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Purpose We report a patient with a human cationic amino acid transporter 2 (CAT-2) defect discovered due to a suspected arginase 1 deficiency observed in newborn screening (NBS). Methods A NBS sample was analyzed using tandem mass spectrometry. Screen results were confirmed by plasma and urine amino acid quantification. Molecular diagnosis was done using clinical exome sequencing. Dimethylated arginines were determined by HPLC and nitrate/nitrite levels by a colorimetric assay. The metabolomic profile was analyzed using 1D nuclear magnetic resonance spectroscopy. Results A Spanish boy of nonconsanguineous parents had high arginine levels in a NBS blood sample. Plasma and urinary cationic amino acids were high. Arginase enzyme activity in erythrocytes was normal and no pathogenic mutations were identified in the ARG1 gene. Massive parallel sequencing detected two loss-of-function mutations in the SLC7A2 gene. Currently, the child receives a protein-controlled diet of 1.2 g/kg/day with protein-and amino-acid free infant formula, 30 g/day, and is asymptomatic. Conclusion We identified a novel defect in human CAT-2 due to biallelic pathogenic variants in the SLC7A2 gene. The characteristic biochemical profile includes high plasma and urine arginine, ornithine, and lysine levels. NBS centers should know of this disorder since it can be detected in arginase 1 deficiency screening.

The cationic amino acid transporter (CAT) protein family transports lysine and arginine in cellular amino acid pools. We hypothesized that CAT expression changes in pig skeletal muscles during rapid pig postnatal development. We aimed to investigate the tissue distribution and changes in the ontogenic expression of CATs in pig skeletal muscles during postnatal development. Six piglets at 1, 12, 26, 45, and 75 days old were selected from six litters, and their longissimus dorsi (LD), biceps femoris (BF), and rhomboideus (RH) muscles, and their stomach, duodenum, jejunum, ileum, colon, liver, kidney, heart, and cerebrum were collected. CAT-1 was expressed in all the 12 tissues investigated. CAT-2 (CAT-2A isoform) expression was highest in the skeletal muscle and liver and lowest in the jejunum, ileum, kidney, and heart. CAT-3 was expressed mainly in the colon and detected in the jejunum, ileum, and cerebrum. The CAT-1 expression was higher in the skeletal muscle of day 1 pigs than in that of older pigs (P 0.05). The CAT-2 mRNA level was lowest at day 1, but increased with postnatal development (P 0.05). There was no significant change in CAT-1 expression among the LD, BF, and RH during postnatal development (P 0.05); however, there was a change in CAT-2 expression. The CAT-2 expression was highest in the LD of 12-, 26-, 45-, and 75-day-old pigs, followed by the BF and RH (P 0.05). These results suggest that CAT-1 and CAT-2 play different roles in pig skeletal muscles during postnatal development.

Many proteins are localized at the vacuolar membrane, but most of them are still poorly described, due to the inaccessibility of this membrane from the extracellular environment. This work focused on the characterization of the CAT2 transporter from S. lycopersicum (SlCAT2) that was previously overexpressed in E. coli and reconstituted in proteoliposomes for transport assay as [3H]Arg uptake. The orientation of the reconstituted transporter has been attempted and current data support the hypothesis that the protein is inserted in the liposome in the same orientation as in the vacuole. SlCAT2 activity was dependent on the pH, with an optimum at pH 7.5. SlCAT2 transport activity was stimulated by the increase of internal osmolality from 0 to 175 mOsmol while the activity was inhibited by the increase of external osmolality. K+, Na+, and Mg2+ present on the external side of proteoliposomes at physiological concentrations, inhibited the transport activity, differently, the cations had no effect when included in the internal proteoliposome compartment. This data highlighted an asymmetric regulation of SlCAT2. Cholesteryl hemisuccinate, included in the proteoliposomal membrane, stimulated the SlCAT2 transport activity. The homology model of the protein was built using, as a template, the 3D structure of the amino acid transporter GkApcT. Putative substrate binding residues and cholesterol binding domains were proposed. Altogether, the described results open new perspectives for studying the response of SlCAT2 and, in general, of plant vacuolar transporters to metabolic and environmental changes.

