The solute carrier SLC7A1 may act as a protein transporter at the blood-brain barrier.

Despite extensive research, targeted delivery of substances to the brain still poses a great challenge due to the selectivity of the blood-brain barrier (BBB). Most molecules require either carrier- or receptor-mediated transport systems to reach the central nervous system (CNS). These transport systems form attractive routes for the delivery of therapeutics into the CNS, yet the number of known brain endothelium-enriched receptors allowing the transport of large molecules into the brain is scarce. Therefore, to identify novel BBB targets, we combined transcriptomic analysis of human and murine brain endothelium and performed a complex screening of BBB-enriched genes according to established selection criteria. As a result, we propose the high-affinity cationic amino acid transporter 1 (SLC7A1) as a novel candidate for transport of large molecules across the BBB. Using RNA sequencing and in situ hybridization assays, we demonstrated elevated SLC7A1 gene expression in both human and mouse brain endothelium. Moreover, we confirmed SLC7A1 protein expression in brain vasculature of both young and aged mice. To assess the potential of SLC7A1 as a transporter for larger proteins, we performed internalization and transcytosis studies using a radiolabelled or fluorophore-labelled anti-SLC7A1 antibody. Our results showed that SLC7A1 internalised a SLC7A1-specific antibody in human colorectal carcinoma (HCT116) cells. Moreover, transcytosis studies in both immortalised human brain endothelial (hCMEC/D3) cells and primary mouse brain endothelial cells clearly demonstrated that SLC7A1 effectively transported the SLC7A1-specific antibody from luminal to abluminal side. Therefore, here in this study, we present for the first time the SLC7A1 as a novel candidate for transport of larger molecules across the BBB.

Research progress on the role of cationic amino acid transporter (CAT) family members in malignant tumors and immune microenvironment.

Amino acids are essential for the survival of all living organisms and living cells. Amino acid transporters mediate the transport and absorption of amino acids, and the dysfunction of these proteins can induce human diseases. Cationic amino acid transporters (CAT family, SLC7A1-4, and SLC7A14) are considered to be a group of transmembrane transporters, of which SLC7A1-3 are essential for arginine transport in mammals. Numerous studies have shown that CAT family-mediated arginine transport is involved in signal crosstalk between malignant tumor cells and immune cells, especially T cells. The modulation of extracellular arginine concentration has entered a number of clinical trials and achieved certain therapeutic effects. Here, we review the role of CAT family on tumor cells and immune infiltrating cells in malignant tumors and explore the therapeutic strategies to interfere with extracellular arginine concentration, to elaborate its application prospects. CAT family members may be used as biomarkers for certain cancer entities and might be included in new ideas for immunotherapy of malignant tumors.

Arginine transportation mechanism through cationic amino acid transporter 1: insights from molecular dynamics studies.

Metabolic and signaling mechanisms in mammalian cells are facilitated by the transportation of L-arginine (Arg) across the plasma membrane through cationic amino acid transporter (CAT) proteins. Due to a lack of argininosuccinate synthase (ASS) activity in various tumor cells such as acute myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, these tumor entities are arginine-auxotrophic and therefore depend on the uptake of the amino acid arginine. Cationic amino acid transporter-1 (CAT-1) is the leading arginine importer expressed in the aforementioned tumor entities. Hence, in the present study, to investigate the transportation mechanism of arginine in CAT-1, we performed molecular dynamics (MD) simulation methods on the modeled human CAT-1. The MM-PBSA approach was conducted to determine the critical residues interacting with arginine within the corresponding binding site of CAT-1. In addition, we found out that the water molecules have the leading role in forming the transportation channel within CAT-1. The conductive structure of CAT-1 was formed only when the water molecules were continuously distributed across the channel. Steered molecular dynamics (SMD) simulation approach showed various energy barriers against arginine transportation through CAT-1, especially while crossing the bottlenecks of the related channel. These findings at the molecular level might shed light on identifying the crucial amino acids in the binding of arginine to eukaryotic CATs and also provide fundamental insights into the arginine transportation mechanisms through CAT-1. Understanding the transportation mechanism of arginine is essential to developing CAT-1 blockers, which can be potential medications for some types of cancers.Communicated by Ramaswamy H. Sarma.

