Independent Amino Acid Transporter Research Articles

Expression of L-Type Amino Acid Transporter 1 is a Predictive Biomarker of Intravesical Recurrence in Patients with Non-Muscle Invasive Bladder Cancer.

PurposeL-type amino acid transporter 1 (LAT1), a Na+-independent amino acid transporter, is highly expressed in various cancer types. We evaluated the prognostic value of LAT1 expression in non-muscle-invasive bladder cancer (NMIBC).Patients and MethodsWe retrospectively reviewed 119 consecutive patients who underwent initial transurethral resection of bladder tumor. Of these, 75 patients with NMIBC were included in this study. Patients were classified into two groups according to the proportion of LAT1-positive cells, as determined by immunohistochemistry. Associations between LAT1 expression and clinicopathological factors were analyzed. Cox multivariate analyses were performed to identify independent predictors of intravesical recurrence (IVR). The LAT1 integrated risk model was compared with the European Organization for Research and Treatment of Cancer (EORTC) risk model to evaluate the predictive ability for IVR based on the c-index.ResultsThe median follow-up was 37 months. Twenty-eight patients (37.3%) had IVR. LAT1 expression was not correlated with any other clinicopathological factors. Patients with high LAT1 expression had a worse IVR-free survival than that of patients with low LAT1 expression (P = 0.038). Cox multivariate analyses indicated that tumor multiplicity and high LAT1 expression were independent predictors of IVR. The LAT1 integrated risk model had a significantly improved performance over the EORTC model for assessing recurrence risk (c-index: 0.695, improvement: 0.091, P = 0.001). When patients were stratified into three groups according to the score calculated by the LAT1 integrated risk model, the 2-year IVR-free survival rates were 93.3% in patients with 0 points, 66.9% for those with 2 points, and 37.5% for those with 4 points.ConclusionHigh LAT1 expression was an independent predictor of IVR in patients with NMIBC. The LAT1 integrated risk model had good predictability for IVR.

\u03b3-Glutamyltransferase enzyme activity of cancer cells modulates L-\u03b3-glutamyl-p-nitroanilide (GPNA) cytotoxicity

L-γ-Glutamyl-p-nitroanilide (GPNA) is widely used to inhibit the glutamine (Gln) transporter ASCT2, but recent studies have demonstrated that it is also able to inhibit other sodium-dependent and independent amino acid transporters. Moreover, GPNA is a well known substrate of the enzyme γ-glutamyltransferase (GGT). Our aim was to evaluate the effect of GGT-mediated GPNA catabolism on cell viability and Gln transport. The GGT-catalyzed hydrolysis of GPNA produced cytotoxic effects in lung cancer A549 cells, resulting from the release of metabolite p-nitroaniline (PNA) rather than from the inhibition of Gln uptake. Interestingly, compounds like valproic acid, verapamil and reversan were able to increase the cytotoxicity of GPNA and PNA, suggesting a key role of intracellular detoxification mechanisms. Our data indicate that the mechanism of action of GPNA is more complex than believed, and further confirm the poor specificity of GPNA as an inhibitor of Gln transport. Different factors may modulate the final effects of GPNA, ranging from GGT and ASCT2 expression to intracellular defenses against xenobiotics. Thus, other strategies - such as a genetic suppression of ASCT2 or the identification of new specific inhibitors - should be preferred when inhibition of ASCT2 function is required.

