L-arginine Uptake Research Articles

Arginine and Citrulline Catabolic Pathways Encoded by the arc Gene Cluster of Lactobacillus brevis ATCC 367.

High concentrations of l-arginine or l-citrulline in the growth medium provided the wine bacterium Lactobacillus brevis with a significant growth advantage. The arginine deiminase pathway (ADI) arc gene cluster of Lactobacillus brevis contains three genes-arcD, arcE1, and arcE2-encoding putative l-arginine/l-ornithine exchangers. Uptake experiments with Lactococcus lactis cells expressing the genes showed that all three transported l-ornithine with affinities in the micromolar range. Similarly, ArcD and ArcE2 transported l-arginine, while ArcE1 transported l-citrulline, an intermediate of the ADI pathway. Chase experiments showed very efficient exchange of l-arginine and l-ornithine by ArcD and ArcE2 and of l-citrulline and l-ornithine by ArcE1. Low affinities (millimolar range) combined with low translocation rates were found for ArcD and ArcE2 with l-citrulline and for ArcE1 with l-arginine. Resting cells of Lactobacillus brevis grown in the presence of l-arginine and l-citrulline rapidly consumed l-arginine and l-citrulline, respectively, while producing ammonia and l-ornithine. About 10% of l-arginine degraded was excreted by the cells as l-citrulline. Degradation of l-arginine and l-citrulline was not subject to carbon catabolite repression by glucose in the medium. At a high medium pH, l-citrulline in the medium was required for induction of the l-citrulline degradation pathway. Pathways are proposed for the catabolic breakdown of l-arginine and l-citrulline that merge at the level of ornithine transcarbamylase in the ADI pathway. l-Arginine uptake is catalyzed by ArcD and/or ArcE2, l-citrulline by ArcE1. l-Citrulline excretion during l-arginine breakdown is proposed to be catalyzed by ArcD and/or ArcE2 through l-arginine/l-citrulline exchange.IMPORTANCELactobacillus brevis, a bacterium isolated from wine, as well as other food environments, expresses a catabolic pathway for the breakdown of l-citrulline in the medium that consists of the l-citrulline/l-ornithine exchanger ArcE1 and part of the catabolic arginine deiminase (ADI) pathway enzymes. The proposed pathways for l-arginine and l-citrulline breakdown provide a mechanism for l-citrulline accumulation in fermented food products that is the precursor of the carcinogen ethyl carbamate.

L‐Arginine Prevents Heme‐Induced Acute Lung Injury (ALI) in a Rat Model of Trauma/Hemorrhage and Resuscitation with Transfusion

Resuscitation with long (> 28 days) stored packed red blood cells (PRBC) promotes acute lung injury (ALI) in our rat model of trauma/hemorrhage. Low nitric oxide (NO) production has been implicated in the development of ALI. Arginase can decrease NO production by decreasing L‐arginine availability for endothelial NO synthase (eNOS). We found that arginase, released from red blood cells (RBC) during prolonged storage, contributes to ALI in our rat model of trauma/hemorrhage. Prolonged storage also increases levels of free hemoglobin (Hb). Heme, a breakdown product of Hb, is metabolized by heme oxygenase to form carbon monoxide (CO). We were the first to report that heme‐derived CO inhibits endothelial nitric oxide (NO) synthase and promotes vasoconstriction in a manner that can be prevented by L‐arginine administration. This study tests the hypothesis that the hemolysis product, heme, promotes ALI in our rat model of trauma/hemorrhage and resuscitation with transfusion in a manner that is exacerbated by limited L‐arginine availability. Fresh PRBC were prepared from rat whole blood. Male Lewis rats were implanted with femoral arterial and venous catheters (trauma), subjected to a 30% hemorrhage and resuscitated with crystalloids and fresh PRBC containing heme alone or with L‐arginine or L‐lysine (competitive inhibitor of L‐arginine uptake). ALI was assessed by pulmonary protein leak (Evans blue dye ‐ EBD) and cytokine‐induced neutrophil chemoattractant‐1 (CINC‐1; rat homologue of human interleukin (IL)‐8) accumulation in bronchoalveolar lavage fluid (BALF). Heme alone caused a 5.6‐fold increase in pulmonary protein leak and 3.5‐fold increase in CINC‐1 accumulation. Co‐administration of L‐arginine decreased heme‐induced pulmonary protein leak by 77% and CINC‐1 accumulation by 32.4%. In contrast, L‐lysine, a competitive inhibitor of L‐arginine uptake, increased heme‐induced pulmonary protein leak by 102% and CINC‐1 accumulation by 106.4%. These data show that the Hb breakdown product, heme, promotes ALI in our rat model of trauma/hemorrhage and resuscitation with transfusion. L‐Arginine co‐administration protects against heme‐induced ALI, while blocking L‐arginine transport by L‐lysine exacerbates heme‐induced ALI. These results suggest that the other hemolysis product, heme, also contributes to ALI in a manner that is greatly exacerbated by decreased L‐arginine bioavailability. We conclude that hemolysis products arginase and heme work in concert to promote ALI during trauma/hemorrhage and resuscitation with transfusion.Support or Funding InformationSupported by grants AHA 0865241F (FKJ), NIH HL074966 (WD) and USAMRC DOD W81XWH‐07‐1‐0717 (RAJ, RMS).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

