SLC1A5 enhances malignant phenotypes through modulating ferroptosis status and immune microenvironment in glioma.

Glioma is the most common type of primary malignant tumor in the central nervous system with limited treatment satisfaction. Finding new therapeutic targets has remained a major challenge. Ferroptosis is a novel and distinct type of programmed cell death, playing a regulatory role in the progression of tumors. However, the role of ferroptosis or ferroptosis-related genes (FRGs) in glioma progression has not been extensively studied. In our study, a novel ferroptosis-related prognostic model, including 7 genes, was established, in which patients classified into the high-risk group had more immuno-suppressive status and worse prognosis. Among these 7 genes, we screened solute carrier family 1 member 5 (SLC1A5), an FRG, as a possible new target for glioma treatment. Our results showed that the expression of SLC1A5 was significantly upregulated in glioblastoma tissues compared with the low-grade gliomas. In addition, SLC1A5 knockdown could significantly inhibit glioma cell proliferation and invasion, and reduce the sensitivity of ferroptosis via the GPX4-dependent pathway. Furthermore, SLC1A5 was found to be related to immune response and SLC1A5 knockdown decreased the infiltration and M2 polarization of tumor-associated macrophages. Pharmacological inhibition of SLC1A5 by V9302 was confirmed to promote the efficacy of anti-PD-1 therapy. Overall, we developed a novel prognostic model for glioma based on the seven-FRGs signature, which could apply to glioma prognostic and immune status prediction. Besides, SLC1A5 in the model could regulate the proliferation, invasion, ferroptosis and immune state in glioma, and be applied as a prognostic biomarker and potential therapeutic target for glioma.

The pro-proliferative effect of interferon-γ in breast cancer cell lines is dependent on stimulation of ASCT2-mediated glutamine cellular uptake.

AIMS: Type 2 diabetes mellitus (T2DM) is a risk factor for breast cancer initiation and progression. Glutamine (GLN) is a critical nutrient for cancer cells. The aim of this study was to investigate the effect of T2DM-associated compounds upon GLN uptake by breast cancer cells. MAIN METHODS: The in vitro uptake of 3H-GLN by breast cancer (MCF-7 and MDA-MB-231) and non-tumorigenic (MCF-12A) cell lines was measured. KEY FINDINGS: 3H-GLN uptake in the three cell lines is mainly Na+-dependent and sensitive to the ASCT2 inhibitor GPNA. IFN-γ increased total and Na+-dependent 3H-GLN uptake in the two breast cancer cell lines, and insulin increased total and Na+-dependent 3H-GLN uptake in the non-tumorigenic cell line. GPNA abolished the increase in 3H-GLN uptake promoted by these T2DM-associated compounds. ASCT2 knockdown confirmed that the increase in 3H-GLN uptake caused by IFN-γ (in breast cancer cells) and by insulin (in non-tumorigenic cells) is ASCT2-dependent. IFN-γ (in MDA-MB-231 cells) and insulin (in MCF-12A cells) increased ASCT2 transcript and protein levels. Importantly, the pro-proliferative effect of IFN-γ in breast cancer cell lines was associated with an increase in 3H-GLN uptake which was GPNA-sensitive, blocked by ASCT2 knockdown and mediated by activation of the PI3K-, STAT3- and STAT1 intracellular signalling pathways. SIGNIFICANCE: IFN-γ and insulin possess pro-proliferative effects in breast cancer and non-cancer cell lines, respectively, which are dependent on an increase in ASCT2-mediated glutamine transport. Thus, an effective inhibition of ASCT2-mediated glutamine uptake may be a therapeutic strategy against human breast cancer in T2DM patients.

ASCT1 and ASCT2: Brother and Sister?

The SLC1 family includes seven members divided into two groups, namely, EAATs and ASCTs, that share similar 3D architecture; the first one includes high-affinity glutamate transporters, and the second one includes SLC1A4 and SLC1A5, known as ASCT1 and ASCT2, respectively, responsible for the traffic of neutral amino acids across the cell plasma membrane. The physiological role of ASCT1 and ASCT2 has been investigated over the years, revealing different properties in terms of substrate specificities, affinities, and regulation by physiological effectors and posttranslational modifications. Furthermore, ASCT1 and ASCT2 are involved in pathological conditions, such as neurodegenerative disorders and cancer. This has driven research in the pharmaceutical field aimed to find drugs able to target the two proteins.This review focuses on structural, functional, and regulatory aspects of ASCT1 and ASCT2, highlighting similarities and differences.