MP83-11 Y+LAT2 (SLC7A6) EXPRESSION IN CASTRATION RESISTANT PROSTATE CANCER

You have accessJournal of UrologyProstate Cancer: Basic Research & Pathophysiology II1 Apr 2017MP83-11 Y+LAT2 (SLC7A6) EXPRESSION IN CASTRATION RESISTANT PROSTATE CANCER Hideo Otsuki, Toru Kimura, Takeo Kosaka, Takashi Yamaga, Jun-ichi Suehiro, and Hiroyuki Sakurai Hideo OtsukiHideo Otsuki More articles by this author , Toru KimuraToru Kimura More articles by this author , Takeo KosakaTakeo Kosaka More articles by this author , Takashi YamagaTakashi Yamaga More articles by this author , Jun-ichi SuehiroJun-ichi Suehiro More articles by this author , and Hiroyuki SakuraiHiroyuki Sakurai More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.2579AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters can be a novel therapeutic strategy. One of leucine transporters, L-type amino acid transporter (LAT) 1 (SLC7A5), a Na+-independent amino acid transporter, has been reported to be selectively expressed in many cancer cells. Recently it has been shown that primary prostate cancer expresses LAT3 (SLC43A1), while castration resistant or highly aggressive prostate cancer expresses LAT1 as a main leucine transporter. In this study, we examine leucine transporters during acquisition of hormone independence. METHODS A new ″LN-abl″ cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is a model of castration resistant prostate cancer (CRPC) with androgen receptor expression. Uptake of 14C leucine was examined in the presence or absence of LAT inhibitors or Na+. Expression of a major leucine transporter was inhibited by siRNA in LN-abl cells. In silico analysis of leucine transporter expression was examined using Oncomine for different progression status of prostate cancers in clinical data sets. RESULTS Cell viability was decreased to 10% in the absence of leucine. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na+-independent. In LN-abl cells, Na+-dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na+-independent uptake was only 0.52, therefore, leucine uptake of LN-abl was largely (~85%) Na+-dependent. y+LAT2 (SLC7A6) expression was confirmed in LN-abl, however, expression of LAT1, LAT3, y+LAT1 (SLC7A7), ATB0+ (SLC6A14), B0AT1 (SLC6A19), B0AT2 (SLC6A15) or b0+AT (SLC7A9) were not observed. Knockdown of y+LAT2 lead to significant leucine uptake inhibition (40%) and cell growth inhibition (20%) in LN-abl cells. In silico analysis revealed that more frequent up-regulation of SLC7A6 (y+LAT2) was observed in hormone resistant or metastatic samples in 2 data sets. In addition, some advanced prostate cancer appeared to express high levels of LAT3, suggesting heterogeneity in leucine transporter expression among different prostate cancers. CONCLUSIONS New CRPC cell line with increased expression of y+LAT2 was established in vitro. This is consistent with the fact that at least some advanced stage prostate cancers express y+LAT2 in Oncomine data. Considering the diversity of leucine transporter expression, target of anti-leucine transporter therapy should be individualized. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e1110 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Hideo Otsuki More articles by this author Toru Kimura More articles by this author Takeo Kosaka More articles by this author Takashi Yamaga More articles by this author Jun-ichi Suehiro More articles by this author Hiroyuki Sakurai More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Prostate Cancer Cells in Different Androgen Receptor Status Employ Different Leucine Transporters

Leucine stimulates cancer cell proliferation through the mTOR pathway, therefore, inhibiting leucine transporters may be a novel therapeutic target for cancer. L-type amino acid transporter (LAT) 1, a Na+ -independent amino acid transporter, is highly expressed in many tumor cells. However, leucine transporter(s) in different stages of prostate cancer, particularly in the stages of castration resistance with androgen receptor (AR) expression, is unclear. LNCaP and DU145 and PC-3 cell lines were used as a model of androgen dependent, and metastatic prostate cancer. A new "LN-cr" cell line was established after culturing LNCaP cells for 6 months under androgen-free conditions, which is considered a model of castration resistant prostate cancer (CRPC) with androgen AR expression. The expression of leucine transporters was investigated with quantitative PCR and immunofluorescence. Uptake of 14 C Leucine was examined in the presence or absence of BCH (a pan-LAT inhibitor), JPH203 (an LAT1-specific inhibitor), or Na+ . Cell growth was assessed with MTT assay. siRNA studies were performed to evaluate the indispensability of y+ LAT2 on leucine uptake and cell viability in LN-cr. Cell viability showed a 90% decrease in the absence of leucine in all four cell lines. LNCaP cells principally expressed LAT3, and their leucine uptake was more than 90% Na+ -independent. BCH, but not JPH203, inhibited leucine uptake, and cell proliferation (IC50BCH :15 mM). DU145 and PC-3 cells predominantly expressed LAT1. Leucine uptake and cell growth were suppressed by BCH or JPH203 in a dose-dependent manner (IC50BCH : ∼20 mM, IC50JPH203 : ∼5 µM). In LN-cr cells, Na+ -dependent uptake of leucine was 3.8 pmol/mgprotein/min, while, Na+ -independent uptake was only 0.52 (P < 0.05). Leucine uptake of LN-cr was largely (∼85%) Na+ -dependent. y+ LAT2 expression was confirmed in LN-cr. Knockdown of y+ LAT2 lead to significant leucine uptake inhibition (40%) and cell growth inhibition (20%). New CRPC cell line with increased expression of y+ LAT2 as a leucine transporter was established in vitro. Anti-leucine transporter therapy could be an important option against prostate cancer. Prostate 77:222-233, 2017. © 2016 Wiley Periodicals, Inc.

