Arginine Transport Research Articles

86 MgSO4 modulates A2A and A2B adenosine receptors, eNOS and iNOS expression, and l -arginine transport in human placental microvascular endothelial cells

Control of vascular tone in the human placenta depends on locally-generated endothelium derived vasoactive molecules. Nitric oxide (NO) is a vasodilator that caused low impedance, high-blood flow circuit facilitating the transfer of oxygen and other nutrients to the developing fetus. Thus, molecules that regulate the synthesis or bioavailability of NO are determinant in this phenomenon. The cationic amino acid l -arginine is the substrate for NO synthases (NOS) and NOS activity seems dependent on the l -arginine transport at the plasma membrane in endothelial cells. The endogenous nucleoside adenosine activates l -arginine transport and NO synthesis in the foetoplacental endothelium via activation of A2A adenosine receptors (A2AAR) in normal pregnancies. Interestingly, maternal foetal plasma concentration of adenosine is increased in preeclampsia, a syndrome associated with reduced vascular reactivity and blood flow. Since magnesium (Mg2+) is required for A2AAR-dependent dilation of rat mesenteric vessels, a potential link between Mg2+ and adenosine receptors in the foetoplacental vasculature is likely. We hypothesize that human placental microvascular endothelial cells (hPMECs) show increased A2AR expression and l -arginine transport when exposed to MgSO4. Objective To evaluate the effect of MgSO4 on eNOS, iNOS, A2AAR, and A2BAR expression, and l -arginine transport in hPMECs. Methods Primary cultures of hPMECs was made under standard conditions (37 °C, 5% CO2) in medium 199 supplemented with 10% newborn and 10% foetal calf serum (passage 3). Cells were incubated (37 °C, 12 h) in the absence or presence of MgSO4 (1.5–4 mM). eNOS, iNOS, A2AAR, and A2BAR protein abundance was assayed by Western blot. Kinetics of l -arginine transport (0–1000 μ M, 3 μ Ci/mL l -[3H]arginine, 1 min, 37 °C) at initial rates was measured. Results Incubation of hPMECs with MgSO4 increased A2AAR (0.53 ± 0.02 fold, EC50 =;0.53 ± 0.24 mM) and iNOS (0.43 ± 0.03 fold, EC50 = 0.81 ± 0.28 mM) protein abundance. However, MgSO4 did not alter the protein abundance of eNOS or A2BAR. Equally, MgSO4 increased (1.7 ± 0.2 fold, EC50 = 2.1 ± 0.3 mM) the maximal transport capacity (Vmax/Km) of l -arginine. Conclusions MgSO4 activates endothelial function via a mechanism requiring A2AAR and iNOS resulting in higher l -arginine transport in hPMECs from normal pregnancies. This phenomenon could be relevant in the endothelial dysfunction seen in preeclampsia where patients are treated with MgSO4. Acknowledgements: Fondo Nacional de Desarrollo Cientifico y Tecnologico (FONDECYT 3160194/1150377/1150344/1150083), Chile. Direccion de Investigacion (2015-0032-I), Universidad San Sebastian, Chile. RS holds Comision Nacional de Investigacion y Desarrollo (CONICYT) PhD and Faculty of Medicine, PUC-PhD fellowships, Chile.

The Trypanosoma cruzi Diamine Transporter Is Essential for Robust Infection of Mammalian Cells.

Trypanosoma cruzi is incapable of synthesizing putrescine or cadaverine de novo, and, therefore, salvage of polyamines from the host milieu is an obligatory nutritional function for the parasite. A high-affinity diamine transporter (TcPOT1) from T. cruzi has been identified previously that recognizes both putrescine and cadaverine as ligands. In order to assess the functional role of TcPOT1 in intact parasites, a Δtcpot1 null mutant was constructed by targeted gene replacement and characterized. The Δtcpot1 mutant lacked high-affinity putrescine-cadaverine transport capability but retained the capacity to transport diamines via a non-saturable, low-affinity mechanism. Transport of spermidine and arginine was not impacted by the Δtcpot1 lesion. The Δtcpot1 cell line exhibited a significant but not total defect in its ability to subsist in Vero cells, although initial infection rates were not affected by the lesion. These findings reveal that TcPOT1 is the sole high-affinity diamine permease in T. cruzi, that genetic obliteration of TcPOT1 impairs the ability of the parasite to maintain a robust infection in mammalian cells, and that a secondary low-affinity uptake mechanism for this key parasite nutrient is operative but insufficient for optimal infection.