The role of the glutamine transporter ASCT2 in antineoplastic therapy.

Cancer cells are metabolically reprogrammed to support their high rates of proliferation, continuous growth, survival, invasion, metastasis, and resistance to cancer treatments. Among changes in cancer cell bioenergetics, the role of glutamine metabolism has been receiving increasing attention. Increased glutaminolysis in cancer cells is associated with increased expression of membrane transporters that mediate the cellular uptake of glutamine. ASCT2 (Alanine, Serine, Cysteine Transporter 2) is a Na+-dependent transmembrane transporter overexpressed in cancer cells and considered to be the primary transporter for glutamine in these cells. The possibility of inhibiting ASCT2 for antineoplastic therapy is currently under investigation. In this article, we will present the pharmacological agents currently known to act on ASCT2, which have been attracting attention in antineoplastic therapy research. We will also address the impact of ASCT2 inhibition on the prognosis of some cancers. We conclude that ASCT2 inhibition and combination of ASCT2 inhibitors with other anti-tumor therapies may be a promising antineoplastic strategy. However, more research is needed in this area.

Functional analysis of OCTN2 and ATB0,+ in normal human airway epithelial cells.

In human, OCTN2 (SLC22A5) and ATB0,+ (SLC6A14) transporters mediate the uptake of L-carnitine, essential for the transport of fatty acids into mitochondria and the subsequent degradation by ß-oxidation. Aim of the present study was to characterize L-carnitine transport in EpiAirway™, a 3D organotypic in vitro model of primary human tracheal-bronchial epithelial cells that form a fully differentiated, pseudostratified columnar epithelium at air-liquid interface (ALI) condition. In parallel, Calu-3 monolayers grown at ALI for different times (8d or 21d of culture) were used as comparison. OCTN2 transporter was equally expressed in both models and functional at the basolateral side. ATB0,+ was, instead, highly expressed and active on the apical membrane of EpiAirway™ and only in early-cultures of Calu-3 (8d but not 21d ALI). In both cell models, L-carnitine uptake on the apical side was significantly inhibited by the bronchodilators glycopyrrolate and tiotropium, that hence can be considered substrates of ATB0,+; ipratropium was instead effective on the basolateral side, indicating its interaction with OCTN2. Inflammatory stimuli, such as LPS or TNFα, caused an induction of SLC6A14/ATB0,+ expression in Calu-3 cells, along with a 2-fold increase of L-carnitine uptake only at the apical side; on the contrary SLC22A5/OCTN2 was not affected. As both OCTN2 and ATB0,+, beyond transporting L-carnitine, have a significant potential as delivery systems for drugs, the identification of these transporters in EpiAirway™ can open new fields of investigation in the study of drug inhalation and pulmonary delivery.

Critical role of ASCT2-mediated amino acid metabolism in promoting leukaemia development and progression.

Amino acid (AA) metabolism is involved in diverse cellular functions, including cell survival and growth, however it remains unclear how it regulates normal hematopoiesis versus leukemogenesis. Here, we report that knockout of Slc1a5 (ASCT2), a transporter of neutral AAs, especially glutamine, results in mild to moderate defects in bone marrow and mature blood cell development under steady state conditions. In contrast, constitutive or induced deletion of Slc1a5 decreases leukemia initiation and maintenance driven by the oncogene MLL-AF9 or Pten deficiency. Survival of leukemic mice is prolonged following Slc1a5 deletion, and pharmacological inhibition of ASCT2 also decreases leukemia development and progression in xenograft models of human acute myeloid leukemia. Mechanistically, loss of ASCT2 generates a global effect on cellular metabolism, disrupts leucine influx and mTOR signaling, and induces apoptosis in leukemic cells. Given the substantial difference in reliance on ASCT2-mediated AA metabolism between normal and malignant blood cells, this in vivo study suggests ASCT2 as a promising therapeutic target for the treatment of leukemia.