Cationic amino acid transporter-1 (CAT-1) promotes fibroblast-like synoviocyte proliferation and cytokine secretion by taking up L-arginine in rheumatoid arthritis.

BACKGROUND: Abnormal proliferation of fibroblast-like synoviocytes (FLSs) in the synovial lining layer is the primary cause of synovial hyperplasia and joint destruction in rheumatoid arthritis (RA). Currently, the relationship between metabolic abnormalities and FLS proliferation is a new focus of investigation. However, little is known regarding the relationship between amino acid metabolism and RA. METHODS: The concentrations of amino acids and cytokines in the synovial fluid of RA (n = 9) and osteoarthritis (OA, n = 9) were detected by LC-MS/MS and CBA assay, respectively. The mRNA and protein expression of cationic amino acid transporter-1 (CAT-1) were determined in FLSs isolated from RA and OA patients by real-time PCR and western blotting. MTT assay, cell cycle, apoptosis, invasion, and cytokine secretion were determined in FLSs knocked down of CAT-1 using siRNA or treated with D-arginine under normoxic and hypoxic culture conditions. A mouse collagen-induced arthritis (CIA) model was applied to test the therapeutic potential of blocking the uptake of L-arginine in vivo. RESULTS: L-rginine was upregulated in the synovial fluid of RA patients and was positively correlated with the elevation of the cytokines IL-1ß, IL-6, and IL-8. Further examination demonstrated that CAT-1 was the primary transporter for L-arginine and was overexpressed on RA FLSs compared to OA FLSs. Moreover, knockdown of CAT-1 using siRNA or inhibition of L-arginine uptake using D-arginine significantly suppressed L-arginine metabolism, cell proliferation, migration, and cytokine secretion in RA FLSs under normoxic and hypoxic culture conditions in vitro but increased cell apoptosis in a dose-dependent manner. Meanwhile, in vivo assays revealed that an L-arginine-free diet or blocking the uptake of L-arginine using D-arginine suppressed arthritis progression in CIA mice. CONCLUSION: CAT-1 is upregulated and promotes FLS proliferation by taking up L-arginine, thereby promoting RA progression.

Carnosine Synthase 1 Contributes to Interferon Gamma-Induced Arginine Depletion via Mitogen-activated Protein Kinase 11 Signaling in Bovine Mammary Epithelial Cells.

Arginine is one of the host semiessential amino acids with diverse biological activities, and arginine depletion is associated with the incidence of many diseases. Arginine depletion induced by diet-derived interferon gamma (IFN-γ) leads to malignant transformation and impaired milk quality in healthy lactating bovine mammary epithelial cells (BMECs). However, the molecular mechanism of IFN-γ-induced arginine depletion is unclear. In this study, the BMEC cell line, mammary alveolar cells-large T antigen cells (MAC-T), was stimulated with IFN-γ (10 ng/mL) for 24 h, and cellular arginine and ornithine quantified by liquid chromatography-tandem mass spectrometry. Carnosine synthase 1 (CARNS1) was identified from RNA-seq data, CARNS1 knockdown was achieved using an shRNA interfering plasmid. The expression levels of CARNS1, argininosuccinate synthetase 1 (ASS1), mitogen-activated protein kinase 11 (p38 MAPK), and phosphorylated (p)-p38, and their cognate genes, were analyzed by Western blotting and real-time quantitative polymerase chain reaction. The results showed that IFN-γ inhibited the biosynthesis of arginine, but enhanced its catalysis via disruption of key enzymes involved in arginine metabolism. IFN-γ also inhibited the expression of CARNS1, ASS1, and cationic amino acid transporter 1, while activating the expression and phosphorylation of p38. However, knockdown of CARNS1 reduced arginine level and ASS1 expression and block of either the IFN-γ receptor IFN-γ receptor 2 or p38 relieved both the expression of Carnosine synthase 1 (CARNS1) and ASS1. In summary, these results indicate that IFN-γ induced arginine depletion through inhibition of CARNS1 signaling via activation of p38 in BMECs. These findings provide a novel insight for IFN-γ-related disease control strategies in dairy cows.