An Arginine Deprivation Response Pathway Is Induced in Leishmania during Macrophage Invasion.

Amino acid sensing is an intracellular function that supports nutrient homeostasis, largely through controlled release of amino acids from lysosomal pools. The intracellular pathogen Leishmania resides and proliferates within human macrophage phagolysosomes. Here we describe a new pathway in Leishmania that specifically senses the extracellular levels of arginine, an amino acid that is essential for the parasite. During infection, the macrophage arginine pool is depleted due to its use to produce metabolites (NO and polyamines) that constitute part of the host defense response and its suppression, respectively. We found that parasites respond to this shortage of arginine by up-regulating expression and activity of the Leishmania arginine transporter (LdAAP3), as well as several other transporters. Our analysis indicates the parasite monitors arginine levels in the environment rather than the intracellular pools. Phosphoproteomics and genetic analysis indicates that the arginine-deprivation response is mediated through a mitogen-activated protein kinase-2-dependent signaling cascade.

Effects of Inflammation on Arginine Transport and Metabolism in Human Primary Myotubes

RationaleSkeletal muscle atrophy among older adults increases the risk for falls and fractures, loss of mobility and independence, physical disabilities, and even mortality. Development of efficacious therapies to attenuate skeletal muscle loss in the older population is necessary. Metabolomics research from our laboratory indicates that skeletal muscle mass (measured as appendicular lean mass divided by height squared) in older adults is associated with arginine metabolism. Arginine supplementation, as part of a multi‐nutrient supplement, has been given to individuals to attenuate skeletal muscle wasting induced by diseases with heightened inflammation (e.g., HIV and cancer cachexia). Older adults in general have chronic increased circulating and intramuscular levels of the proinflammatory cytokine tumor necrosis factor alpha (TNFα) that is associated with skeletal muscle atrophy. The effect of TNFα on skeletal muscle arginine metabolism is largely unknown.ObjectiveTo investigate the effects of age and TNFα on skeletal muscle arginine metabolism.MethodsSkeletal muscle biopsies were collected from 11 younger (21–39 years old) and 5 older (68–80 years old) males and females (YM, YF, OM, OF) after an overnight fast. To assess inflammatory signaling and arginine metabolism, the gene expression of TNFα, signal transducer and activator of transcription 3 (STAT3), interleukin‐6 (IL‐6), the IL‐6 receptors (IL‐6R and IL‐6ST), and cationic amino acid transporter 1 and 2 (CAT‐1 and CAT‐2) in whole tissue lysate was measured using quantitative PCR. To investigate the effects of TNFα specifically, primary skeletal muscle progenitor cells from 6 YM were cultured in growth media for 7 days, switched to differentiation media (DM) for 3 days, then incubated in DM containing 10 ng/mL TNFα for 2 days. Gene expression of the arginine transporters and metabolic enzymes in the TNFα treated primary human myotubes was measured using quantitative PCR.ResultsThere was no difference between young and old adults for CAT‐1 or CAT‐2 gene expression in whole skeletal muscle tissue lysate. There was also no difference in the gene expression of TNFα, STAT3, IL‐6, or the IL‐6 receptors. Incubation with TNFα decreased CAT‐1 gene expression slightly (−1.1 fold change, p=0.045) and increased CAT‐2 gene expression (11.2‐fold change, p=0.002) in primary human myotubes.ConclusionsThe proinflammatory cytokine TNFα increased gene expression of the arginine transporter CAT‐2 in primary human myotubes, indicating that older adults with elevated TNFα may have altered arginine transport, and potentially altered metabolism, in skeletal muscle. Ongoing research in our laboratory is assessing the effects of increased arginine availability in the skeletal myotubes in a proinflammatory culture. Future research is needed to determine if altering dietary arginine consumption in older adults can attenuate the atrophic effects of TNFα and thus, be a possible therapy to reduce skeletal muscle atrophy.Support or Funding InformationCornell Start‐up

Basic concepts of Mediterranean lifestyle and their impacts in the genomic aspects.