The role of ASCT2 in cancer: A review.

Reorganization of cellular metabolism is one of the hallmarks of cancer and many tumors show high glucose uptake and glutamine addiction. Glutamine is imported by the SLC family transporters from the microenvironment, and ASCT2 (encoded by the SLC1A5 gene) is recognized as a primary transporter. Of note, ASCT2 is overexpressed in different cancers and is closely related to poor prognosis. Nonetheless, the mechanisms regulating ASCT2 activity has not been elucidated. Moreover, several inhibitors of ASCT2 have emerged and shown a surprising antitumor effect. In conclusion, this review describes the function, regulatory mechanism, and inhibitors of ASCT2 in cancer, suggesting that high expression of ASCT2 is a promising prognostic marker and a potential drug target.

[Effect of ASCT2 gene knock-down by shRNA on biological behaviors of colorectal cancer cells].

OBJECTIVE: To investigate the effect of ASCT2 gene (glutamine transporter) knock-down by shRNA on biological behaviors of colorectal cancer cells. METHODS: shRNA was transfected into colorectal cancer cells Lovo and SW480 to knockdown ASCT2 mediated by Lipofectamine 2000. Reverse transcription-PCR and Western blot were used to examine the mRNA and protein expression of ASCT2. MTT and transwell assay were used to determine the proliferation and invasiveness of Lovo and SW480 cells. Radioactive-tracer was used to detect the uptake of glutamine. RESULTS: ASCT2 mRNA and protein levels were significantly down-regulated by shRNA in Lovo and SW480 cells(P<0.01). MTT and transwell assays showed that ASCT2 knock-down could significantly inhibit the proliferation of Lovo and SW480 cells (A490) and decrease the number of invasive Lovo and SW480 cells from the membrane (both P<0.01). The number of membrane Lovo cells in shASCT group and control group was 46.3±5.9 and 197.7±9.1, respectively while the number of membrane SW480 cells in shASCT group and control group was 29.7±3.8 and 139.0±9.5, respectively. Radioactive-tracer showed that shASCT2 transfection could significantly reduce the uptake of glutamine, with an inhibition rate of 79.15% in Lovo and 67.22% in SW480 cells (both P<0.01). CONCLUSIONS: ASCT2 plays an oncogenic role in colonic cancer, and its promotion mechanism may be associated with glutamine metabolism. ASCT2 may be a novel therapeutic target of colonic cancer.

Nutritional Stress Induced by Tryptophan-Degrading Enzymes Results in ATF4-Dependent Reprogramming of the Amino Acid Transporter Profile in Tumor Cells.

Tryptophan degradation is an immune escape strategy shared by many tumors. However, cancer cells' compensatory mechanisms remain unclear. We demonstrate here that a shortage of tryptophan caused by expression of indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) resulted in ATF4-dependent upregulation of several amino acid transporters, including SLC1A5 and its truncated isoforms, which in turn enhanced tryptophan and glutamine uptake. Importantly, SLC1A5 failed to be upregulated in resting human T cells kept under low tryptophan conditions but was enhanced upon cognate antigen T-cell receptor engagement. Our results highlight key differences in the ability of tumor and T cells to adapt to tryptophan starvation and provide important insights into the poor prognosis of tumors coexpressing IDO and SLC1A5. Cancer Res; 76(21); 6193-204. ©2016 AACR.

Syncytin proteins incorporated in placenta exosomes are important for cell uptake and show variation in abundance in serum exosomes from patients with preeclampsia.