The activity of IKCa and BKCa channels contributes to insulin-mediated NO synthesis and vascular tone regulation in human umbilical vein.

Insulin regulates the l-arginine/nitric oxide (NO) pathway in human umbilical vein endothelial cells (HUVECs), increasing the plasma membrane expression of the l-arginine transporter hCAT-1 and inducing vasodilation in umbilical and placental veins. Placental vascular relaxation induced by insulin is dependent of large conductance calcium-activated potassium channels (BKCa), but the role of KCa channels on l-arginine transport and NO synthesis is still unknown. The aim of this study was to determine the contribution of KCa channels in both insulin-induced l-arginine transport and NO synthesis, and its relationship with placental vascular relaxation. HUVECs, human placental vein endothelial cells (HPVECs) and placental veins were freshly isolated from umbilical cords and placenta from normal pregnancies. Cells or tissue were incubated in absence or presence of insulin and/or tetraethylammonium, 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole, iberiotoxin or NG-nitro-l-arginine methyl ester. l-Arginine uptake, plasma membrane polarity, NO levels, hCAT-1 expression and placenta vascular reactivity were analyzed. The inhibition of intermediate-conductance KCa (IKCa) and BKCa increases l-arginine uptake, which was related with protein abundance of hCAT-1 in HUVECs. IKCa and BKCa activities contribute to NO-synthesis induced by insulin but are not directly involved in insulin-stimulated l-arginine uptake. Long term incubation (8 h) with insulin increases the plasma membrane hyperpolarization and hCAT-1 expression in HUVECs and HPVECs. Insulin-induced relaxation in placental vasculature was reversed by KCa inhibition. The results show that the activity of IKCa and BKCa channels are relevant for both physiological regulations of NO synthesis and vascular tone regulation in the human placenta, acting as a part of negative feedback mechanism for autoregulation of l-arginine transport in HUVECs.

Human cationic amino acid transporters are not affected by direct nitros(yl)ation.

A direct inhibiting effect of NO on the function of CAT-1 and -2A has been postulated to occur via nitrosylation of cysteine residues in the transporters. Neither the NO donor SNAP nor a mixture of SIN-1 and Spermine NONOate, that generates the strong nitrosating agent N2O3, reduced CAT-mediated L-arginine transport. Direct nitros(yl)ation does either not occur in CATs or does not affect their transport function. A regulatory effect of NO or nitrosating agents on CAT-mediated transport under physiological conditions seems, therefore, unlikely.

Role of insulin, adenosine, and adipokine receptors in the foetoplacental vascular dysfunction in gestational diabetes mellitus.

Gestational diabetes mellitus (GDM) is a disease of pregnancy associated with maternal and foetal hyperglycaemia and altered foetoplacental vascular function. Human foetoplacental microvascular and macrovascular endothelium from GDM pregnancy show increased maximal l-arginine transport capacity via the human cationic amino acid transporter 1 (hCAT-1) isoform and nitric oxide (NO) synthesis by the endothelial NO synthase (eNOS). These alterations are paralleled by lower maximal transport activity of the endogenous nucleoside adenosine via the human equilibrative nucleoside transporter 1 (hENT1) and activation of adenosine receptors. A causal relationship has been described for adenosine-activation of A2A adenosine receptors, hCAT-1, and eNOS activity (i.e. the Adenosine/l-Arginine/Nitric Oxide, ALANO, signalling pathway). Insulin restores these alterations in GDM via activation of insulin receptor A (IR-A) form in the macrovascular but IR-A and IR-B forms in the microcirculation of the human placenta. Adipokines are secreted from adipocytes influencing the foetoplacental metabolic and vascular function. Various adipokines are dysregulated in GDM, with adiponectin and leptin playing major roles. Abnormal plasma concentration of these adipokines and the activation or their receptors are involved in the pathophysiology of GDM. However, involvement of adipokines, adenosine, and insulin receptors and membrane transporters in the aetiology of this disease of pregnancy is unknown. This review focuses on the pathophysiology of insulin and adenosine receptors and l-arginine and adenosine membranes transporters giving an overview of the key adipokines leptin and adiponectin in the foetoplacental vasculature in GDM. This article is part of a Special Issue entitled: Membrane Transporters and Receptors in Pregnancy Metabolic Complications edited by Luis Sobrevia.