SLC6A14 Is a Genetic Modifier of Cystic Fibrosis That Regulates Pseudomonas aeruginosa Attachment to Human Bronchial Epithelial Cells.

ABSTRACTCystic fibrosis (CF) is caused by mutations in the CFTR gene and is associated with progressive and ultimately fatal infectious lung disease. There can be considerable variability in disease severity among individuals with the same CFTR mutations, and recent genome-wide association studies have identified secondary genetic factors that contribute to this. One of these modifier genes is SLC6A14, which encodes an amino acid transporter. Importantly, variants of this gene have been associated with age at first acquisition of Pseudomonas aeruginosa. In this study, we aimed to determine the function of SLC6A14 in airway epithelia and how it might affect colonization by P. aeruginosa. We show that SLC6A14 is expressed in respiratory epithelial cells and transports l-arginine out of the airway surface liquid (ASL). Exposure of airway epithelia to flagellin from P. aeruginosa led to upregulation of SLC6A14 expression and increased SLC6A14-dependent uptake of l-arginine from the ASL. In support of the hypothesis that l-arginine affects P. aeruginosa attachment, we showed that l-arginine supplementation promoted P. aeruginosa attachment to an abiotic surface in a dose-dependent manner. In a coculture model, we found that inhibition of SLC6A14-dependent l-arginine transport enhanced P. aeruginosa attachment. In Slc6a14−/y (knockout) mice, P. aeruginosa attachment to lung tissue was also significantly enhanced. Together, these findings suggest that SLC6A14 activity plays a role in the modification of the initial stages of airway infection by altering the level of l-arginine in the ASL, which in turn affects the attachment of P. aeruginosa.

Human umbilical vein endothelium-derived exosomes play a role in foetoplacental endothelial dysfunction in gestational diabetes mellitus

Gestational diabetes mellitus (GDM) characterizes by foetoplacental endothelial dysfunction. Human umbilical vein endothelial cells (HUVECs) from women with GDM show increased L-arginine transport via the human cationic amino acid transporter 1 (hCAT-1). Moreover, expression of endothelial nitric oxide synthase (eNOS) and nitric oxide synthesis are increased. Exosomes are increased in maternal plasma from GDM. We evaluated the role of foetoplacental endothelial exosomes on endothelial dysfunction in GDM. Exosomes were isolated from HUVECs from normal (ExN) and GDM (ExGDM) pregnancies. HUVECs were exposed (8h) to ExN or ExGDM and used for wound recovery assay (up to 8h), L-arginine transport, hCAT-1 and eNOS expression and activity, reactive oxygen species (ROS) generation, and 44 and 42kDa mitogen activated protein kinases (p44/42mapk) and protein kinase B/Akt (Akt) activation. Wound recovery was slower in GDM compared with normal pregnancies and was recovered by ExN. However, ExGDM delayed wound recovery in cells from normal pregnancies. GDM-increased L-arginine transport, hCAT-1 and eNOS expression and activity, and p44/42mapk activation were blocked by ExN, but ExGDM increased these parameters and ROS generation, and reduced eNOS phosphorylation at threonine495 in cells from normal pregnancies. Inhibition of p44/42mapk, but not Akt reversed GDM-increased L-arginine uptake. In conclusion foetoplacental endothelial-released exosomes play a role in the maintenance of a GDM phenotype in HUVECs. It is suggested that ExN and ExGDM cargo are different with differential effects in cells from normal or GDM pregnancies. This phenomenon could contribute to the understanding of mechanisms behind foetoplacental endothelial dysfunction in GDM pregnancies.