Chenopodium quinoa Wild is a &ldquo, pseudocereal&rdquo, grain which attracts a lot of attention in the scientific community as it has a positive effect on health. Here, we investigate the presence of biologically active O-prenylated phenylpropanoids in the ethanol extract of commercially available quinoa seeds. We claim that 4&prime, Geranyloxyferulic acid (GOFA) was the only phytochemical product found that belongs to quinoa&rsquo, s group secondary metabolites. We studied the changes in the oxidative and inflammatory status of the cellular environment in HCT 116 cell line processed with quinoa extract and its component GOFA, the implementation was done through the analysis of the antioxidant enzymes (SOD and CAT), the pro-inflammatory components (iNOS, IL-6 and TNF-&alpha, ), and the products of intermediary metabolism (ONOO&minus, O2&minus, ). Moreover, the l-arginine uptake was proposed as a target of the tested compounds. We demonstrated that the GOFA, through a decrease of the CAT-2B expression, leads to a reduction of the l-arginine uptake, downregulating the harmful iNOS and restoring the altered redox state. These results propose a new molecular target involved in the reduction of the critical inflammatory process responsible for the cancer progression.

We investigated the effects of hepatic ischemia/reperfusion (I/R) injury on asymmetric dimethylarginine (ADMA, a nitric oxide synthase inhibitor), protein methyltransferase (PRMT) and dimethylarginine dimethylaminohydrolase (DDAH) (involved, resp., in ADMA synthesis and degradation), and the cationic transporter (CAT). Male Wistar rats were subjected to 30 or 60 min hepatic ischemia followed by 60 min reperfusion. ADMA levels in serum and bile were determined. Tissue ADMA, DDAH activity, DDAH-1 and CAT-2 protein, DDAH-1 and PRMT-1 mRNA expression, GSH/GSSG, ROS production, and lipid peroxidation were detected. ADMA was found in bile. I/R increased serum and bile ADMA levels while an intracellular decrease was detected after 60 min ischemia. Decreased DDAH activity, mRNA, and protein expression were observed at the end of reperfusion. No significant difference was observed in GSH/GSSG, ROS, lipid peroxidation, and CAT-2; a decrease in PRMT-1 mRNA expression was found after I/R. Liver is responsible for the biliary excretion of ADMA, as documented here for the first time, and I/R injury is associated with an oxidative stress-independent alteration in DDAH activity. These data are a step forward in the understanding of the pathways that regulate serum, tissue, and biliary levels of ADMA in which DDAH enzyme plays a crucial role.

Systemic inflammation and oxidative stress are features of normal pregnancy and, in excess, contribute to the pathogenesis of preeclampsia. Inflammatory cell activation stimulates uptake of arginine (the precursor for nitric oxide) by transport system y + , expression of one of its genes (CAT-2) together with inducible nitric oxide synthase, leading to nitric oxide production. We investigated whether these changes occur in peripheral blood mononuclear cells in normal pregnancy and are exaggerated in preeclampsia. Samples from matched trios of nonpregnant, normal pregnant, and preeclamptic women were studied. Arginine transport was characterized, and the expression of inducible nitric oxide synthase and cell-specific nitric oxide production were measured. Arginine uptake by system y + was significantly increased ( P P P

DMD (Duchenne muscular dystrophy) is an incurable rapidly worsening neuromuscular degenerative disease caused by the absence of dystrophin. In skeletal muscle a lack of dystrophin disrupts the recruitment of neuronal NOS (nitric oxide synthase) to the sarcolemma thus affecting NO (nitric oxide) production. Utrophin is a dystrophin homologue, the expression of which is greatly up-regulated in the sarcolemma of dystrophin-negative fibres from mdx mice, a mouse model of DMD. Although cardiomyopathy is an important cause of death, little is known about the NO signalling pathway in the cardiac muscle of DMD patients. Thus we used cardiomyocytes and hearts from two month-old mdx and mdx:utrophin−/− (double knockout) mice (mdx:utr) to study key steps in NO signalling: L-arginine transporters, NOS and sGC (soluble guanylyl cyclase). nNOS did not co-localize with dystrophin or utrophin to the cardiomyocyte membrane. Despite this nNOS activity was markedly decreased in both mdx and mdx:utr mice, whereas nNOS expression was only decreased in mdx:utr mouse hearts, suggesting that utrophin up-regulation in cardiomyocytes maintains nNOS levels, but not function. sGC protein levels and activity remained at control levels. Unexpectedly, L-arginine transporter expression and function were significantly increased, suggesting a novel biochemical compensatory mechanism of the NO pathway and a potential entry site for therapeutics.