High-Throughput Assay Development for Cystine-Glutamate Antiporter (xc-) Highlights Faster Cystine Uptake than Glutamate Release in Glioma Cells.

The cystine-glutamate antiporter (system xc -) is a Na+-independent amino acid transporter that exchanges extracellular cystine for intracellular glutamate. It is thought to play a critical role in cellular redox processes through regulation of intracellular glutathione synthesis via cystine uptake. In gliomas, system xc - expression is universally up-regulated while that of glutamate transporters down-regulated, leading to a progressive accumulation of extracellular glutamate and excitotoxic cell death of the surrounding non-tumorous tissue. Additionally, up-regulation of system xc - in activated microglia has been implicated in the pathogenesis of several neurodegenerative disorders mediated by excess glutamate. Consequently, system xc - is a new drug target for brain cancer and neuroinflammatory diseases associated with excess extracellular glutamate. Unfortunately no potent and selective small molecule system xc - inhibitors exist and to our knowledge, no high throughput screening (HTS) assay has been developed to identify new scaffolds for inhibitor design. To develop such an assay, various neuronal and non-neuronal human cells were evaluated as sources of system xc -. Human glioma cells were chosen based on their high system xc - activity. Using these cells, [14C]-cystine uptake and cystine-induced glutamate release assays were characterized and optimized with respect to cystine and protein concentrations and time of incubation. A pilot screen of the LOPAC/NINDS libraries using glutamate release demonstrated that the logistics of the assay were in place but unfortunately, did not yield meaningful pharmacophores. A larger, HTS campaign using the 384-well cystine-induced glutamate release as primary assay and the 96-well 14C-cystine uptake as confirmatory assay is currently underway. Unexpectedly, we observed that the rate of cystine uptake was significantly faster than the rate of glutamate release in human glioma cells. This was in contrast to the same rates of cystine uptake and glutamate release previously reported in normal human fibroblast cells.

Abstract 5286: Expression of cancer type amino acid transporter LAT1 is a prognosis prediction factor in breast carcinoma: comparison between triple-negative and non-triple-negative types

Abstract Purpose: Amino acid transporters play important roles in cell survival and proliferation. L-Type amino acid transporter 1 (LAT1) is one of the major Na+-independent neutral amino acid transporters. LAT1 has an aspect of oncofetal protein. Recently, high levels of LAT1 expression have been reported in various tumors, such as prostate cancer, pancreas cancer, bile duct cancer, lung cancer and stomach cancer.We revealed LAT1 expression is an independent prognosis marker in stomach cancer, prostate cancer and pancreas cancer. The purpose of the present study was to investigate the LAT1 expression in breast carcinoma and relationship between LAT1 expression and prognosis. Materials &amp; Methods: We enrolled 290 patients with invasive breast carcinoma surgically resected between 2006 and 2010 at Kitasato University Hospital and Toho University Omori Medical Center, Japan. We investigated LAT1 expression in the enrolled breast carcinoma cases by immunohistochemical staining. All cases were divided into two groups, 78 cases of triple negative carcinoma (TNBC) and 212 cases of non-triple negative carcinoma (NTNBC), with immunohistochemistry of estrogen and progesterone receptors and Her2. After LAT1 expression was compared between TNBC and NTNBC, correlations among LAT1 expression, Ki-67 labeling index (LI), CD98 expression, and clinicopathological findings was assessed. Results: TNBC showed significantly higher LAT1 expression than NTNBC. TNBC also showed significantly higher CD98 expression rather than NTNBC. LAT1 expression of extraductal invasive cancer cells was significantly higher than those of intraductal carcinoma cells. A significantly positive correlation was observed between LAT1 expression and CD98 expression. Further, a positive correlation between LAT1 expression and Ki-67 LI was found. Log-rank test in overall breast cancers revealed that high LAT1 expression group showed significantly poor prognosis, compared to low LAT1 group. In NTNBC, high LAT1expression cases possessed poorer prognosis than low LAT1 expression cases. Conclusion: In invasive ductal carcinoma of breast, LAT1 expression has a potential for a prognosis prediction factor. LAT1 offers a potential target for anti-cancer therapy by its inhibitor JPH203 which has been developed by us. Citation Format: Masaaki Ichinoe, Tetsuo Mikami, Kiyomi Hana, Nobuyuki Yanagisawa, Hitoshi Endou, Isao Okayasu, Yoshiki Murakumo. Expression of cancer type amino acid transporter LAT1 is a prognosis prediction factor in breast carcinoma: comparison between triple-negative and non-triple-negative types. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5286. doi:10.1158/1538-7445.AM2015-5286