L-arginine availability and arginase activity: Characterization of amino acid permease 3 in Leishmania amazonensis.

BackgroundLeishmania uses the amino acid L-arginine as a substrate for arginase, enzyme that produces urea and ornithine, last precursor of polyamine pathway. This pathway is used by the parasite to replicate and it is essential to establish the infection in the mammalian host. L-arginine is not synthesized by the parasite, so its uptake occurs through the amino acid permease 3 (AAP3). AAP3 is codified by two copies genes (5.1 and 4.7 copies), organized in tandem in the parasite genome. One copy presents the expression regulated by L-arginine availability.Methodology/Principal findingsRNA-seq data revealed 14 amino acid transporters differentially expressed in the comparison of La-WT vs. La-arg- promastigotes and axenic amastigotes. The 5.1 and 4.7 aap3 transcripts were down-regulated in La-WT promastigotes vs. axenic amastigotes, and in La-WT vs. La-arg- promastigotes. In contrast, transcripts of other transporters were up-regulated in the same comparisons. The amount of 5.1 and 4.7 aap3 mRNA of intracellular amastigotes was also determined in sample preparations from macrophages, obtained from BALB/c and C57BL/6 mice and the human THP-1 lineage infected with La-WT or La-arg-, revealing that the genetic host background is also important. We also determined the aap3 mRNA and AAP3 protein amounts of promastigotes and axenic amastigotes in different environmental growth conditions, varying pH, temperature and L-arginine availability. Interestingly, the increase of temperature increased the AAP3 level in plasma membrane and consequently the L-arginine uptake, independently of pH and L-arginine availability. In addition, we demonstrated that besides the plasma membrane localization, AAP3 was also localized in the glycosome of L. amazonensis promastigotes and axenic amastigotes.Conclusions/SignificanceIn this report, we described the differential transcriptional profiling of amino acids transporters from La-WT and La-arg- promastigotes and axenic amastigotes. We also showed the increased AAP3 levels under amino acid starvation or its decrease in L-arginine supplementation. The differential AAP3 expression was determined in the differentiation of promastigotes to amastigotes conditions, as well as the detection of AAP3 in the plasma membrane reflecting in the L-arginine uptake. Our data suggest that depending on the amino acid pool and arginase activity, Leishmania senses and could use an alternative route for the amino acid transport in response to stress signaling.

Astragaloside II promotes intestinal epithelial repair by enhancing L-arginine uptake and activating the mTOR pathway

Astragaloside II (AS II) extracted from Astragalus membranaceus has been reported to promote tissue wound repair. However, the effect of AS II on inflammatory bowel disease is unknown. We investigated the effects and mechanism of AS II on intestinal wound healing in both in vitro and in vivo models. Human intestinal Caco-2 cells were treated with multiple concentrations of AS II to assess cell proliferation, scratch wound closure, L-arginine uptake, cationic amino acid transporter activity, and activation of the mTOR signaling pathway. These effects were also measured in a mouse model of colitis. AS II promoted wound closure and increased cell proliferation, L-arginine uptake, CAT1 and CAT2 protein levels, total protein synthesis, and phosphorylation of mTOR, S6K, and 4E-BP1 in Caco-2 cells. These effects were suppressed by lysine or rapamycin treatment, suggesting that the enhanced arginine uptake mediates AS II-induced wound healing. Similar results were also observed in vivo. Our findings indicate that AS II can contribute to epithelial barrier repair following intestinal injury, and may offer a therapeutic avenue in treating irritable bowel disease.