Inducible nitric oxide (NO) synthase (iNOS) is a stress response protein upregulated in inflammatory conditions, and NO may suppress cellular proliferation. We hypothesized that preventing l-arginine (l-arg) uptake in endothelial cells would prevent lipopolysaccharide/tumor necrosis factor-α (LPS/TNF)-induced, NO-mediated suppression of cellular proliferation. Bovine pulmonary arterial endothelial cells (bPAEC) were treated with LPS/TNF or vehicle (control), and either 10 mM l-leucine [l-leu; a competitive inhibitor of l-arg uptake by the cationic amino acid transporter (CAT)] or its vehicle. In parallel experiments, iNOS or arginase II were overexpressed in bPAEC using an adenoviral vector (AdiNOS or AdArgII, respectively). LPS/TNF treatment increased the expression of iNOS, arginase II, CAT-1, and CAT-2 mRNA in bPAEC, resulting in greater NO and urea production than in control bPAEC, which was prevented by l-leu. LPS/TNF treatment resulted in fewer viable cells than in controls, and LPS/TNF-stimulated bPAEC treated with l-leu had more viable cells than LPS/TNF treatment alone. LPS/TNF treatment resulted in cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase expression, which was attenuated by l-leu. AdiNOS reduced viable cell number, and treatment of AdiNOS transfected bPAEC with l-leu preserved cell number. AdArgII increased viable cell number, and treatment of AdArgII transfected bPAEC with l-leu prevented the increase in cell number. These data demonstrate that iNOS expression in pulmonary endothelial cells leads to decreased cellular proliferation, which can be attenuated by preventing cellular l-arg uptake. We speculate that CAT activity may represent a novel therapeutic target in inflammatory lung diseases characterized by NO overproduction.

We have recently shown that inhibition of nitric oxide (NO) synthesis by asymmetrical dimethylarginine (ADMA) accelerated endothelial cell (EC) senescence which was prevented by coincubation with L-arginine; however the effect of long-term treatment of l-arginine alone on senescence of ECs have not been investigated. Human ECs were cultured in medium containing different concentrations of L-arginine until senescence. L-Arginine paradoxically accelerated senescence indicated by inhibiting telomerase activity. Moreover, L-arginine decreased NO metabolites, increased peroxynitrite, and 8-iso-prostaglandin F(2alpha) formation. In old cells, the mRNA expression of human amino acid transporter (hCAT)2B, the activity and protein expression of arginase II were upregulated indicated by enhanced urea, L-ornithine, and L-arginine consumption. Inhibition of arginase activity, or transfection with arginase II siRNA prevented L-arginine-accelerated senescence. The most possible explanation for the paradoxical acceleration of senescence by L-arginine so far may be the translational and posttranslational activation of arginase II.

Background l -arginine transport mediated by type-2 cationic amino acid transporter (CAT-2) isozymes is one crucial mechanism that regulates nitric oxide (NO) production via inducible nitric oxide synthase (iNOS). We sought to investigate the effects of heme oxygenase-1 (HO-1) overexpression on CAT-2 isozymes, e.g., CAT-2, CAT-2A, and CAT-2B. Materials and Methods Adult male Sprague Dawley rats were allocated to receive lipopolysaccharide (LPS), normal saline, hemin (a HO-1 inducer), tin protoporphyrin (SnPP, a HO-1 inhibitor), LPS plus hemin, or LPS plus hemin plus SnPP. After maintaining for 6 h, rats were sacrificed and the expression and activity of individual enzyme was evaluated. Results LPS increased HO activity, HO-1 concentration, NO production, l -arginine transport, and concentrations of iNOS, CAT-2, and CAT-2B in rat lungs and kidney. LPS also increased HO activity, HO-1 concentration, NO production, l -arginine transport, and iNOS concentration but decreased CAT-2 and CAT-2B concentrations in rat liver. LPS increased CAT-2A concentration in rat liver but did not affect CAT-2A concentration in rat lungs and kidney. Hemin further increased HO activity and induced HO-1 overexpression in the lungs, kidney, and liver from LPS-treated rats. In addition, the effects of LPS on NO production, l -arginine transport, and concentrations of iNOS and CAT-2 isozymes were significantly attenuated by hemin. SnPP, on the other hand, reversed the effects of hemin. Conclusions HO-1 overexpression significantly attenuates endotoxin-induced increases in NO production and l -arginine transport. Induction of HO-1 overexpression also significantly attenuates the effects of endotoxin on the expression of iNOS and CAT-2 isozymes in septic rats.