Exosomes are extracellular vesicles that mediate intercellular communication and are involved in several biological processes. The objective of our study was to determine whether endogenous retrovirus group WE, member l (ERVWE1)/syncytin-1 and endogenous retrovirus group FRD, member 1 (ERVFRDE1)/syncytin-2, encoded by human endogenous retrovirus (HERV) envelope (env) genes, are present at the surface of exosomes produced by placenta-derived villous cytotrophoblasts and whether they play a role in cellular uptake of exosomes. In addition, we sought to determine whether these proteins are present in various abundances in serum-derived exosomes from normal pregnant women vs. women with preeclampsia (PE). Isolated exosomes were analyzed for their content by Western blot, a bead-associated flow cytometry approach, and a syncytin-2 ELISA. Binding and uptake were tested through confocal and electron microscopy using the BeWo choriocarcinoma cell line. Quality control of exosome preparations consisted of detection of exosomal and nonexosomal markers. Exosome-cell interactions were compared between cells incubated in the presence of control exosomes, syncytin-1 or syncytin-2-deprived exosomes, or exosomes solely bearing the uncleaved forms of these HERV env proteins. From our data, we conclude that villous cytotrophoblast exosomes are positive for both env proteins and are rapidly taken up by BeWo cells in a syncytin-1- and syncytin-2-dependent manner and that syncytin-2 is reduced in serum-derived exosomes from women with PE when compared to exosomes from normal pregnant women.

In vivo D-serine hetero-exchange through alanine-serine-cysteine (ASC) transporters detected by microelectrode biosensors.

D-serine, a co-agonist of N-methyl D-aspartate (NMDA) receptors, has been implicated in neurological and psychiatric disorders such as cerebral ischemia, lateral amyotrophic sclerosis, or schizophrenia. D-serine signaling represents an important pharmacological target for treating these diseases; however, the biochemical mechanisms controlling extracellular D-serine levels in vivo are still unclear. D-serine heteroexchange through small neutral amino acid transporters has been shown in cell cultures and brain slices and could provide a biochemical mechanism for the control of D-serine extracellular concentration in vivo. Alternatively, exocytotic D-serine release has also been proposed. In this study, the dynamics of D-serine release and clearance were explored in vivo on a second-by-second time scale using microelectrode biosensors. The rate of D-serine clearance in the rat frontal cortex after a microionophoretic injection revealed a transporter-mediated uptake mechanism. D-serine uptake was blocked by small neutral l-amino acids, implicating alanine-serine-cysteine (ASC) transporters, in particular high affinity Asc-1 and low affinity ASCT2 transporters. Interestingly, changes in alanine, serine, or threonine levels resulted in D-serine release through ASC transporters. Asc-1, but not ASCT2, appeared to release D-serine in response to changes in amino acid concentrations. Finally, neuronal silencing by tetrodotoxin increased D-serine extracellular concentration by an ASC-transporter-dependent mechanism. Together, these results indicate that D-serine heteroexchange through ASC transporters is present in vivo and may constitute a key component in the regulation of D-serine extracellular concentration.

NMDA receptor activation: two targets for two co-agonists.

Neuronal N-methyl-D-aspartate receptors (NMDARs) play a critical role in synaptic plasticity. Their activation requires not only binding of their ligand glutamate and membrane depolarization but also the presence of a co-agonist, glycine or D-serine. An increasing body of experimental evidence suggests that different populations of NMDARs could be gated by different co-agonists. Here we discuss how the spatial distribution of co-agonist sources and uptake mechanisms, together with diffusional properties of the synaptic environment, could shape NMDAR co-agonist supply and therefore NMDAR-dependent plasticity.

Molecular determinants for functional differences between alanine-serine-cysteine transporter 1 and other glutamate transporter family members.

The ASCTs (alanine, serine, and cysteine transporters) belong to the solute carrier family 1 (SLC1), which also includes the human glutamate transporters (excitatory amino acid transporters, EAATs) and the prokaryotic aspartate transporter GltPh. Despite the high degree of amino acid sequence identity between family members, ASCTs function quite differently from the EAATs and GltPh. The aim of this study was to mutate ASCT1 to generate a transporter with functional properties of the EAATs and GltPh, to further our understanding of the structural basis for the different transport mechanisms of the SLC1 family. We have identified three key residues involved in determining differences between ASCT1, the EAATs and GltPh. ASCT1 transporters containing the mutations A382T, T459R, and Q386E were expressed in Xenopus laevis oocytes, and their transport and anion channel functions were investigated. A382T and T459R altered the substrate selectivity of ASCT1 to allow the transport of acidic amino acids, particularly l-aspartate. The combination of A382T and T459R within ASCT1 generates a transporter with a similar profile to that of GltPh, with preference for l-aspartate over l-glutamate. Interestingly, the amplitude of the anion conductance activated by the acidic amino acids does not correlate with rates of transport, highlighting the distinction between these two processes. Q386E impaired the ability of ASCT1 to bind acidic amino acids at pH 5.5; however, this was reversed by the additional mutation A382T. We propose that these residues differences in TM7 and TM8 combine to determine differences in substrate selectivity between members of the SLC1 family.