The Mediterranean lifestyle has traditionally been associated with a healthier life pattern, less prone to chronic illnesses, especially cardiovascular diseases. Among the different lifestyle variables, the concept of the Mediterranean diet (MedDiet) stands out as the healthiest aspect of the Mediterranean lifestyle. Moreover, although big differences in diet can be observed between the various countries on the Mediterranean coast, the Mediterranean lifestyle is also characterized by greater physical activity, mainly consisting of frequent walks encouraged by the warm and pleasant climate in comparison to central and northern Europe. So-called social support is also greater in the Mediterranean lifestyle with a closer and stronger family network and friends proving to be beneficial for health. However, there is great controversy about the Mediterranean siesta, mealtimes and sleep patterns. With regard to the MedDiet and despite the heterogeneity in the MedDiet definition, there is a commonly recognized pattern of consumption characterized by: a) high consumption of vegetables, fruits, cereals, legumes, nuts, and olive oil; b) moderate to high fish consumption; c) low consumption of red meats, and meat products; d) poultry and dairy products in moderate to small amounts and. There also seems to be a pattern of alcoholic and alcohol-free beverage consumption that varies depending on age. Environmental factors mainly contribute to this Mediterranean lifestyle, although genetic influences also interact. It is known that there are genetic variants that are associated with the intake of certain foodstuffs and among them the most relevant gene is the lactase gene (LCT) and its association with milk consumption. Thus, single nucleotide polymorphisms (SNPs) in the minichromosome maintenance complex component 6 (MCM6) gene are associated with differential transcriptional activation of the promoter of the neighboring lactase (LCT) gene and, thereby, influence lactase persistence (LP) in adulthood. The rs4988235 SNP, located at -13910 bp upstream from the LCT gene (-13910C>T) within intron 13 of the MCM6, has been the most studied SNP in relation to LP, milk intake and obesity-related diseases. Curiously, there is a north/south gradient in the prevalence of this gene variant, LP being less prevalent in the Mediterranean countries. Fruit, vegetable, sweets, sugared drinks, bitter drinks, etc. have been associated with variations in the different genes related to taste perception. There is increasingly detailed knowledge on the genes associated with the perception of sweet, bitter, sour, umami and salty tastes and their association with both food consumption, and the different health/disease phenotypes are being investigated. We have found associations between the perception of different tastes and the degree of adherence to the MedDiet. Moreover, diet itself may explain why certain gene variants are more prevalent in some countries than in others, as in the case of mutations in the MTHFR gene, which are more prevalent in Mediterranean countries than in northern Europe. In contrast, mutations in the APOE (E4 allele) are more frequent in northern Europe than in Mediterranean countries. To better understand these gene-diet interactions and their impact on the intermediate and final phenotypes of cardiovascular disease, we shall review several results from the PREDIMED (PREvencion con DIeta MEDiterranea) study. We have detected interesting gene-diet interactions in which greater adherence to the MedDiet, or to some of its typical foods, is able to reverse the adverse effects that the risk alleles have on the specific phenotypes.Although in nutrigenetics, the interactions of SNP candidates with total liquid intake in the diet are less well known and difficult to study, there are classic examples in monogenic diseases that point to their importance. Outstanding among these examples is that of cystinuria, an autosomic recessive genetic disorder characterized by an impairment in the transport of cystine, ornithine, lysine, and arginine. Of these, only cystine is insoluble enough to cause stone formation. It can lead to significant morbidity in affected patients due to the often large and recurrent resulting kidney stones. Incidence of cystinuria in the Spanish Mediterranean population is high and has a specific genetic pattern. Mutations in the amino acid exchanger System b(0,+), the two subunits of which are encoded by SLC3A1 and SLC7A9, predominantly underlie this disease. Our group showed a low prevalence of mutations in the SLC3A1 gene and a higher prevalence of mutations in the SLC7A9 gene, in contrast to that found in other countries. However, the wide variation of phenotypical traits suggests that further investigation into other genetic and/

Induction of inducible nitric oxide synthase expression in ammonia-exposed cultured astrocytes is coupled to increased arginine transport by upregulated y(+)LAT2 transporter.