Abstract 204: Sodium Sensing Network in Hypertension-Induced Cardiac Hypertrophy

Hypertension is related to alterations of sodium homeostasis that promote abnormal accumulation of water in the intravascular compartment leading to blood pressure elevation. This process can been associated with increased heart mass resulting in cardiac hypertrophy. At cellular level, the concentration and active transport of Na+ is regulated by Na+,K+-ATPase enzyme (NK). In vitro studies have shown a correlation between high blood pressure, NK and SIK activities. Increases in intracellular Na+ are paralleled by elevations in intracellular Ca2+ via NCX1, leading to the activation of SIK1 by a Ca2+/calmodulin-dependent kinase. Activation of SIK1 results in the dephosphorylation of the NK α-subunit and an increase in its catalytic activity. Accordingly, the aim of the present study was to evaluate the cardiac Na+ sensing network changes associated with hypertension in aging hypertensive rats. Systolic and diastolic blood pressures, determined by the tail-cuff method, were significantly higher in 3, 13 and 21-month old SHR than in age-matched WKY. In the SHR, ageing was associated with increases in cardiac mass, gene expression of hypertrophic (α-skeletal muscle actin and β-myosin heavy chain) markers as well as inflammatory cytokine (IL-6). The decreased expression of heart SIK isoforms, SIK1 and SIK3, was associated with downregulation of transcription factors Snail2, Zeb1, MFAT5c and KLF4, in 13 and 21-month old SHR. Cardiac α2-isoform of NK was found to be decreased in 21 month old SHR. Whereas, NKα1, NHE3 and NCX1 showed no differences between age-matched SHR and WKY. Na+-dependent ASCT2 and Na+-independent LAT1 amino acid transporter transcript levels of were significantly higher in 21 month old SHR than in WKY. In the aged SHR, cardiac hypertrophy was associated with dysregulation of several elements of the intracellular Na+ sensing network. Downregulation of SIK1 and transcription factors is correlated with expression of NKα2, suggesting impaired sodium handling. It is proposed that, cardiac hypertrophy is not exclusively the consequence of mechanical stress but also of other factors associated with elevated blood pressure such as abnormal cell sodium homeostasis.Supported by grant PIC/IC/83204/2007.

Functional upregulation of system xc− by fibroblast growth factor-2

The cystine/glutamate antiporter (system xc−) is a Na +-independent amino acid transport system. Disruption of this system may lead to multiple effects in the CNS including decreased cellular glutathione. Since multiple neurological diseases involve glutathione depletion, and disruption of growth factor signaling has also been implicated in these diseases, it is possible that some growth factors effects are mediated by regulation of system xc−. We tested the growth factors fibroblast growth factor-2 (FGF-2), insulin-like growth factor-1 (IGF-1), neuregulin-1 (NRG), neurotrophin-4 (NT-4), and brain derived neurotrophic factor (BDNF) on system xc− mediated 14C-cystine uptake in mixed neuronal and glial cortical cultures. Only FGF-2 significantly increased cystine uptake. The effect was observed in astrocyte-enriched cultures, but not in cultures of neurons or microglia. The increase was blocked by the system xc− inhibitor (s)-4-carboxyphenylglycine, required at least 12 h FGF-2 treatment, and was prevented by the protein synthesis inhibitor cycloheximide. Kinetic analysis indicated FGF-2 treatment increased the V max for cystine uptake while the K m remained the same. Quantitative PCR showed an increase in mRNA for xCT, the functional subunit of system xc−, beginning at 3 h of FGF-2 treatment, with a dramatic increase after 12 h. Blocking FGFR1 with PD 166866 blocked the FGF-2 effect. Treatment with a PI3-kinase inhibitor (LY-294002) or a MEK/ERK inhibitor (U0126) for 1 h prior to and during the FGF-2 treatment, each partially blocked the increased cystine uptake. The upregulation of system xc− by FGF-2 may be responsible for some of the known physiological actions of FGF-2. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.