Involvement of OCTN2 in the transport of acetyl-L-carnitine across the inner blood-retinal barrier.

PURPOSE: To elucidate the mechanisms of acetyl-L-carnitine transport across the inner blood-retinal barrier (inner BRB). METHODS: In vivo integration plot and retinal uptake index (RUI) analyses were used to examine acetyl-L-[(3)H]carnitine transport in the retina across the inner BRB in rats. RUI was determined from the ratio of acetyl-L-[(3)H]carnitine and [(14)C]n-butanol, a freely diffusible internal reference, in the retina divided by the same ratio in the solution injected in the carotid artery. The transport mechanism was characterized in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells), as an in vitro inner BRB model. RESULTS: The apparent influx permeability clearance (K(in)) per gram retina of acetyl-L-[(3)H]carnitine was found to be 2.31 microL/(minute . g retina). The K(in) of acetyl-L-[(3)H]carnitine was 3.7-fold greater than that of [(3)H]D-mannitol, a nonpermeable paracellular marker. Acetyl-L-[(3)H]carnitine uptake by the retina was found to be significantly inhibited by L-carnitine and acetyl-L-carnitine, supporting a carrier-mediated influx transport of acetyl-L-carnitine at the inner BRB. L-[(3)H]carnitine and acetyl-L-[(3)H]carnitine uptake by TR-iBRB2 cells was Na(+)- and concentration-dependent, with a K(m) of 29 and 26 microM, respectively. These forms of transport were significantly inhibited by organic cation/carnitine transporter (OCTN) substrates and inhibitors such as L-carnitine and acetyl-L-carnitine, tetraethylammonium, quinidine, and betaine. These transport properties are consistent with those of carnitine transport by OCTN2. OCTN2 was predominantly expressed in TR-iBRB2 cells and isolated rat retinal vascular endothelial cells. CONCLUSIONS: The findings suggest that OCTN2 is involved in the transport of acetyl-L-carnitine from the circulating blood to the retina across the inner BRB.

Manganese disrupts astrocyte glutamine transporter expression and function.

Glutamine (Gln) plays an important role in brain energy metabolism and as a precursor for the synthesis of neurotransmitter glutamate and GABA. Previous studies have shown that astrocytic Gln transport is impaired following manganese (Mn) exposure. The present studies were performed to identify the transport routes and the respective Gln transporters contributing to the impairment. Rat neonatal cortical primary astrocytes treated with Mn displayed a significant decrease in Gln uptake mediated by the principle Gln transporting systems, N and ASC. Moreover, systems N, ASC and L were less efficient in Gln export after Mn treatment. Mn treatment caused a significant reduction of both in mRNA expression and protein levels of SNAT3 (system N), SNAT2 (system A) and LAT2 (system L), and lowered the protein but not mRNA expression of ASCT2 (system ASC). Mn exposure did not affect the expression of the less abundant systems N transporter SNAT5 and the system L transporter LAT1, at either the mRNA or protein level. Hence, Mn-induced decrease of inward and outward Gln transport can be largely ascribed to the loss of the specific Gln transporters. Consequently, deregulation of glutamate homeostasis and its diminished availability to neurons may lead to impairment in glutamatergic neurotransmission, a phenomenon characteristic of Mn-induced neurotoxicity.

Mapping of glutathione and its precursor amino acids reveals a role for GLYT2 in glycine uptake in the lens core.