One of the aspects of ammonia toxicity to brain cells is increased production of nitric oxide (NO) by NO synthases (NOSs). Previously we showed that ammonia increases arginine (Arg) uptake in cultured rat cortical astrocytes specifically via y(+)L amino acid transport system, by activation of its member, a heteromeric y(+)LAT2 transporter. Here, we tested the hypothesis that up-regulation of y(+)LAT2 underlies ammonia-dependent increase of NO production via inducible NOS (iNOS) induction, and protein nitration. Treatment of rat cortical astrocytes for 48 with 5 mM ammonium chloride ('ammonia') (i) increased the y(+)L-mediated Arg uptake, (ii) raised the expression of iNOS and endothelial NOS (eNOS), (iii) stimulated NO production, as manifested by increased nitrite+nitrate (Griess) and/or nitrite alone (chemiluminescence), and consequently, (iv) evoked nitration of tyrosine residues of proteins in astrocytes. Except for the increase of eNOS, all the above described effects of ammonia were abrogated by pre-treatment of astrocytes with either siRNA silencing of the Slc7a6 gene coding for y(+)LAT2 protein, or antibody to y(+)LAT2, indicating their strict coupling to y(+)LAT2 activity. Moreover, induction of y(+)LAT2 expression by ammonia was sensitive to Nf-κB inhibitor, BAY 11-7085, linking y(+)LAT2 upregulation to the Nf-κB activation in this experimental setting as reported earlier and here confirmed. Importantly, ammonia did not affect y(+)LAT2 expression nor y(+)L-mediated Arg uptake activity in the cultured cerebellar neurons, suggesting astroglia-specificity of the above described mechanism. The described coupling of up-regulation of y(+)LAT2 transporter with iNOS in ammonia-exposed astrocytes may be considered as a mechanism to ensure NO supply for protein nitration. Ammonia (NH4(+)) increases the expression and activity of the L-arginine (Arg) transporter (Arg/neutral amino acids [NAA] exchanger) y(+)LAT2 in cultured rat cortical astrocytes by a mechanism involving activation (nuclear translocation) of the transcription factor nuclear factor-Nuclear factor-κB (Nf-κB-p65). Up-regulation of y(+)LAT2 transporter is coupled with increased inducible nitric oxide synthase (iNOS) expression, which leads to increase nitric oxide (NO) synthesis and protein nitration.

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.

Physiological implications of arginine metabolism in plants.

Nitrogen is a limiting resource for plant growth in most terrestrial habitats since large amounts of nitrogen are needed to synthesize nucleic acids and proteins. Among the 21 proteinogenic amino acids, arginine has the highest nitrogen to carbon ratio, which makes it especially suitable as a storage form of organic nitrogen. Synthesis in chloroplasts via ornithine is apparently the only operational pathway to provide arginine in plants, and the rate of arginine synthesis is tightly regulated by various feedback mechanisms in accordance with the overall nutritional status. While several steps of arginine biosynthesis still remain poorly characterized in plants, much wider attention has been paid to inter- and intracellular arginine transport as well as arginine-derived metabolites. A role of arginine as alternative source besides glutamate for proline biosynthesis is still discussed controversially and may be prevented by differential subcellular localization of enzymes. Apparently, arginine is a precursor for nitric oxide (NO), although the molecular mechanism of NO production from arginine remains unclear in higher plants. In contrast, conversion of arginine to polyamines is well documented, and in several plant species also ornithine can serve as a precursor for polyamines. Both NO and polyamines play crucial roles in regulating developmental processes as well as responses to biotic and abiotic stress. It is thus conceivable that arginine catabolism serves on the one hand to mobilize nitrogen storages, while on the other hand it may be used to fine-tune development and defense mechanisms against stress. This review summarizes the recent advances in our knowledge about arginine metabolism, with a special focus on the model plant Arabidopsis thaliana, and pinpoints still unresolved critical questions.

Resolution and quantification of arginine, monomethylarginine, asymmetric dimethylarginine, and symmetric dimethylarginine in plasma using HPLC with internal calibration.

