and potassium oxalate, followed by acid hydrolysis on a Tracerlab FX(FN) synthesis module. [(18)F]-L-FPHCys and [(18)F]-D-FPHCys were isolated in 20 ± 5% radiochemical yield and >98% radiochemical and enantiomeric purity in 65 min. Competitive uptake studies in A375 and HT29 tumor cells suggest that L- and D-[(18)F]FPHCys are taken up by the L-transporter system. [(18)F]-L-FPHCys and [(18)F]-D-FPHCys displayed good stability In Vivo without incorporation into protein at least 2 h postinjection. Biodistribution studies demonstrate good uptake in A375 tumor-bearing rodents with tumor to blood ratios of 3.5 and 5.0 for [(18)F]-L-FPHCys and [(18)F]-D-FPHCys, respectively, at 2 h postinjection.

Characterization of amino acid transport system L in HTB-41 human salivary gland epidermoid carcinoma cells.

BACKGROUND: The amino acid transport system L is a major nutrient transport system that is responsible for transport of neutral amino acids, including several essential amino acids. The current study attempted to investigate the expression and functional characterization of amino acid transport system L in HTB-41 human submaxillary salivary gland epidermoid carcinoma cells. MATERIALS AND METHODS: RT-PCR analysis, Western blot analysis and amino acid transport measurements were used. RESULTS: The HTB-41 cells expressed the L-type amino acid transporter 1 (LAT1) together with its associating protein heavy chain of 4F2 antigen (4F2hc) in the plasma membrane, whereas the HTB-41 cells did not express the L-type amino acid transporter 2 (LAT2). The uptakes of [14C]L-leucine were Na+-independent and completely inhibited by a system L selective inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The affinity of [14C]L-leucine uptake and the inhibition profile of [14C]L-leucine uptake by various L-amino acids in the HTB-41 cells were comparable with those for the LAT1 expressed in Xenopus oocytes. CONCLUSION: The transport of neutral amino acids including several essential amino acids into the HTB-41 human submaxillary salivary gland epidermoid carcinoma cells are mediated by LAT1.

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.

The L-amino acid carrier inhibitor 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH) reduces L-dopa-elicited responses in dopaminergic neurons of the substantia nigra pars compacta.

It is not yet clear how L-dopa, that is the most effective drug for the treatment of Parkinson's disease, enters into the dopaminergic neurons to be transformed into dopamine. It is suggested that L-dopa is mainly transported into cells by a group of L-amino acid carriers named "System L". Since these carriers are selectively inhibited by 2-aminobicyclo[2.2.1]heptane-2-carboxylic acid (BCH), we have applied this compound to electrophysiologically recorded dopaminergic neurons of the rat substantia nigra pars compacta to examine the possible modulation of the effects of L-dopa by System L. We have observed that BCH reduced, in a concentration-dependent manner, the membrane hyperpolarization/outward current caused by L-dopa. Interestingly, the actions of dopamine were not changed by this System L inhibitor, suggesting that the reducing effects on L-dopa are not due to a BCH-induced unspecific block of dopamine-mediated events. Therefore, our electrophysiological data that an l-type amino acid carrier, possibly System L, is involved in the transport of L-dopa into dopaminergic neurons.

Functional activity of a large neutral amino acid transporter (LAT) in rabbit retina: a study involving the in vivo retinal uptake and vitreal pharmacokinetics of L-phenyl alanine.

PURPOSE: The purpose of this study is to elucidate the functional activity of large neutral amino acid transporter (LAT) in rabbit retina and to delineate its role in the retinal uptake and intravitreal pharmacokinetics of L-phenylalanine (L-Phe). METHODS: In vivo retinal uptake of L-Phe and L-alanine (L-Ala) was determined in the presence and absence of specific transport inhibitors following intravitreal administration. L and D isomers of amino acids were employed as inhibitors to determine the stereo-selectivity of LAT. Reverse transcription-polymerase chain reaction (RT-PCR) was carried out for LAT isoforms (LAT1 and LAT2). Vitreal disposition of L-Phe following administration in rabbit vitreous was studied in the presence of an other competing LAT substrate D-methionine using microdialysis. RESULTS: Retinal uptake of L-Phe was significantly inhibited in presence of a specific LAT inhibitor, 2-aminobicyclo-[2,2,1]-heptane-2-carboxylic acid (BCH), L isomers of large neutral amino acids and LAT1 specific but not by LAT2 specific and charged amino acids. No significant inhibition of L-Ala retinal uptake was observed with LAT substrates. LAT isoforms (LAT1 and LAT2) were identified by RT-PCR in rabbit retina. The mean residence time (MRT) and area under curve (AUC) values of L-Phe following intravitreal administration were significantly increased in the presence of D-methionine, a LAT substrate. CONCLUSIONS: This study demonstrates the functional activity and molecular expression of large neutral amino acid transporter in the rabbit retina. Furthermore, based on these studies it can be concluded that LAT is involved in the retinal uptake and intravitreal elimination of L-Phe.

Brasilicardin A, a natural immunosuppressant, targets amino Acid transport system L.

Lymphocytes in T cell activation require extracellular nutrients to provide energy for cellular proliferation and effector functions. Therefore, inhibitors of nutrient transporters are expected to be a new class of immunosuppressant. Here, we report that the molecular target of brasilicardin A (BraA), an immunosuppressive compound, is the amino acid transporter system L. BraA inhibited the cell-cycle progression of murine T cell lymphocyte CTLL-2 cells in G1 phase, and potently inhibited the uptake of amino acids that are substrates for amino acid transport system L. Moreover, BraA stimulated the GCN2 activation and, subsequently, the phosphorylation of eIF2alpha. These results suggest that the immunosuppressive activity of BraA is induced by amino acid deprivation via the inhibition of system L and that the amino acid transporter is a target for immunosuppressant.