Abstract 101: Tissue-specific Deletion of Collectrin in the Proximal Tubular Epithelium Increases Arterial Pressure and Augments Salt-sensitivity

Background: Collectrin ( Tmem27 ) is a key regulator of blood pressure (BP) and modulator of the bioavailability of nitric oxide (NO) and superoxide. It is highly expressed in the kidney in the proximal tubule (PT), collecting duct, and throughout the vascular endothelium. We reported that collectrin plays a critical role as a chaperone for the reabsorption of all amino acids (AAs) in the PT, and for the uptake of the cationic AA L-arginine (L-Arg) in endothelial cells. Global collectrin knockout ( Tmem27 Y/- ) mice display baseline hypertension (HTN), augmented salt-sensitive hypertension (SSH), and decreased renal blood flow. Objective and Methods: To determine the PT-specific effect of collectrin on BP homeostasis and salt sensitivity, we used the Cre -loxP approach and PEPCK-Cre to generate a mouse line lacking collectrin specifically in the PT-- PEPCK-Cre + Tmem27 Y/Flox mice. PEPCK-Cre - Tmem27 Y/Flox mice were used as control. Radiotelemetry was used to measure BP for 2 weeks at baseline and 2 weeks on high salt diet (HSD). Renal blood flow at baseline and on HSD was measured using contrast enhanced ultrasound in the same mice. Results: Successful deletion of collectrin in the PT was confirmed by assessing mRNA levels using real-time RT-PCR, immunohistochemistry staining of renal tissues using anti-collectrin antibody, and quantitation of protein from kidney cortex by Western analysis. Compared to control PEPCK-Cre - Tmem27 Y/Flox mice (n=6), PEPCK-Cre + Tmem27 Y/Flox mice (n=6) displayed significantly higher systolic BP (SBP) at baseline (120.0 ± 2.5 vs 131.6 ± 2.9 mm Hg; p = 0.014) and after HSD (135.3 ± 2.6 vs 151.5 ± 5.2 mm Hg; p = 0.019). Renal blood flow was not different between groups, at baseline nor after HSD. Conclusion: Collectrin in the PT plays an important role in blood pressure homeostasis and response to sodium intake, independent of renal blood flow. Increasing proximal tubular collectrin activity may be a novel therapeutic strategy for the treatment of hypertension and salt-sensitivity.

A Dynamic Metabolic Flux Analysis of Myeloid-Derived Suppressor Cells Confirms Immunosuppression-Related Metabolic Plasticity

Recent years have witnessed an increasing interest at understanding the role of myeloid-derived suppressor cells (MDSCs) in cancer-induced immunosuppression, with efforts to inhibit their maturation and/or their activity. We have thus modelled MDSCs central carbon metabolism and bioenergetics dynamic, calibrating the model using experimental data on in vitro matured mice bone marrow cells into MDSCs. The model was then used to probe the cells metabolic state and dynamics, performing a dynamic metabolic flux analysis (dMFA) study. Indeed, MDSCs maturation correlates with a high glycolytic flux contributing to up to 95% of the global ATP turnover rate, while most of the glucose-derived carbon enters the TCA cycle. Model simulations also reveal that pentose phosphate pathway and oxidative phosphorylation activities were kept at minimal levels to ensure NADPH production and anabolic precursors synthesis. Surprisingly, MDSCs immunosuppressive activity, i.e. L-arginine uptake, metabolism and endogenous synthesis, only consumes sparse quantities of energy-rich nucleotides (ATP and NADPH). Therefore, model simulations suggest that MDSCs exhibit a heterogeous metabolic profile similar to tumour cells. This behavior is probably an indirect immunosuppressive mechanism where MDSCs reduce the availability of carbon sources in the tumour periphery microenvironment, which could explain the dysfuntion and death of immune effector cells.

L-Arginine Uptake by Cationic Amino Acid Transporter Promotes Intra-Macrophage Survival of Leishmania donovani by Enhancing Arginase-Mediated Polyamine Synthesis.

The survival of intracellular protozoan parasite, Leishmania donovani, the causative agent of Indian visceral leishmaniasis (VL), depends on the activation status of macrophages. l-Arginine, a semi-essential amino acid plays a crucial regulatory role for activation of macrophages. However, the role of l-arginine transport in VL still remains elusive. In this study, we demonstrated that intra-macrophage survival of L. donovani depends on the availability of extracellular l-arginine. Infection of THP-1-derived macrophage/human monocyte-derived macrophage (hMDM) with Leishmania, resulted in upregulation of l-arginine transport. While investigating the involvement of the transporters, we observed that Leishmania survival was greatly impaired when the transporters were blocked either using inhibitor or siRNA-mediated downregulation. CAT-2 was found to be the main isoform associated with l-arginine transport in L. donovani-infected macrophages. l-arginine availability and its transport regulated the host arginase in Leishmania infection. Arginase and inducible nitric oxide synthase (iNOS) expression were reciprocally regulated when assayed using specific inhibitors and siRNA-mediated downregulation. Interestingly, induction of iNOS expression and nitric oxide production were observed in case of inhibition of arginase in infected macrophages. Furthermore, inhibition of l-arginine transport as well as arginase resulted in decreased polyamine production, limiting parasite survival inside macrophages. l-arginine availability and transport regulated Th1/Th2 cytokine levels in case of Leishmania infection. Upregulation of l-arginine transport, induction of host arginase, and enhanced polyamine production were correlated with increased level of IL-10 and decreased level of IL-12 and TNF-α in L. donovani-infected macrophages. Our findings provide clear evidence for targeting the metabolism of l-arginine and l-arginine-metabolizing enzymes as an important therapeutic and prophylactic strategy to treat VL.