CAT1/SLC7A1 acts as a cellular receptor for bovine leukemia virus infection.

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle, which is closely related to human T-cell leukemia viruses. BLV has spread worldwide and causes a serious problem for the cattle industry. The cellular receptor specifically binds with viral envelope glycoprotein (Env), and this attachment mediates cell fusion to lead virus entry. BLV Env reportedly binds to cationic amino acid transporter 1 (CAT1)/solute carrier family 7 member 1 (SLC7A1), but whether the CAT1/SLC7A1 is an actual receptor for BLV remains unknown. Here, we showed that CAT1 functioned as an infection receptor, interacting with BLV particles. Cells expressing undetectable CAT1 levels were resistant to BLV infection but became highly susceptible upon CAT1 overexpression. CAT1 exhibited specific binding to BLV particles on the cell surface and colocalized with the Env in endomembrane compartments and membrane. Knockdown of CAT1 in permissive cells significantly reduced binding to BLV particles and BLV infection. Expression of CAT1 from various species demonstrated no species specificity for BLV infection, implicating CAT1 as a functional BLV receptor responsible for its broad host range. These findings provide insights for BLV infection and for developing new strategies for treating BLV and preventing its spread.-Bai, L., Sato, H., Kubo, Y., Wada, S., Aida, Y. CAT1/SLC7A1 acts as a cellular receptor for bovine leukemia virus infection.

Cytoplasmic R-peptide of murine leukemia virus envelope protein negatively regulates its interaction with the cell surface receptor.

Cytoplasmic tails of envelope (Env) glycoproteins of many retroviruses inhibit their membrane fusion activity. The cytoplasmic 16-amino acid peptide of ecotropic murine leukemia virus (E-MLV) Env protein, called the R-peptide, also inhibits the membrane fusion activity of the Env protein. However, the molecular mechanism of the inhibition has not been elucidated yet. In this study, we found that R-peptide-containing Env protein of E-MLV binds to the cell surface receptor cationic amino acid transporter-1 (CAT-1) with weaker affinity than R-peptide-truncated Env protein. Consistent with this result, R-peptide-containing Env protein had less efficient inhibition of E-MLV vector infection than R-peptide-truncated Env protein. R-peptide truncation has been reported to induce conformational change in the surface subunit of E-MLV Env protein that interacts with the receptor. Taken together, our findings indicate that R-peptide truncation induces conformational change in the receptor-binding domain of the E-MLV Env protein and facilitates the Env-receptor interaction.

Profound Decrease in Glomerular Arginine Transport by CAT (Cationic Amino Acid Transporter)-1 Contributes to the FLT-1 (FMS-Like Tyrosine Kinase 1) Induced Preeclampsia in the Pregnant Mice.