NG,NG‐dimethyl‐l‐arginine (asymmetric dimethylarginine, ADMA),NG‐monomethyl‐l‐arginine (l‐NMMA) and NG,N G’‐dimethyl‐l‐arginine (symmetric dimethylarginine, SDMA) are released during hydrolysis of proteins containing methylated arginine residues. ADMA and l‐NMMA inhibit nitric oxide synthase by competing with l‐arginine substrate. All three methylarginine derivatives also inhibit arginine transport. To enable investigation of methylarginines in diseases involving impaired nitric oxide synthesis, we developed a high‐performance liquid chromatography (HPLC) assay to simultaneously quantify arginine, ADMA, l‐NMMA and SDMA. Our assay requires 12 μL of plasma and is ideal for applications where sample availability is limited. We extracted arginine and methylarginines with mixed‐mode cation‐exchange columns, using synthetic monoethyl‐l‐arginine as an internal standard. Metabolites were derivatized with ortho‐phthaldialdeyhde and 3‐mercaptopropionic acid, separated by reverse‐phase HPLC and quantified with fluorescence detection. Standard curve linearity was ≥0.9995 for all metabolites. Inter‐day coefficient of variation (CV) values were ≤5% for arginine, ADMA and SDMA in human plasma and for arginine and ADMA in mouse plasma. The CV value for l‐NMMA was higher in human (10.4%) and mouse (15.8%) plasma because concentrations were substantially lower than ADMA and SDMA. This assay provides unique advantages of small sample volume requirements, excellent separation of target metabolites from contaminants and validation for both human and mouse plasma samples. © 2015 The Authors Biomedical Chromatography published by John Wiley & Sons, Ltd.

The oral and gut microbiomes are perturbed in rheumatoid arthritis and partly normalized after treatment.

We carried out metagenomic shotgun sequencing and a metagenome-wide association study (MGWAS) of fecal, dental and salivary samples from a cohort of individuals with rheumatoid arthritis (RA) and healthy controls. Concordance was observed between the gut and oral microbiomes, suggesting overlap in the abundance and function of species at different body sites. Dysbiosis was detected in the gut and oral microbiomes of RA patients, but it was partially resolved after RA treatment. Alterations in the gut, dental or saliva microbiome distinguished individuals with RA from healthy controls, were correlated with clinical measures and could be used to stratify individuals on the basis of their response to therapy. In particular, Haemophilus spp. were depleted in individuals with RA at all three sites and negatively correlated with levels of serum autoantibodies, whereas Lactobacillus salivarius was over-represented in individuals with RA at all three sites and was present in increased amounts in cases of very active RA. Functionally, the redox environment, transport and metabolism of iron, sulfur, zinc and arginine were altered in the microbiota of individuals with RA. Molecular mimicry of human antigens related to RA was also detectable. Our results establish specific alterations in the gut and oral microbiomes in individuals with RA and suggest potential ways of using microbiome composition for prognosis and diagnosis.

Augmented endothelial l-arginine transport ameliorates pressure-overload-induced cardiac hypertrophy.

What is the central question of this study? What is the potential role of endothelial NO production via overexpression of the l-arginine transporter, CAT1, as a mitigator of cardiac hypertrophy? What is the main finding and its importance? Augmentation of endothelium-specific l-arginine transport via CAT1 can attenuate pressure-overload-dependent cardiac hypertrophy and fibrosis. Our findings support the conclusion that interventions that improve endothelial l-arginine transport may provide therapeutic utility in the setting of myocardial hypertrophy. Such modifications may be introduced by exercise training or locally delivered gene therapy, but further experimental and clinical studies are required. Endothelial dysfunction has been postulated to play a central role in the development of cardiac hypertrophy, probably as a result of reduced NO bioavailability. We tested the hypothesis that increased endothelial NO production, mediated by increased l-arginine transport, could attenuate pressure-overload-induced cardiac hypertrophy. Echocardiography and blood pressure measurements were performed 15 weeks after transverse aortic constriction (TAC) in wild-type (WT) mice (n = 12) and in mice with endothelium-specific overexpression of the l-arginine transporter, CAT1 (CAT+; n = 12). Transverse aortic constriction induced greater increases in heart weight to body weight ratio in WT (by 47%) than CAT+ mice (by 25%) compared with the respective controls (P ≤ 0.05). Likewise, the increase in left ventricular wall thickness induced by TAC was significantly attenuated in CAT+ mice (P = 0.05). Cardiac collagen type I mRNA expression was greater in WT mice with TAC (by 22%; P = 0.03), but not in CAT+ mice with TAC, compared with the respective controls. Transverse aortic constriction also induced lesser increases in β-myosin heavy chain mRNA expression in CAT+ mice compared with WT (P ≤ 0.05). Left ventricular systolic pressure after TAC was 36 and 39% greater in WT and CAT+ mice, respectively, compared with the respective controls (P ≤ 0.001). Transverse aortic constriction had little effect on left ventricular end-diastolic pressure in both genotypes. Taken together, these data indicate that augmenting endothelial function by overexpression of l-arginine transport can attenuate pressure-overload-induced cardiac hypertrophy.