Uptake and metabolism of arginine impact Plasmodium development in the liver

Prior to infecting erythrocytes and causing malaria symptoms, Plasmodium parasites undergo an obligatory phase of invasion and extensive replication inside their mammalian host’s liver cells that depends on the parasite’s ability to obtain the nutrients it requires for its intra-hepatic growth and multiplication. Here, we show that L-arginine (Arg) uptake through the host cell’s SLC7A2-encoded transporters is essential for the parasite’s development and maturation in the liver. Our data suggest that the Arg that is taken up is primarily metabolized by the arginase pathway to produce the polyamines required for Plasmodium growth. Although the parasite may hijack the host’s biosynthesis pathway, it relies mainly upon its own arginase-AdoMetDC/ODC pathway to acquire the polyamines it needs to develop. These results identify for the first time a pivotal role for Arg-dependent polyamine production during Plasmodium’s hepatic development and pave the way to the exploitation of strategies to impact liver infection by the malaria parasite through the modulation of Arg uptake and polyamine synthesis.

Effects of simvastatin on CAT-1-mediated arginine transport and NO level under high glucose conditions in conditionally immortalized rat inner blood-retinal barrier cell lines (TR-iBRB)

ObjectiveHyperglycemia causes the breakdown of the blood-retinal barrier by impairing endothelial nitric oxide synthase (eNOS) function. Statins have many pleiotropic effects such as improving endothelial barrier permeability and increasing eNOS mRNA stability. The objective of this study was to determine effect of simvastatin on l-arginine transport and NO production under high-glucose conditions in conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB). MethodsChanges in l-arginine transport uptake and, expression levels of cationic amino acid transporter 1 (CAT-1) and eNOS mRNA were investigated after pre-treatment with simvastatin and NOS inhibitors (l-NMMA and l-NAME) under high-glucose conditions using TR-iBRB, an in vitro model of iBRB. The NO level released from TR-iBRB cells was examined using Griess reagents. ResultsUnder high glucose conditions, [3H]l-arginine uptake was decreased in TR-iBRB cells. Simvastatin pretreatment elevated [3H]l-arginine uptake, the expression levels of CAT-1 and eNOS mRNA, and NO production under high-glucose conditions. Moreover, the co-treatment with simvastatin and NOS inhibitors reduced [3H]l-arginine uptake compared to pretreatment with simvastatin alone. ConclusionOur results suggest that, in the presence of high-glucose levels, increased l-arginine uptake due to simvastatin treatment was associated with increased CAT-1 and eNOS mRNA levels, leading to higher NO production in TR-iBRB cells. Thus, simvastatin might be a good modulator for diabetic retinopathy therapy by increasing of the l-arginine uptake and improving endothelial function in retinal capillary endothelial cells.

Renal Collectrin Protects against Salt-Sensitive Hypertension and Is Downregulated by Angiotensin II.