PURPOSE: To correlate the distribution of glutathione (GSH) and its precursor amino acids (cysteine, glycine, and glutamate) with the expression of their respective amino acid transporters in the rat lens. METHODS: Whole rat lenses were fixed, cryoprotected, and cryosectioned in either an equatorial or axial orientation. Sections were double labeled with cystine, glycine, glutamate, GSH, GLYT1, or GLYT2 antibodies, and the membrane marker wheat germ agglutinin (WGA). Sections were imaged by confocal laser scanning microscopy. Cystine, glycine, glutamate, and GSH labeling were quantified by using image-analysis software and intensity profiles plotted as a function of distance from the lens periphery. Western blot analysis was used to verify regional differences in amino acid transporter expression. RESULTS: Cystine and glycine labeling in equatorial sections was most intense in the outer cortex, was diminished in the inner cortex, but was increased again in the core relative to the inner cortex. Glutamate and GSH labeling was most intense in the outer cortex and was diminished in the inner cortex to a minimum that was sustained throughout the core. The distribution of cystine and glutamate levels correlated well with the expression patterns observed previously for the cystine/glutamate exchanger (Xc-) and the glutamate transporter (EAAT4/5), respectively. Although high levels of glycine labeling in the outer cortex correlated well with the expression of the glycine transporter GLYT1, the absence of GLYT1 in the core, despite an increase of glycine in this region, suggests an alternative glycine uptake system such as GLYT2 exists in the core. Equatorial sections labeled with GLYT2 antibodies, showed that labeling in the outer cortex was predominantly cytoplasmic, but progressively became more membranous with distance into the lens. In the inner cortex and core, GLYT2 labeling was localized around the entire membrane of fiber cells. Western blot analysis confirmed GLYT2 to be expressed in the outer cortex, inner cortex, and core of the lens. Axial sections labeled for glycine revealed a track of high-intensity glycine labeling that extended from the anterior pole through to the core that was associated with the sutures. CONCLUSIONS: The mapping of GSH and its precursor amino acids has shown that an alternative glycine uptake pathway exists in mature fiber cells. Although GLYT1 and -2 are likely to mediate glycine uptake in cortical fiber cells, GLYT2 alone appears responsible for the accumulation of glycine in the center of the lens. Enhancing the delivery of glycine to the core via the sutures may represent a pathway to protect the lens against the protein modifications associated with age-related nuclear cataract.

Differential regulation of homocysteine transport in vascular endothelial and smooth muscle cells.

OBJECTIVE: We previously reported that homocysteine (Hcy) inhibits endothelial cell (EC) growth and promotes vascular smooth muscle cell (VSMC) proliferation. This study characterized and directly compared Hcy transport in cultured human aortic ECs (HAECs) and smooth muscle cells (HASMCs). METHODS AND RESULTS: Hcy (10 micromol/L) was transported into both cell types in a time-dependent fashion but was approximately 4-fold greater in HASMCs, and is nonstereoenantiomer specific. Hcy transport in HAECs had a Michaelis-Menten constant (Km) of 39 micromol/L and a maximal transport velocity (Vmax) of 873 pmol/mg protein/min. In contrast, Hcy transport in HASMCs had a lower affinity (Km = 106 micromol/L) but a higher transport capacity (Vmax = 4192 pmol/mg protein/min). Competition studies revealed that the small neutral amino acids tyrosine, cysteine, glycine, serine, alanine, methionine, and leucine inhibited Hcy uptake in both cell types, but the inhibition was greater for tyrosine, serine, glycine, and alanine in HAECs. Sodium-depletion reduced Hcy transport to 16% in HAECs and 56% in HASMCs. Increases in pH from 6.5 to 8.2 or lysosomal inhibitors blocked Hcy uptake only in HAECs. In addition, Hcy shares carrier systems with cysteine, in a preferable order of alanine-serine-cysteine (ASC) > aspartate and glutamate (X(AG)) = large branched-chain neutral amino acids (L) transporter systems in HAECs and ASC > L > X(AG) in HASMCs. The sodium-dependent system ASC plays a predominant role for Hcy transport in vascular cells. CONCLUSIONS: Transport system ASC predominantly mediates Hcy transport in EC and is lysosomal dependent.