Identification and functional characterization of a novel arginine/ornithine transporter, a member of a cationic amino acid transporter subfamily in the Trypanosoma cruzi genome.

BackgroundTrypanosoma cruzi, the etiological agent of Chagas disease, is auxotrophic for arginine. It obtains this amino acid from the host through transporters expressed on the plasma membrane and on the membranes of intracellular compartments. A few cationic amino acid transporters have been characterized at the molecular level, such as the novel intracellular arginine/ornithine transporter, TcCAT1.1, a member of the TcCAT subfamily that is composed of four almost identical open reading frames in the T. cruzi genome.MethodsThe functional characterization of the TcCAT1.1 isoform was performed in two heterologous expression systems. TcCAT subfamily expression was evaluated by real-time PCR in polysomal RNA fractions, and the cellular localization of TcCAT1.1 fused to EGFP was performed by confocal and immunoelectron microscopy.ResultsIn the S. cerevisiae expression system, TcCAT1.1 showed high affinity for arginine (Km = 0.085 ± 0.04 mM) and low affinity for ornithine (Km = 1.7 ± 0.2 mM). Xenopus laevis oocytes expressing TcCAT1.1 showed a 7-fold increase in arginine uptake when they were pre-loaded with arginine, indicating that transport is enhanced by substrates on the trans side of the membrane (trans-stimulation). Oocytes that were pre-loaded with [3H]-arginine displayed a 16-fold higher efflux of [3H]-arginine compared with that of the control. Analysis of polysomal RNA fractions demonstrated that the expression of members of the arginine transporter TcCAT subfamily is upregulated under nutritional stress and that this upregulation precedes metacyclogenesis. To investigate the cellular localization of the transporter, EGFP was fused to TcCAT1.1, and fluorescence microscopy and immunocytochemistry revealed the intracellular labeling of vesicles in the anterior region, in a network of tubules and vesicles.ConclusionsTcCAT1.1 is a novel arginine/ornithine transporter, an exchanger expressed in intracellular compartments that is physiologically involved in arginine homeostasis throughout the T. cruzi life cycle. The properties and estimated kinetic parameters of TcCAT1.1 can be extended to other members of the TcCAT subfamily.

Ammonia reduces intracellular ADMA level in cultured astrocytes and endothelial cells: possible involvement of increased y+LAT2-mediated efflux