Endothelial dysfunction because of nitric oxide inactivation has been suggested to play a role in the pathogenesis of preeclampsia. During pregnancy, L-arginine transport by CAT-1 (cationic amino acid transporter 1), the only transporter for eNOS (endothelial nitric oxide synthase) is inhibited. We hypothesize that maternal arginine deficiency contributes to the development of preeclampsia. Adenovirus-mediated overexpression of sFlt-1 (soluble fms-like tyrosine kinase 1) in virgin and pregnant mice resulted in glomerular endotheliosis, hypertension, and albuminuria. L-arginine prevented the increase in blood pressure and albuminuria in Flt-1 pregnant but not in Flt-1 virgin mice. Flt-1 augmented arginine transport in pregnant but not in virgin dames. Ex vivo inhibition of CAT-2 leaving exclusively CAT-1 activity, decreased arginine transport velocities in Flt-1 animals more prominently in pregnant dames. Phosphorylated CAT-1/CAT-1 increased in pregnant, sFlt-1-pregnant, and sFlt-1 virgin mice. CAT-2 increased in Flt-1-pregnant and Flt-1-virgin dames. L-arginine augmented arginine transport in pregnant and Flt-pregnant mice and prevented the increase in pCAT-1 and CAT-2 expression. Glomerular cGMP (cyclic guanosine monophosphate) generation as a measure of eNOS activity was decreased in all Flt-1 treated animals. L-arginine abolished the decrease in cGMP levels only in Flt-1-pregnant mice. In conclusion, glomerular endothelial NO generation is compromised in Flt-1-pregnant mice because of CAT-1 inhibition induced by a combined effect of pregnancy and preeclampsia which involves: phosphorylation of CAT-1 and induction of CAT-2. These processes contribute to the clinical syndrome of preeclampsia in mice and are prevented by L-arginine.

DCAF1 is involved in HCV replication through regulation of miR-122.

Hepatitis C virus (HCV) is a worldwide threaten to human health with a high ratio of chronic infections. Recently, we found that Vpr-mediated regulation of HCV replication depends on the host protein DDB1-Cul4 associate factor 1 (DCAF1), implying that DCAF1 might be involved in the replication of HCV. In this study, we demonstrated that DCAF1 knockdown reduced HCV replication both in the infectious (JFH1) and replicon (Con1) systems. Further investigation showed a negative regulation of HCV internal ribosome entry site (IRES)-mediated translation by DCAF1. Considering the positive effects on the replication of the HCV replicon, we speculated that DCAF1 affected the balance between HCV RNA replication and protein translation. Since miR-122 is involved in the regulation of this balance, we investigated the influence of DCAF1 on miR-122 expression. By measuring the expression of miR-122, pre-miR-122 and its target CAT-1 mRNA, we found that miR-122 was downregulated following DCAF1 knockdown. Furthermore, overexpression of miR-122 rescued HCV replication impairment induced by DCAF1 knockdown. In conclusion, our study suggests that DCAF1 is involved in HCV replication through regulation of miR-122 and thus provides new insights into the interaction between HCV and the host cell.

Developmental changes of l-arginine transport at the blood-brain barrier in rats.

l-Arginine is required for regulating synapse formation/patterning and angiogenesis in the developing brain. We hypothesized that this requirement would be met by increased transporter-mediated supply across the blood-brain barrier (BBB). Thus, the purpose of this work was to test the idea that elevation of blood-to-brain l-arginine transport across the BBB in the postnatal period coincides with up-regulation of cationic acid transporter 1 (CAT1) expression in developing brain capillaries. We found that the apparent brain-to-plasma concentration ratio (Kp, app) of l-arginine after intravenous administration during the first and second postnatal weeks was 2-fold greater than that at the adult stage. Kp, app of l-serine was also increased at the first postnatal week. In contrast, Kp, app of d-mannitol, a passively BBB-permeable molecule, did not change, indicating that increased transport of l-arginine and l-serine is not due to BBB immaturity. Double immunohistochemical staining of CAT1 and a marker protein, glucose transporter 1, revealed that CAT1 was localized on both luminal and abluminal membranes of brain capillary endothelial cells during the developmental and adult stages. A dramatic increase in CAT1 expression in the brain was seen at postnatal day 7 (P7) and day 14 (P14) and the expression subsequently decreased as the brain matured. In accordance with this, intense immunostaining of CAT1 was observed in brain capillaries at P7 and P14. These findings strongly support our hypothesis and suggest that the supply of blood-born l-arginine to the brain via CAT1 at the BBB plays a key role in meeting the elevated demand for l-arginine in postnatal brain.

Maternal insulin therapy does not restore foetoplacental endothelial dysfunction in gestational diabetes mellitus.