Collectrin, encoded by the Tmem27 gene, is a transmembrane glycoprotein with approximately 50% homology with angiotensin converting enzyme 2, but without a catalytic domain. Collectrin is most abundantly expressed in the kidney proximal tubule and collecting duct epithelia, where it has an important role in amino acid transport. Collectrin is also expressed in endothelial cells throughout the vasculature, where it regulates L-arginine uptake. We previously reported that global deletion of collectrin leads to endothelial dysfunction, augmented salt sensitivity, and hypertension. Here, we performed kidney crosstransplants between wild-type (WT) and collectrin knockout (Tmem27Y/- ) mice to delineate the specific contribution of renal versus extrarenal collectrin on BP regulation and salt sensitivity. On a high-salt diet, WT mice with Tmem27Y/- kidneys had the highest systolic BP and were the only group to exhibit glomerular mesangial hypercellularity. Additional studies showed that, on a high-salt diet, Tmem27Y/- mice had lower renal blood flow, higher abundance of renal sodium-hydrogen antiporter 3, and lower lithium clearance than WT mice. In WT mice, administration of angiotensin II for 2 weeks downregulated collectrin expression in a type 1 angiotensin II receptor-dependent manner. This downregulation coincided with the onset of hypertension, such that WT and Tmem27Y/- mice had similar levels of hypertension after 2 weeks of angiotensin II administration. Altogether, these data suggest that salt sensitivity is determined by intrarenal collectrin, and increasing the abundance or activity of collectrin may have therapeutic benefits in the treatment of hypertension and salt sensitivity.

Cellular transport of l-arginine determines renal medullary blood flow in control rats, but not in diabetic rats despite enhanced cellular uptake capacity.

Diabetes mellitus is associated with decreased nitric oxide bioavailability thereby affecting renal blood flow regulation. Previous reports have demonstrated that cellular uptake of l-arginine is rate limiting for nitric oxide production and that plasma l-arginine concentration is decreased in diabetes. We therefore investigated whether regional renal blood flow regulation is affected by cellular l-arginine uptake in streptozotocin-induced diabetic rats. Rats were anesthetized with thiobutabarbital, and the left kidney was exposed. Total, cortical, and medullary renal blood flow was investigated before and after renal artery infusion of increasing doses of either l-homoarginine to inhibit cellular uptake of l-arginine or Nω-nitro- l-arginine methyl ester (l-NAME) to inhibit nitric oxide synthase. l-Homoarginine infusion did not affect total or cortical blood flow in any of the groups, but caused a dose-dependent reduction in medullary blood flow. l-NAME decreased total, cortical and medullary blood flow in both groups. However, the reductions in medullary blood flow in response to both l-homoarginine and l-NAME were more pronounced in the control groups compared with the diabetic groups. Isolated cortical tubular cells displayed similar l-arginine uptake capacity whereas medullary tubular cells isolated from diabetic rats had increased l-arginine uptake capacity. Diabetics had reduced l-arginine concentrations in plasma and medullary tissue but increased l-arginine concentration in cortical tissue. In conclusion, the reduced l-arginine availability in plasma and medullary tissue in diabetes results in reduced nitric oxide-mediated regulation of renal medullary hemodynamics. Cortical blood flow regulation displays less dependency on extracellular l-arginine and the upregulated cortical tissue l-arginine may protect cortical hemodynamics in diabetes.

R59949, a diacylglycerol kinase inhibitor, inhibits inducible nitric oxide production through decreasing transplasmalemmal L-arginine uptake in vascular smooth muscle cells.

Although diacylglycerol kinase (DGK) is known to be expressed in vascular smooth muscle cell, its functional significance remains to be clarified. We hypothesized that DGK is involved in the pathway of cytokine-induced nitric oxide (NO) production in vascular smooth muscle cells. The purpose of this study was to investigate the effects of R59949, a diacylglycerol kinase inhibitor, on inducible nitric oxide production in vascular smooth muscle cell. Cultured rat aortic smooth muscle cells (RASMCs) were used to elucidate the effects of R59949 on basal and interleukin-1β (IL-1β)-induced NO production. The effects of R59949 on protein and mRNA expression of induced nitric oxide synthase (iNOS) and on transplasmalemmal L-arginine uptake were also evaluated using RASMCs. Treatment of RASMCs with R59949 (10μM) inhibited IL-1β (10ng/ml)-induced NO production but not basal NO production. Neither protein nor mRNA expression level of iNOS after stimulation with IL-1β was significantly affected by R59949. Estimated enzymatic activities of iNOS in RASMCs were comparable in the absence and presence of R59949. Stimulation of RASMCs with IL-1β caused a marked increase in transplasmalemmal L-arginine uptake into RASMCs. L-Arginine uptake in the presence of IL-1β was markedly inhibited by R59949, while basal L-arginine uptake was not significantly affected by R59949. Both IL-1β-induced NO production and L-arginine uptake were abolished in the presence of cycloheximide (1μM). The results indicate that R59949 inhibits inducible NO production through decreasing transplasmalemmal L-arginine uptake. DGK is suggested to be involved in cytokine-stimulated L-arginine transport and regulate its intracellular concentration in vascular smooth muscle cell.