Role of the l-amino Acid Transporter-1 (LAT-1) in Mouse Trophoblast Cell Invasion

LAT-1 (L-type amino acid transporter 1) is a system L, Na+-independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and it's over expression is related to tumor invasion. LAT-1 is highly expressed in the trophoblast giant cells (TGCs) at the time of implantation. Since trophoblast giant cells are highly invasive during the process of endometrial implantation and placentation, LAT-1 may play a role in the invasive phenotype. Our objectives were to identify the effects of increased and decreased LAT-1 expression on mouse trophoblast invasion. We therefore examined the role of amino acid deprivation, pharmacologic blockade specific to leucine transport and gene silencing (siRNA) on LAT-1 expression and trophoblast cell invasion. We utilized mouse primary trophoblast stem (TS) cells. LAT-1 mRNA expression was quantified by real time qPCR, protein by Western blotting and cell invasion was measured in Transwell plates through Matrigel. Amino acid transport using uptake of tritiated leucine. Under limited leucine availability and/or pharmacologic blockage, LAT-1 gene expression was significantly increased, p<0.05. This was associated with a 3-fold increase in cell invasion, p<0.05. In contrast, following siRNA-mediated gene silencing decreased LAT-1 expression (both mRNA and protein) was associated with decreased cell invasion and decreased leucine uptake, p<0.05. Upregulation of LAT-1 gene expression via limited amino acid availability or following pharmacologic blockade of transport leads to an increase in mouse trophoblast stem cell invasiveness. Downregulation of LAT-1 expression via genetic silencing leads to inhibition of invasiveness. These results demonstrate that LAT-1 plays an important role in trophoblast invasion.

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.

Structure and mechanism of a Na+-independent amino acid transporter.

Amino acid, polyamine, and organocation (APC) transporters are secondary transporters that play essential roles in nutrient uptake, neurotransmitter recycling, ionic homeostasis, and regulation of cell volume. Here, we present the crystal structure of apo-ApcT, a proton-coupled broad-specificity amino acid transporter, at 2.35 angstrom resolution. The structure contains 12 transmembrane helices, with the first 10 consisting of an inverted structural repeat of 5 transmembrane helices like the leucine transporter LeuT. The ApcT structure reveals an inward-facing, apo state and an amine moiety of lysine-158 located in a position equivalent to the sodium ion site Na2 of LeuT. We propose that lysine-158 is central to proton-coupled transport and that the amine group serves the same functional role as the Na2 ion in LeuT, thus demonstrating common principles among proton- and sodium-coupled transporters.

LAT-1 Expression in Pre- and Post-implantation Embryos and Placenta

Objectives LAT-1 (L-type amino acid transporter 1) is a system L, Na +-independent amino acid transporter responsible for transport of large neutral amino acids. Dysregulated expression of LAT-1 is characteristic of many primary human cancers and is related to tumor invasion. Primary rat hepatocytes in culture increase LAT-1 mRNA in response to amino acid depletion. Transformed hepatic cell lines demonstrate constitutive expression of LAT-1. These observations suggest that LAT-1 expression confers a growth and survival advantage under limited amino acid availability. LAT-1 is highly expressed in the placenta. It has been shown previously that amino acids are fundamental regulators of cell function and energy metabolism in pre-implantation embryos. Our objectives were to analyze qualitatively and quantitatively LAT-1 expression in pre-implantation stages of mouse embryo development and to identify cell types expressing LAT-1 in post-implantation stages. Methods LAT-1 was quantified by real-time qPCR. Localization of expression was by laser capture microdissection, in situ hybridization and immunohistochemistry. Results Our results show increasing mRNA levels of LAT-1 as the embryo develops from zygote to blastocyst with highest levels at hatching blastocyst. Expression studies of LAT-1 on microdissected samples from developing mouse placenta show highest levels of LAT-1 mRNA in trophoblast giant cells (TGCs) at the time of implantation (E7.5), followed by maternal decidua, ectoplacental cone and epiblast. At later stages of development (E9.5 and E11.5) no differential expression of LAT-1 was observed. In situ hybridization and immunohistochemistry also showed differential expression of LAT-1 mRNA and protein, respectively, with darkest staining in TGCs at E7.5. By E9.5 and E11.5 mRNA expression was no longer preferentially localized to TGCs, hybridization was equal across the different cell types and regions. LAT-1 protein expression, however, still showed highest intensity of staining in TGCs at E9.5 and E11.5. Conclusions Since trophoblast giant cells are invasive cells that displace and phagocytose the uterine epithelial cells, these data suggest that LAT-1 may play a role in the invasive phenotype. The mechanism of LAT-1 regulation during placentation, therefore, might provide valuable clues to its role in tumor progression and invasion.