Pregnant women diagnosed with gestational diabetes mellitus subjected to diet (GDMd) that do not reach normal glycaemia are passed to insulin therapy (GDMi). GDMd associates with increased human cationic amino acid transporter 1 (hCAT-1)-mediated transport of L-arginine and nitric oxide synthase (NOS) activity in foetoplacental vasculature, a phenomenon reversed by exogenous insulin. Whether insulin therapy results in reversal of the GDMd effect on the foetoplacental vasculature is unknown. We assayed whether insulin therapy normalizes GDMd-associated foetoplacental endothelial dysfunction. Primary cultures of human umbilical vein endothelial cells (HUVECs) from GDMi pregnancies were used to assay L-arginine transport kinetics, NOS activity, p44/42mapk and protein kinase B/Akt activation, and umbilical vein rings reactivity. HUVECs from GDMi or GDMd show increased hCAT-1 expression and maximal transport capacity, NOS activity, and eNOS, and p44/42mapk, but not Akt activator phosphorylation. Dilation in response to insulin or calcitonin-gene related peptide was impaired in umbilical vein rings from GDMi and GDMd pregnancies. Incubation of HUVECs in vitro with insulin (1 nmol/L) restored hCAT-1 and eNOS expression and activity, and eNOS and p44/42mapk activator phosphorylation. Thus, maternal insulin therapy does not seem to reverse GDMd-associated alterations in human foetoplacental vasculature.

Minimal dose of milk protein concentrate to enhance the anabolic signalling response to a single bout of resistance exercise; a randomised controlled trial.

BACKGROUND: Resistance training is a potent stimulus to induce muscle hypertrophy. Supplemental protein intake is known to enhance gains in muscle mass through activation of the mammalian target of rapamycin complex 1 (mTORC1) pathway, which initiates protein translation. While the optimal dose of high quality protein to promote post exercise anabolism in young or older men has been investigated, little is known about the minimum doses of protein required to potentiate the resistance exercise activation of anabolic signalling in middle aged men. METHODS: Twenty healthy men (46.3 ± 5.7 years, BMI: 23.9 ± 6.6 kg/m2) completed a single bout of unilateral resistance exercise consisting of 4 sets of leg extension and press at 80% of 1 repetition maximum. Participants were randomised to consume either formulated milk product containing 9 g milk protein (FMP) or an isoenergetic carbohydrate placebo (CHO) immediately post exercise, in a double blind fashion. A single muscle biopsy was collected at pre-exercise baseline and then bilateral biopsies were collected 90 and 240 min after beverage consumption. RESULTS: P70S6KThr389 phosphorylation was increased with exercise irrespective of group, P70S6KThr421/Ser424 was increased with exercise only in the FMP group at 240 min. Likewise, rpS6 Ser235/236 phosphorylation was increased with exercise irrespective of group, rpS6 Ser240/244 increased to a greater extent following exercise in the FMP group. mRNA expression of the amino acid transporter, LAT1/ SLC7A5 increased with both exercise and beverage consumption irrespective of group. PAT1/ SLC36A1, CAT1/ SLC7A1 and SNAT2/ SLC38A2 mRNA increased only after exercise regardless of group. CONCLUSIONS: Nine grams of milk protein is sufficient to augment some measures of downstream mTORC1 signalling after resistance exercise but does not potentiate exercise induced increases in amino acid transporter expression. Formulated products containing nine grams of milk protein would be expected stimulate muscle anabolism after resistance exercise. TRIAL REGISTRATION: New Zealand Clinical Trials Registry ACTRN12615001375549. Registered: 17 December, 2015.

Vascular Endothelial Growth Factor Augments Arginine Transport and Nitric Oxide Generation via a KDR Receptor Signaling Pathway.