The glutamine/amino acid transporter (ASCT2) reconstituted in liposomes: electrical nature of the glutamine/glutamate antiport.

The glutamine/amino acid transporter solubilized from rat renal apical plasma membrane (brush-border membrane) with C12E8 and reconstituted into liposomes has been previously identified as the ASCT2 transporter. The reconstituted transporter catalyses an antiport reaction in which extraliposomal glutamine and Na+ are cotransported in exchange with intraliposomal neutral amino acids. Differently from other neutral amino acid transporters, ASCT2 accepts also glutamate as substrate, as demonstrated by the glutamine/glutamate antiport measured in proteoliposomes. The electrical nature of the homologous glutamine/glutamine antiport and of the heterologous glutamine/glutamate antiport has been investigated by imposing a K+ diffusion potential (positive outside) across the proteoliposomal membrane in the presence of valinomycin. The membrane potential did not affect the glutamine/glutamine antiport whereas it stimulated about two fold the glutamine/glutamate antiport rate.

Functional and molecular analysis of D-serine transport in retinal Müller cells.

D-serine, an endogenous co-agonist of NMDA receptors in vertebrate retina, may modulate glutamate sensitivity of retinal neurons. This study determined at the functional and molecular level the transport process responsible for D-serine in retinal Müller cells. RT-PCR and immunoblotting showed that serine racemase (SR), the synthesizing enzyme for D-serine, is expressed in the rMC-1 Müller cell line and primary cultures of mouse Müller cells (1 degrees MCs). The relative contributions of different amino acid transport systems to d-serine uptake were determined based on differential substrate specificities and ion dependencies. D-serine uptake was obligatorily dependent on Na+, eliminating Na+-independent transporters (asc-1 and system L) for D-serine in Müller cells. The Na+:substrate stoichiometry for the transport process was 1:1. D-serine transport was inhibited by alanine, serine, cysteine, glutamine, and asparagine, but not anionic amino acids or cationic amino acids, suggesting that D-serine transport in Müller cells occurs via ASCT2 rather than ASCT1 or ATB0,+. The expression of mRNAs specific for ASCT1, ASCT2, and ATB0,+ was analyzed by RT-PCR confirming the expression of ASCT2 (and ASCT1) mRNA, but not ATB0,+, in Müller cells. Immunoblotting detected ASCT2 in neural retina and in 1 degrees MCs; immunohistochemistry confirmed these data in retinal sections and in cultures of 1 degrees MCs. The efflux of D-serine via ASCT2 by ASCT2 substrates was demonstrable using the Xenopus laevis oocyte heterologous expression system. These data provide the first molecular evidence for SR and ASCT2 expression in a Müller cell line and in 1 degrees MCs and suggest that D-serine, synthesized in Müller cells by SR, is effluxed via ASCT2 to regulate NMDA receptors in adjacent neurons.

The transport of glutamine into mammalian cells.

Glutamine has many important functions in mammalian cells, and glutamine transport across cell membranes has accordingly been extensively studied. In the past few years a number of important glutamine transport proteins have been sequenced and their molecular properties have been characterised. In general, four major transporters are important physiologically. These are known as (i) SNAT3 (System N) which is important in glutamine uptake in periportal cells in liver and in across the basolateral membrane of renal proximal tubule cells and is also involved in glutamine release by liver perivenous cells and by astrocytes; a variant of this protein catalyses glutamine release from skeletal muscle. (ii) SNAT1 (a specific System A sub-type) which is important in glutamine uptake by neuronal cells (iii) ASCT2 which is essential for glutamine uptake by rapidly growing epithelial cells and tumour cells in culture and (iv) the recently discovered brush border membrane transporter B0 AT1 (SLC6A19). Recent studies considered both the importance of ASCT2 in tumour cell growth and the regulation of ASCT2 expression. In SK-Hep hepatoma cells, knockdown of ASCT2 using antisense mRNA has been shown to cause apoptosis. Expression of the ASCT2 transporter in HepG2 hepatoma cells is stimulated by glutamine by a pathway involving the promoter element AGGTGAATGACTT which binds FXR/RXR dimers.