Toxic effects of ammonia in the brain are partly related to impaired NO production which depending on the dose/time of ammonia exposure, may be either increased or decrease. Asymmetric dimethylarginine (ADMA), is endogenous NOSs inhibitor and symmetric dimethylarginine (SDMA) is arginine (Arg) transport inhibitor (Teerlink et al., 2009). Previously we reported an increase of ADMA and SDMA concentration in brain of rats with acute liver failure (Milewski et al., 2014), but distribution of the ADMA/SDMA surplus between the particular intra and extracellular compartments has not been studied. Here, we measured the intracellular concentration of ADMA, SDMA and ADMA/SDMA/NO precursor Arg, in cultured cortical astrocytes and rat brain endothelium cells (RBE-4) treated or not with ammonia. In RBE-4 cells not treated with ammonia the ADMA concentration was twice higher and the Arg/ADMA ratio was much lower than in astrocytes, confirming the well documented role of ADMA in endothelial NOS inhibition (Pope et al., 2009). Treatment for 48 h with 5 mM ammonia led to an almost 50% reduction of ADMA and SDMA concentration in both cell type. Since ammonia-dependent Arg transport in astrocytes is specifically mediated by the heteromeric Arg/Gln transporter y+LAT2 (Zielinska et al., 2012), we speculated that this may also hold for both Arg derivatives. Indeed, silencing of the y+LAT2 gene diminished the reduction of intracellular ADMA concentration caused by ammonia treatment in astrocytes. Moreover, the y+LAT2-dependent component of ammonia-evoked Arg uptake was reduced in the presence of ADMA in the medium. The results suggest that increased ADMA (and possibly SDMA) efflux mediated by upregulated y+LAT2 may be one of the ways in which ammonia interferes with intra-astrocytic ADMA content and, subsequently, NO synthesis. Studies are underway to establish if the same sequence of changes holds for ammonia-treated cerebral endothelial cells.

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 17β-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.

Mitochondrial transporters for ornithine and related amino acids: a review

Among the members of the mitochondrial carrier family, there are transporters that catalyze the translocation of ornithine and related substrates, such as arginine, homoarginine, lysine, histidine, and citrulline, across the inner mitochondrial membrane. The mitochondrial carriers ORC1, ORC2, and SLC25A29 from Homo sapiens, BAC1 and BAC2 from Arabidopsis thaliana, and Ort1p from Saccharomyces cerevisiae have been biochemically characterized by transport assays in liposomes. All of them transport ornithine and amino acids with side chains terminating at least with one amine. There are, however, marked differences in their substrate specificities including their affinity for ornithine (KM values in the mM to μM range). These differences are most likely reflected by minor differences in the substrate binding sites of these carriers. The physiological role of the above-mentioned mitochondrial carriers is to link several metabolic pathways that take place partly in the cytosol and partly in the mitochondrial matrix and to provide basic amino acids for mitochondrial translation. In the liver, human ORC1 catalyzes the citrulline/ornithine exchange across the mitochondrial inner membrane, which is required for the urea cycle. Human ORC1, ORC2, and SLC25A29 are likely to be involved in the biosynthesis and transport of arginine, which can be used as a precursor for the synthesis of NO, agmatine, polyamines, creatine, glutamine, glutamate, and proline, as well as in the degradation of basic amino acids. BAC1 and BAC2 are implicated in some processes similar to those of their human counterparts and in nitrogen and amino acid metabolism linked to stress conditions and the development of plants. Ort1p is involved in the biosynthesis of arginine and polyamines in yeast.

Arginine dependence of acute myeloid leukemia blast proliferation: a novel therapeutic target

AbstractAcute myeloid leukemia (AML) is one of the most common acute leukemias in adults and children, yet significant numbers of patients relapse and die of disease. In this study, we identify the dependence of AML blasts on arginine for proliferation. We show that AML blasts constitutively express the arginine transporters CAT-1 and CAT-2B, and that the majority of newly diagnosed patients' blasts have deficiencies in the arginine-recycling pathway enzymes argininosuccinate synthase and ornithine transcarbamylase, making them arginine auxotrophic. BCT-100, a pegylated human recombinant arginase, leads to a rapid depletion in extracellular and intracellular arginine concentrations, resulting in arrest of AML blast proliferation and a reduction in AML engraftment in vivo. BCT-100 as a single agent causes significant death of AML blasts from adults and children, and acts synergistically in combination with cytarabine. Using RNA sequencing, 20 further candidate genes which correlated with resistance have been identified. Thus, AML blasts are dependent on arginine for survival and proliferation, as well as depletion of arginine with BCT-100 of clinical value in the treatment of AML.