Evidence from gene knockout studies implicates Asc‐1 as the primary transporter mediating d‐serine reuptake in the mouse CNS

In the mammalian central nervous system, transporter-mediated reuptake may be critical for terminating the neurotransmitter action of D-serine at the strychnine insensitive glycine site of the NMDA receptor. The Na(+) independent amino acid transporter alanine-serine-cysteine transporter 1 (Asc-1) has been proposed to account for synaptosomal d-serine uptake by virtue of its high affinity for D-serine and widespread neuronal expression throughout the brain. Here, we sought to validate the contribution of Asc-1 to D-serine uptake in mouse brain synaptosomes using Asc-1 gene knockout (KO) mice. Total [(3)H]D-serine uptake in forebrain and cerebellar synaptosomes from Asc-1 knockout mice was reduced to 34 +/- 5% and 22 +/- 3% of that observed in wildtype (WT) mice, respectively. When the Na(+) dependent transport components were removed by omission of Na(+) ions in the assay buffer, D-serine uptake in knockout mice was reduced to 8 +/- 1% and 3 +/- 1% of that measured in wildtype mice in forebrain and cerebellum, respectively, suggesting Asc-1 plays a major role in the Na(+) independent transport of D-serine. Potency determination of D-serine uptake showed that Asc-1 mediated rapid high affinity Na(+) independent uptake with an IC(50) of 19 +/- 1 microm. The remaining uptake was mediated predominantly via a low affinity Na(+) dependent transporter with an IC(50) of 670 +/- 300 microm that we propose is the glial alanine-serine-cysteine transporter 2 (ASCT2) transporter. The results presented reveal that Asc-1 is the only high affinity D-serine transporter in the mouse CNS and is the predominant mechanism for D-serine reuptake.

Structure, tissue expression pattern, and function of the amino acid transporter rat PAT2

The second member of the PAT (proton-coupled amino acid transporter) family of H +-coupled, pH-dependent, Na +-independent amino acid transporters was isolated from a rat lung cDNA library. The cDNA for rat PAT2 is 2396 bp in length, including 70 bp of 5 ′UTR and a poly(A) tail. The transporter gene, consisting of 10 exons, is located on rat chromosome 10q22. The cDNA codes for a protein of 481 amino acids with 72% identity (over 449 amino acids) with rat PAT1. Tissue expression studies demonstrate that mRNA abundance is generally low with highest levels being detected in lung and spleen, with lower levels in the brain, heart, kidney, and skeletal muscle. Functional expression in either mammalian cells or Xenopus laevis oocytes demonstrates that rat PAT2 mediates pH-dependent, Na +-independent uptake of glycine, proline, and α(methyl)aminoisobutyric acid (MeAIB). In conclusion PAT2 has a limited tissue distribution, higher affinity (Michaelis–Menten constant for glycine uptake between 0.49 and 0.69 mM), and distinct substrate specificity compared to PAT1.

Regulation of L-cystine transport and intracellular GSH level by a nitric oxide donor in primary cultured rabbit conjunctival epithelial cell layers.