Deprivation of L-Arginine Induces Oxidative Stress Mediated Apoptosis in Leishmania donovani Promastigotes: Contribution of the Polyamine Pathway.

The growth and survival of intracellular parasites depends on the availability of extracellular nutrients. Deprivation of nutrients viz glucose or amino acid alters redox balance in mammalian cells as well as some lower organisms. To further understand the relationship, the mechanistic role of L-arginine in regulation of redox mediated survival of Leishmania donovani promastigotes was investigated. L-arginine deprivation from the culture medium was found to inhibit cell growth, reduce proliferation and increase L-arginine uptake. Relative expression of enzymes, involved in L-arginine metabolism, which leads to polyamine and trypanothione biosynthesis, were downregulated causing decreased production of polyamines in L-arginine deprived parasites and cell death. The resultant increase in reactive oxygen species (ROS), due to L-arginine deprivation, correlated with increased NADP+/NADPH ratio, decreased superoxide dismutase (SOD) level, increased lipid peroxidation and reduced thiol content. A deficiency of L-arginine triggered phosphatidyl serine externalization, a change in mitochondrial membrane potential, release of intracellular calcium and cytochrome-c. This finally led to DNA damage in Leishmania promastigotes. In summary, the growth and survival of Leishmania depends on the availability of extracellular L-arginine. In its absence the parasite undergoes ROS mediated, caspase-independent apoptosis-like cell death. Therefore, L-arginine metabolism pathway could be a probable target for controlling the growth of Leishmania parasites and disease pathogenesis.

The Vascular Endothelium in Chronic Kidney Disease: A Novel Target for Aerobic Exercise.

Endothelial dysfunction occurs in chronic kidney disease (CKD) and increases the risk for cardiovascular disease. The mechanisms of endothelial dysfunction seem to evolve throughout kidney disease progression, culminating in reduced L-arginine transport and impaired nitric oxide bioavailability in advanced disease. This review examines the hypothesis that aerobic exercise may reverse endothelial dysfunction by improving endothelial cell L-arginine uptake in CKD.

L-Arginine Transport and Nitric Oxide Production in Kinin Receptor B1-/- Endothelial Cells.

Kinins are important vasoactive peptides, but the role of the B1 receptor subtype in the vascular control is poorly understood. This study analyzed the nitric oxide (NO) release, L-arginine (L-Arg) uptake and the expression of the cationic amino acid transporter (CAT) -1 in endothelial cells obtained from B1 receptor knockout (B1-/-) and wild type (WT) mice. NO production was assessed through a fluorescent dye in living cells stimulated with acetylcholine. L-Arg uptake was determined indirectly in the culture medium by HPLC, in the presence or absence of the CAT-1 blocker N-ethylmaleimide (NEM). CAT-1 mRNA levels and protein expression were determined by qPCR and western blot, respectively. NO release was significantly reduced in B1-/- when compared to WT cells. This result was accompanied by a decreased rate in the L-Arg uptake by B1-/- cells. Incubation with NEM impaired the L-Arg uptake in WT, but had no effect in B1-/- cells. Protein expression and mRNA levels for CAT-1 were reduced in B1-/- in comparison to WT cells. These findings suggest an important role of the endothelial B1 receptor in the vascular control by interfering with CAT-1 expression, L-Arg uptake and NO release.

Cardio-protective effects of combined l-arginine and insulin: Mechanism and therapeutic actions in myocardial ischemia-reperfusion injury