Differential expression and functional characterization of system L amino acid transporters in human normal osteoblast cells and osteogenic sarcoma cells.

BACKGROUND: The amino acid transport system L is a major nutrient transport system responsible for Na(+)-independent transport of neutral amino acids, including several essential amino acids. The system L is divided into two major subgroups, the L-type amino acid transporter 1 (LAT1) and the L-type amino acid transporter 2 (LAT2). In malignant tumors, the LAT1 is highly expressed to support tumor cell growth. In the present study, the expressions and functions of the system L amino acid transporters were examined and compared in both FOB human osteoblast cells and Saos2 human osteogenic sarcoma cells. MATERIALS AND METHODS: The expressions and functions of the system L amino acid transporters in both FOB and Saos2 cells were examined using RT-PCR, Western blot analysis and amino acid transport measurement. RESULTS: RT-PCR and Western blot analysis revealed that the FOB and Saos2 cells expressed LAT1 and LAT2, together with their associated protein 4F2hc, but the expression of LAT2 in the Saos2 cells was very weak. The uptakes of [14C]L-leucine by FOB and Saos2 cells were Na(+)-independent and were completely inhibited by the system L selective inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH). The affinity and the inhibition profiles of [14C]L-leucine uptake by various amino acids in the FOB and Saos2 cells were comparable with those for the LAT2 and LAT1 expressed in Xenopus oocytes, respectively. The majority of [14C]L-leucine uptake is, therefore, mediated by LAT2 and LAT1 in FOB and Saos2 cells, respectively. CONCLUSION: These results suggest that the transport of neutral amino acids, including several essential amino acids into the FOB and Saos2 cells, are mainly mediated by LAT2 and LAT1, respectively. Moreover, the specific inhibition of LAT1 in tumor cells might be a new rationale for antitumor therapy.

Distribution and pharmacology of alanine-serine-cysteine transporter 1 (asc-1) in rodent brain.

A polyclonal antibody against the Na+-independent alanine-serine-cysteine transporter 1 (asc-1) was raised and the specificity of the antibody verified by Western blots performed on membranes prepared from HEK293 cells transiently transfected with the cloned murine asc-1. The antibody was then used to localize the transporter in the brain of two rodent species by using immunohistochemistry at the light and electron microscopical level. asc-1-immunoreactivity (asc-1-ir) was widely distributed throughout the mouse and rat brain. Areas with high levels of asc-1-ir included hypothalamus, the medial septal area, globus pallidus, entopeduncular nucleus, cingulate and retrosplenial cortices. Moderate asc-1-ir was observed in several areas including layers III and V of the neocortex, thalamus, nucleus accumbens, caudate putamen, bed nucleus of stria terminalis, all amygdaloid nuclei, hippocampus (CA1-CA3 and hilus of the dentate gyrus), as well as several brainstem nuclei. asc-1-ir was observed as punctuate staining consistent with varicosities matching neuronal cell bodies and dendritic fields. At the ultrastructural level, asc-1-ir was mainly confined to presynaptic terminals. Immunostaining in either glial cell bodies or perivascular sites was not observed and white matter was completely devoid of asc-1-ir. Furthermore, the pharmacology of the Na+-independent uptake site for [3H]d-serine in rat brain synaptosomal P2 fractions was compared with the substrate specificity of the cloned human asc-1 transporter and a high degree of correlation was demonstrated. We conclude that asc-1-ir is widespread in the brain and limited to neuronal structures and that asc-1 may contribute to synaptic clearance of d-serine in brain.

Synthesis of syn- and anti-1-amino-3-[18F]fluoromethyl-cyclobutane-1-carboxylic acid (FMACBC), potential PET ligands for tumor detection.

syn- and anti-1-amino-3-[18F]fluoromethyl-cyclobutane-1-carboxylic acid (FMACBC, 16 and 17), analogues of anti-1-amino-3-[18F]fluorocyclobutyl-1-carboxylic acid (FACBC), were prepared to evaluate the contributions of C-3 substitution and configuration on the uptake of these radiolabeled amino acids in a rodent model of brain tumors. Radiofluorinated targets [18F]16 and [18F]17 were prepared by no-carrier-added radiofluorination from their corresponding methanesulfonyl esters 12 and 13, respectively, with decay-corrected radiochemical yields of 30% for [18F]16 and 20% for [18F]17. In amino acid transport assays performed in vitro using 9L gliosarcoma cells, both [18F]16 and [18F]17 were substrates for L type amino acid transport, while [18F]17 but not [18F]16 was a substrate for A type transport. Biodistribution studies in normal Fischer rats with [18F]16 and [18F]17 showed high uptake of radioactivity (>2.0% dose/g) in the pancreas while other tissues studied, including liver, heart, lung, kidney, blood, muscle, and testis, showed relatively low uptake of radioactivity (<1.0% dose/g). In rats implanted intracranially with 9L gliosarcoma cells, the retention of radioactivity in tumor tissue was high at 5, 60, and 120 min after intravenous injection of [18F]16 and [18F]17 while the uptake of radioactivity in brain tissue contralateral to the tumor remained low (<0.3% dose/g). Ratios of tumor uptake to normal brain uptake for [18F]16 were 7.5:1, 7:1, and 5:1 at 5, 60, and 120 min, respectively, while for [18F]17 the ratios were 7.5:1, 9:1, and 9:1 at the same time points. This work demonstrates that like anti-[18F]FACBC, [18F]16 and [18F]17 are excellent candidates for imaging brain tumors.