BACKGROUND/AIMS: Vascular endothelial growth factor (VEGF) is an endothelium-specific peptide that stimulates angiogenesis via two receptor tyrosine kinases, Flt-1 and KDR. Endothelial nitric oxide synthase (eNOS) plays a major role in VEGF signaling. Delivery of arginine to membrane bound eNOS by the cationic amino acid transporter-1 (CAT-1) has been shown to modulate eNOS activity. The current studies were designed to test the hypothesis that VEGF enhances eNOS activity via modulation of arginine transport by CAT-1. METHODS: Using radio-labeled arginine, {[3H] L-arginine} uptake was determined in human umbilical vein endothelial cells (HUVEC) following incubation with VEGF with and without silencing the VEGF receptors Flt-1 or KDR. Subsequently, western blotting for CAT-1, PKCα, ERK 1/2, JNK, and their phosphorylated forms were performed. NO generation was measured by the Griess reaction. RESULTS: VEGF (50 and 100 ng/ml) significantly augmented endothelial arginine transport in a time dependent manner, an effect which was prevented by Sunitinib (2 µM), a multi targeted receptor tyrosine kinase inhibitor. The increase in arginine transport velocities by VEGF was not affected by silencing Flt-1 while silencing KDR abrogated VEGF effect. Furthermore, incubating cells with 50 and 100 ng of VEGF for 30 minutes significantly augmented CAT-1 abundance. The expression of PKC-α, JNK, and ERK1/2 and their phosphorylated forms were unchanged following incubation of HUVEC with VEGF. The concentration of NO2/NO3 following incubation with VEGF was significantly higher than from untreated cells. This increase was significantly attenuated by silencing KDR. CONCLUSIONS: VEGF increases arginine transport via modulation of CAT-1 in endothelial cells. This effect is exclusively dependent on KDR rather than Flt-1.

L-arginine attenuates Interleukin-1ß (IL-1ß) induced Nuclear Factor Kappa-Beta (NF-κB) activation in Caco-2 cells.

BACKGROUND: Specific nutrients like L-arginine (L-Arg) ameliorate intestinal inflammation, however the exact mechanisms of this effect are unclear. We hypothesized the anti-inflammatory effects of L-Arg require active transport and metabolism by inducible nitric oxide synthase (iNOS) to generate nitric oxide (NO). To test this hypothesis we examined the effects of L-Arg, L-Arg transport activity, NO production and iNOS inhibitor on IL-1ß-mediated NF-κB-activation in Caco-2 cells. METHODS: Caco-2 cells were cultured, transfected with a NF-κB promoter luciferase vector, incubated ± L-Arg, ± IL-1ß and luciferase activity was measured. Using siRNA we inhibited the L-Arg cationic amino acid transporter system y+ (CAT1) expression and examined its effects on L-Arg transport activity and IL-1ß-mediated NF-κB-activation. Finally, the effects of sodium nitroprusside (SNP, a NO donor) and Nω-nitro-L-arginine (NNA, an iNOS inhibitor) on IL-1ß-mediated NF-κB-activation were examined. RESULTS: IL-1ß increased NF-κB luciferase activity (8-fold) and NF-κB expression (mRNA and protein), both of these were significantly decreased by L-Arg. System y+ CAT1 siRNA decreased CAT1 expression, L-Arg transport activity and attenuated the inhibitory effects of L-Arg on NF- κB activity. SNP attenuated the IL-1ß-induced increase in NF-κB luciferase activity and expression, whereas NNA diminished the inhibitory effects of L-Arg on IL-1ß-inducible NF- κB luciferase activity. CONCLUSION: The inhibitory effects of L-Arg on IL-1ß-mediated NF-κB-activation in Caco-2 cells involve L-Arg transport activity by CAT1, regulation of IL-1ß-mediated increases in NF-κB expression, changes in iNOS expression and NO production. Our data suggest the inhibitory effects of L-Arg on NF-κB activation are mediated in part by iNOS since SNP preserves and NNA attenuates the effects of L-Arg on IL-1ß-mediated NF-κB-activation and expression.

The transporter and permeability interactions of asymmetric dimethylarginine (ADMA) and L-arginine with the human blood-brain barrier in vitro.