Reduced nitric oxide (NO) bioavailability plays a central role in the pathogenesis of myocardial ischemia-reperfusion injury (I-R), and reduced l-arginine transport via cationic amino acid transporter-1 is a key contributor to the reduced NO levels. Insulin can increase NO levels by increasing the transport of its substrate l-arginine but insulin alone exerts minimal cardiac protection in I-R. We hypothesized that combined insulin and l-arginine may provide cardioprotective effects in the setting of myocardial I-R. The effect of supplemental insulin, l-arginine and the combination was examined in cardiomyocytes exposed to hypoxia/reoxygenation and in isolated perfused mouse hearts undergoing ischemia/reperfusion. When compared to controls, cardiomyocytes treated upon reoxygenation with 1nM insulin+1mM l-arginine exhibited significant (all P<0.05) improvements in NO generation and mitochondrial membrane potential, with a concomitant fall in reactive oxygen species production and LDH release. Insulin also increased l-arginine uptake following hypoxia-reoxygenation (P<0.05; n=4–6). In langendorff perfused isolated mouse hearts, combined l-arginine-insulin treatment upon reperfusion significantly (all P<0.05; n=9–11) improved recovery of left ventricular developed pressure, rate pressure product and end diastolic pressure following ischemia, independent of any changes in post-ischemic coronary flow, together with significantly lower LDH release. The observed improvements were greater than l-arginine or insulin treatment alone. In isolated cardiomyocytes (n=3–5), 1nM insulin caused cationic amino acid transporter-1 to redistribute to the cellular membrane from the cytosol and the effects of insulin on l-arginine uptake were partially dependent on the PI3K/Akt pathway. l-arginine-insulin treatment may be a novel strategy to ameliorate I-R injury.

Mycoplasma gallisepticum inactivated by targeting the hydrophobic domain of the membrane preserves surface lipoproteins and induces a strong immune response.

An innovative approach for inactivation of Mycoplasma gallisepticum using the hydrophobic photoinduced alkylating probe 1, 5-iodonaphthylazide (INA) is described. Treatment of washed M. gallisepticum mid-exponential culture (0.2 mg cell protein /mL) with INA followed by irradiation with far-ultraviolet light (310–380 nm) completely abolished viability. Transmission electron microscopy showed that the majority of the inactivated M. gallisepticum were comparable in size to intact cells, but that part of the INA-treated M. gallisepticum preparation also contained low density cells and membrane vesicles. Confocal microscopy revealed that untreated M. gallisepticum cells were internalized by chicken red blood cells (c-RBCs), whereas the INA-inactivated cells remained attached to the outer surface of the c-RBCs. INA treatment of M. gallisepticum resulted in a complete inactivation of F0F1 –ATPase and of the L-arginine uptake system, but the cytoplasmatic soluble NADH2 dehydrogenase was only partially affected. Western blot analysis of the lipoprotein fraction showed that the INA-treated M. gallisepticum retained their lipoproteins. Following subcutaneous injection of M. gallisepticum INA-bacterin, 100% and 68.8% of chickens were positive by the rapid serum agglutination test and enzyme-linked immunosorbent assay respectively, 2 weeks post-injection. These data suggest that the photoinducible alkylating agent INA inactivates M. gallisepticum but preserves its surface lipoproteins and thus has the potential to be used as a general approach for the inactivation of mycoplasmas for vaccine development.

L-arginine stimulates CAT-1-mediated arginine uptake and regulation of inducible nitric oxide synthase for the growth of chick intestinal epithelial cells.

L-arginine (L-Arg) uptake is mediated by members of cationic amino acid transporter (CAT) family and may coincide with the induction of nitric oxide synthases (NOS). The present study was conducted to investigate the extracellular concentrations of L-Arg regulating the CAT-1, CAT-4 and inducible NOS (iNOS) in chick intestinal epithelial cells. The cells were cultured for 4days in Arg-free Dulbecco's modified Eagle's medium containing 10, 100, 200, 400, or 600μM L-Arg. Cell viability, nitric oxide (NO) concentrations, uptake and metabolism of L-[3H]-Arg as well as expression of CAT-1, CAT-4, and iNOS were determined. Our results showed that L-Arg enhances cell growth with a maximal response at 10-400μM. Addition of 100, 200, or 400μM L-Arg increased the L-[3H]-Arg uptake, which was associated with greater conversion of L-[3H]-citrulline and NO production in comparison with 10μM L-Arg group. Increasing extracellular concentrations of L-Arg from 10 to 400μM dose dependently increased the levels of CAT-1 mRNA and protein, while no effect on CAT-4 mRNA abundance was found. Furthermore, supplementation of 100, 200, or 400μM L-Arg upregulated the expression of iNOS mRNA, and the relative protein levels for iNOS in 200 and 400μM L-Arg groups were higher than those in 10 and 100μM L-Arg groups. Collectively, we conclude that the CAT-1 isoform plays a role in L-Arg uptake, and L-Arg-mediated elevation of NO via iNOS promotes the growth of chick intestinal epithelial cells.