Metabolism and transport of cysteine are critical for maintenance of the intracellular glutathione (GSH) level. In this study, transport mechanisms of L-cystine and regulation of GSH biosynthesis in the absence or presence of NO-induced oxidant stress were investigated in primary cultured rabbit conjunctival epithelial cells (RCECs). RCECs were grown in membrane filters to exhibit tight barrier properties. Uptake and transepithelial transport of L-cystine were determined in the presence or absence of extracellular Na(+). Uptake was determined at 10 minutes after (14)C-L-cystine instillation into apical (a) or basolateral (b) bathing fluid. The effect of nitric oxide (NO) on L-cystine uptake, cellular GSH level, and expression level of two subunits of the rate-limiting enzyme gamma-glutamylcysteine synthetase (GCS) was examined after a 24-hour incubation of primary cultured RCECs with an NO donor, S-nitroso-N-acetylpenicillamine (SNAP; N-acetyl-3-(nitrosothio)-D-valine. Cellular uptake of L-cystine by RCECs occurred through both Na(+)-dependent and -independent mechanisms. Uptake from apical fluid was higher than that from basolateral fluid, except for the highest concentration of L-cystine tested (200 microM). Transepithelial permeability (P(app)) of L-cystine (at 2.5 microM) was three times higher in the a-to-b direction than in the b-to-a direction in the presence of Na(+), whereas the reverse was true in the absence of Na(+). Na(+)-dependent L-cystine uptake from apical fluid was significantly elevated in primary cultured RCECs treated for 24 hours with various concentrations (0.1-2.0 mM) of SNAP, with maximum uptake observed at 1 mM. A similar pattern of SNAP-induced increase of Na(+)-independent L-cystine uptake from apical fluid was observed, whereas no significant difference was observed for basolateral uptake. Concomitantly, a significant elevation of intracellular GSH (up to fivefold versus the control) was recorded, with the highest increase occurring at 0.1 to 0.25 mM SNAP. A parallel increase in the expression levels of both catalytic and regulatory subunits of GCS was observed by Western blot analysis of lysates from RCECs treated with 0.25 mM SNAP for 24 hours. L-Cystine is transported by both Na(+)-dependent and -independent amino acid transport systems in RCECs. At low substrate concentrations, L-cystine uptake was higher from apical than basolateral fluid. Permeability studies indicated net absorption of L-cystine across RCECs. SNAP caused significant increases in both L-cystine uptake and intracellular GSH level, which occurred concomitantly with elevation of both catalytic and regulatory subunits of GCS. Understanding sulfur amino acid precursor-dependent cellular mechanisms of GSH homeostasis would be of value in devising GSH-based treatment for conjunctival or other ocular disorders.

Epithelial cells isolated from chicken jejunum: an experimental model for the study of the functional properties of amino acid transport system b 0,+

The transport of lysine has been investigated in epithelial cells isolated from chicken jejunum. The kinetics of lysine transport and the pattern of interaction with zwitterionic amino acids were consistent with system b 0,+ activity, the broad-spectrum and Na +-independent amino acid transporter. The half-saturation constant for lysine entry ( K m±S.E.) was 0.029±0.002 mM and the flux was not affected significantly by Na + replacement with choline. Lysine influx was inhibited by l-leucine both in Na + and choline medium with inhibition constants ( K i±S.E.) 0.068±0.006 mM (in Na +) and 0.065±0.009 mM (in choline). Other inhibitory amino acids ( K i±S.E.) were (mM): l-tyrosine (0.073±0.018), l-methionine (0.15±0.015), l-cystine (0.42±0.04), l-cysteine (1.1±0.07), l-isoleucine (1.1±0.09), l-glutamine (1.8±0.16) and l-valine (2.5±0.13). Lysine exit was trans-accelerated (approx. 20 fold) by 2 mM l-lysine and l-leucine. The flux was resistant to pretreatment of the cells with p-chloromercuriphenylsulfonate (0.2 mM), which is an inhibitor of system y +L, the broad-spectrum and cation-modulated transporter.

The heterodimeric amino acid transporter 4F2hc/LAT1 is associated in Xenopus oocytes with a non-selective cation channel that is regulated by the serine/threonine kinase sgk-1.

System L is the major Na(+)-independent amino acid transporter of mammalian cells. It is constituted of the type II membrane protein 4F2hc (CD98) which is covalently linked to the polytopic membrane protein LAT1 via a disulfide bridge. The transporter is known to be regulated by the mineral corticoid aldosterone in Xenopus A6 cells. To understand the regulation of the transporter, the 4F2hc/LAT1 heterodimer was functionally expressed in Xenopus laevis oocytes and its transport properties were analysed using flux measurements and the two-electrode voltage-clamp technique. Expression of 4F2hc/LAT1 resulted in a rapid increase in a Na(+)-independent neutral amino acid antiport activity and simultaneously gave rise to a cation conductance. The cation channel was non-rectifying and non-selective, conducting Li(+) > Cs(+) = Na(+) > K(+). After replacement of Na(+) by NMDG, however, the currents were suppressed almost completely. The cation channel was not inhibited by amiloride, Ba2(+), TEA, Hoe293B, flufenamic acid or substrates of the system L amino acid transporter. Significant inhibition, however, was observed in the presence of La3(+), Gd3(+) and quinidine. Channel activity was upregulated by coexpression of 4F2hc/LAT1 with the aldosterone-regulated protein kinase sgk-1. The cation conductance was sensitive to changes in the redox potential, being inhibited following incubation of the oocytes with DTE for 30 min. Mutation of either of the disulfide bridge-constituting cysteines to serine resulted in a loss of ion channel activity whereas amino acid transport was unaffected. It is concluded that the 4F2hc/LAT1 heterodimer regulates a closely associated cation channel or even constitutes a cation channel itself.