The blood-brain barrier (BBB) is a biological firewall that carefully regulates the cerebral microenvironment by acting as a physical, metabolic and transport barrier. This selectively permeable interface was modelled using the immortalised human cerebral microvascular endothelial cell line (hCMEC/D3) to investigate interactions with the cationic amino acid (CAA) L-arginine, the precursor for nitric oxide (NO), and with asymmetric dimethylarginine (ADMA), an endogenously derived analogue of L-arginine that potently inhibits NO production. The transport mechanisms utilised by L-arginine are known but they are not fully understood for ADMA, particularly at the BBB. This is of clinical significance giving the emerging role of ADMA in many brain and cerebrovascular diseases and its potential as a therapeutic target. We discovered that high concentrations of ADMA could induce endothelial dysfunction in the hCMEC/D3s BBB permeability model, leading to an increase in paracellular permeability to the paracellular marker FITC-dextran (40kDa). We also investigated interactions of ADMA with a variety of transport mechanisms, comparing the data with L-arginine interactions. Both molecules are able to utilise the CAA transport system y(+). Furthermore, the expression of CAT-1, the best known protein from this group, was confirmed in the hCMEC/D3s. It is likely that influx systems, such as y(+)L and b(0,+), have an important physiological role in ADMA transport at the BBB. These data are not only important with regards to the brain, but apply to other microvascular endothelia where ADMA is a major area of investigation.

Induced arginine transport via cationic amino acid transporter-1 is necessary for human T-cell proliferation.

Availability of the semiessential amino acid arginine is fundamental for the efficient function of human T lymphocytes. Tumor-associated arginine deprivation, mainly induced by myeloid-derived suppressor cells, is a central mechanism of tumor immune escape from T-cell-mediated antitumor immune responses. We thus assumed that transmembranous transport of arginine must be crucial for T-cell function and studied which transporters are responsible for arginine influx into primary human T lymphocytes. Here, we show that activation via CD3 and CD28 induces arginine transport into primary human T cells. Both naïve and memory CD4(+) T cells as well as CD8(+) T cells specifically upregulated the human cationic amino acid transporter-1 (hCAT-1), with an enhanced and persistent expression under arginine starvation. When hCAT-1 induction was suppressed via siRNA transfection, arginine uptake, and cellular proliferation were impaired. In summary, our results demonstrate that hCAT-1 is a key component of efficient T-cell activation and a novel potential target structure to modulate adaptive immune responses in tumor immunity or inflammation.

Estradiol augments while progesterone inhibits arginine transport in human endothelial cells through modulation of cationic amino acid transporter-1.

Decreased generation of nitric oxide (NO) by endothelial NO synthase (eNOS) characterizes endothelial dysfunction (ECD). Delivery of arginine to eNOS by cationic amino acid transporter-1 (CAT-1) was shown to modulate eNOS activity. We found in female rats, but not in males, that CAT-1 activity is preserved with age and in chronic renal failure, two experimental models of ECD. In contrast, during pregnancy CAT-1 is inhibited. We hypothesize that female sex hormones regulate arginine transport. Arginine uptake in human umbilical vein endothelial cells (HUVEC) was determined following incubation with either 17ß-estradiol (E2) or progesterone. Exposure to E2 (50 and 100 nM) for 30 min resulted in a significant increase in arginine transport and reduction in phosphorylated CAT-1 (the inactive form) protein content. This was coupled with a decrease in phosphorylated MAPK/extracellular signal-regulated kinase (ERK) 1/2. Progesterone (1 and 100 pM for 30 min) attenuated arginine uptake and increased phosphorylated CAT-1, phosphorylated protein kinase Cα (PKCα), and phosphorylated ERK1/2 protein content. GO-6976 (PKCα inhibitor) prevented the progesterone-induced decrease in arginine transport. Coincubation with both progesterone and estrogen for 30 min resulted in attenuated arginine transport. While estradiol increases arginine transport and CAT-1 activity through modulation of constitutive signaling transduction pathways involving ERK, progesterone inhibits arginine transport and CAT-1 via both PKCα and ERK1/2 phosphorylation, an effect that predominates over estradiol.