Importance of Host Cell Arginine Uptake in Francisella Phagosomal Escape and Ribosomal Protein Amounts*[S

Upon entry into mammalian host cells, the pathogenic bacterium Francisella must import host cell arginine to multiply actively in the host cytoplasm. We identified and functionally characterized an arginine transporter (hereafter designated ArgP) whose inactivation considerably delayed bacterial phagosomal escape and intracellular multiplication. Intramacrophagic growth of the ΔargP mutant was fully restored upon supplementation of the growth medium with excess arginine, in both F. tularensis subsp. novicida and F. tularensis subsp. holarctica LVS, demonstrating the importance of arginine acquisition in these two subspecies. High-resolution mass spectrometry revealed that arginine limitation reduced the amount of most of the ribosomal proteins in the ΔargP mutant. In response to stresses such as nutritional limitation, repression of ribosomal protein synthesis has been observed in all kingdoms of life. Arginine availability may thus contribute to the sensing of the intracellular stage of the pathogen and to trigger phagosomal egress. All MS data have been deposited in the ProteomeXchange database with identifier PXD001584 (http://proteomecentral.proteomexchange.org/dataset/PXD001584).

Cystinuria: current concepts and future directions.

Cystinuria, an autosomic recessive genetic disorder is an uncommon cause of nephrolithiasis characterized by an impairment of transport of cystine, ornithine, lysine, and arginine (COLA). Of these, only cystine is insoluble enough to cause stone formation. Although a classification exists that categorizes the disease depending on chromosomal mutation, this does not currently alter management which consists of increased fluid intake, urine alkalinization, reduced sodium intake and, if warranted, cystine-binding thiol drugs. Cystine stones are relatively resistant to fragmentation. Intrinsic characteristics on imaging may help in planning surgical treatment. Finally, advances in crystal growth inhibition are encouraging as they may provide a new tool to treat this condition which although uncommon, is treatable and has been associated with lower quality of life and renal function compared to other stone formers.

Studies of conformational changes of an arginine-binding protein from Thermotoga maritima in the presence and absence of ligand via molecular dynamics simulations with the coarse-grained UNRES force field.

The arginine-binding protein (ArgBP) from the hyperthermophilic eubacterium Thermotoga maritima (TmArgBP) is responsible for arginine transport through the bacterial cell membrane. The protein binds a single molecule of L-arginine, which results in conformational changes due to hinge bending. Thereby, TmArgBP acquires one of two possible conformations: open (without the presence of the arginine ligand) and closed (in the presence of the arginine ligand). Here we report a molecular dynamics study of the influence of the presence or absence of the ligand on the dynamics of TmArgBP, using the coarse-grained UNRES force field. The results of our studies indicate that binding of the arginine ligand promotes a closed conformation, which agrees with experimental data. However, the sensitivity of the TmArgBP conformation to the presence of arginine decreases and the protein becomes more flexible with increasing temperature, which might be related to the functionality of this protein in the thermophilic organism T. maritima.

Arginine increases development of in vitro-produced porcine embryos and affects the protein arginine methyltransferase-dimethylarginine dimethylaminohydrolase-nitric oxide axis.

Culture systems promote development at rates lower than the in vivo environment. Here, we evaluated the embryo's transcriptome to determine what the embryo needs during development. A previous mRNA sequencing endeavour found upregulation of solute carrier family 7 (cationic amino acid transporter, y+ system), member 1 (SLC7A1), an arginine transporter, in in vitro- compared with in vivo-cultured embryos. In the present study, we added different concentrations of arginine to our culture medium to meet the needs of the porcine embryo. Increasing arginine from 0.12 to 1.69mM improved the number of embryos that developed to the blastocyst stage. These blastocysts also had more total nuclei compared with controls and, specifically, more trophectoderm nuclei. Embryos cultured in 1.69mM arginine had lower SLC7A1 levels and a higher abundance of messages involved with glycolysis (hexokinase 1, hexokinase 2 and glutamic pyruvate transaminase (alanine aminotransferase) 2) and decreased expression of genes involved with blocking the tricarboxylic acid cycle (pyruvate dehydrogenase kinase, isozyme 1) and the pentose phosphate pathway (transaldolase 1). Expression of the protein arginine methyltransferase (PRMT) genes PRMT1, PRMT3 and PRMT5 throughout development was not affected by arginine. However, the dimethylarginine dimethylaminohydrolase 1 (DDAH1) and DDAH2 message was found to be differentially regulated through development, and the DDAH2 protein was localised to the nuclei of blastocysts. Arginine has a positive effect on preimplantation development and may be affecting the nitric oxide-DDAH-PRMT axis.