Transport of N(G)-nitro-L-arginine across intestinal brush border membranes by Na+ -dependent and Na+-independent amino acid transporters.

To clarify the transport mechanism of NG-nitro-L-arginine (L-NNA), a potent NO-synthase inhibitor, across intestinal brush border membranes (BBM). Dog intestinal BBM vesicles were used. The time course of L-NNA uptake showed a Na+ -dependent overshoot phenomenon. Concentration-dependence curves of L-NNA initial uptake were saturable in the presence and absence of Na+, indicating participation of Na+ -dependent and Na+ -independent carrier-mediated transport systems. The calculated kinetic parameters of L-NNA initial uptake indicate that the former is a low-affinity high-capacity system and the latter is a high-affinity low-capacity one, similar to those in neutral amino acid transport. Neutral and basic amino acids showed cis-inhibitory and trans-stimulatory effects on L-NNA uptake in the presence or absence of Na+. N(G)-Nitro-L-arginine methyl ester, another potent NO-synthase inhibitor, also had both effects, which were smaller than with amino acids. The present study clearly indicates that transport of L-NNA across the intestinal BBM occurs in the same manner as neutral amino acid transport. However, it is affected by both neutral and basic amino acids in the presence or absence of Na+ differently from that across plasma membranes of nonepithelial cells, because B0,+ and b0,+ amino acid transporters function partly in L-NNA transport across intestinal BBM.

18] Cut-open recording techniques

Publisher Summary This chapter describes a new technique for voltage-clamp recording of Xenopus oocytes that enables fast and stable monitoring of transmembrane current, as well as intracellular perfusion by cutting a part of the plasma membrane to open the cytoplasm to the recording chamber, which is filled with an artificial intracellular solution. The original method was developed and described by E. Stefani and colleagues, and this chapter has made a modification by exchanging intracellular solution using a push-pull cannula. This technique has certain characteristic features. High-frequency response and relatively low current noise is the first feature. Fast activation and deactivation of ionic currents can be precisely evaluated. Stable recording conditions that lasts for several hours is a marked advantage over the conventional and patch-clamp recording. Control of the ionic composition of both the internal and external media is another feature. Although it is possible to study the action of compounds on the internal surface of oocytes using inside-out patches or by injecting drugs under a whole-cell clamp, the cut-open configuration enables the desired manipulation of the internal milieu of oocytes easily. These features allow reliable measurements of gating currents and fast ionic currents of Shaker K + channels in single and unsubtracted traces. Moreover, the easy access to the intracellular milieu is suitable for the study of channel modulation by second messengers and drugs. This method has been applied for the studies of transient K + channels, rapid delayed rectifier K + channels, voltage-dependent Ca 2+ channels, Na + -glucose co-transporters, and Na + -independent amino acid transporters.

Consideration of the analgesic efficacy of gabapentin

Gabapentin (1-[aminomethyl]-cyclohexaneacetic acid; Neurontin, Park-Davis, Morris Plains, NJ) is a new anticonvulsant approved by the Food and Drug Administration for adults in the treatment of partial seizures, with or without secondary generalization. Gabapentin has been on the market since 1994, and it demonstrates good anticonvulsant efficacy and extremely low toxicity. The mechanisms of action of gabapentin remain unclear, although it binds to a specific receptor in the brain, inhibits voltage-dependent sodium (Na) currents, interacts with a central nervous system L-type Na+-independent amino acid transporter, and modulates several neurotransmitters actions and release. Other actions of gabapentin have been tested in different neurologic and psychiatric disorders. Gabapentins analgesic properties have been known since 1995. In this chapter, we review some of the known and documented mechanisms of action of gabapentin, targeting the drugs possible efficacy in the treatment of acute and chronic pain states of nociceptive and neurogenic origin.