Background: The decrease in glomerular filtration rate (GFR), which is characteristic of obstructive uropathy, was suggested to be associated with attenuated nitric oxide (NO) generation. Since availability of L-arginine, the sole precursor for NO, governs NO synthesis, we aimed to determine the role of glomerular arginine transport in rats subjected to 24 h of bilateral ureteral ligation (BUO). Methods: Glomerular arginine transport was measured by uptake of radiolabeled arginine ([3H]-L-arginine), cationic amino acid transporters (CAT)-1 and -2 and arginases I and II mRNA expression were determined using reverse transcription-polymerase chain reaction. CAT-1, arginase I, and arginase II protein contents were evaluated by Western blotting. Results:L-Arginine transport by freshly harvested glomeruli from BUO rats was significantly augmented than in controls. The aforementioned findings were associated with a significant increase in glomerular CAT-1 mRNA expression, while CAT-2 mRNA was unchanged. Western blotting demonstrated a significant increase in CAT-1 abundance in BUO. Expression of both glomerular arginase I and II mRNA and protein content were significantly elevated in BUO. Conclusions: BUO induces an increase in glomerular arginine transport via upregulation of CAT-1, probably due to increase in arginine utilization by a non-NO pathway.

Purpose. The transcellular arginine transportation via cationic amino acid transporter (CAT) is the rate-limiting step in nitric oxide (NO) synthesis, which is crucial in intraocular inflammation. In this study, CAT isoforms and inducible nitric oxide synthase (iNOS) expression was investigated in endotoxin-induced uveitis (EIU).Methods.EIU was induced in Lewis rats by lipopolysaccharide (LPS) injection. In the treatment group, the rats were injected intraperitoneally with the proteasome inhibitor bortezomib before EIU induction. After 24 hours, leukocyte quantification, NO measurement of the aqueous humor, and histopathological examination were evaluated. The expression of CAT isoforms and iNOS was determined by reverse transcription-polymerase chain reaction, western blotting, and immunofluorescence staining. Nuclear factor-kappa B (NF-κB) binding activity was evaluated by electrophoretic mobility shift assay. The mouse macrophage cell line RAW 264.7 was used to validate thein vivofindings.Results. LPS significantly stimulated iNOS, CAT-2A, and CAT-2B mRNA and protein expression but did not affect CAT-1 in EIU rats and RAW 264.7 cells. Bortezomib attenuated inflammation and inhibited iNOS, CAT-2A, and CAT-2B expression through NF-κB inhibition.Conclusions.CAT-2 and iNOS, but not CAT-1, are specifically involved in EIU. NF-κB is essential in the induction of CAT-2 and iNOS in EIU.

Leishmaniasis is a vector-borne disease found in many countries worldwide. The causative agent of the disease, Leishmania spp., lives as an obligate intracellular parasite within mammalian hosts. Since tissue macrophages are major target cells for parasite replication, the outcome of infection depends largely on the activation status of these cells. L-arginine is a crucial amino acid required for both nitric oxide (NO)-mediated parasite killing and polyamine-mediated parasite replication. This review highlights the significance of L-arginine as a factor determining the outcomes of Leishmania infection in vitro and its influences on host immune responses in vivo. Various therapeutic approaches targeting L-arginine metabolic pathways during infections with Leishmania are also discussed.

L-arginine or its metabolites may be important pathogenetic factors in ischemic acute renal failure (iARF) in rats. It was found that the L-arginine-nitric oxide synthase-nitric oxide system plays an important role in the renal hemodynamic alterations in the early stages of diabetes. The iARF in diabetic rats is much more severe than the normal rats exposed to a same ischemia time. The purpose of the present study was to evaluated L-arginine uptake and its transporters and nitric oxide synthase isoform expression in tubuli and glomeruli of STZ-induced diabetic rats with iARF.iARF was induced by right nephrectomy and left renal artery clamping for 60 min followed by a 60 min reflow period. iARF was induced in STZ diabetes rats two weeks after intraperitoneal streptozotocin (60 mg/kg body weight) and in normal control rats. L-arginine uptake, L-arginine transporters (CAT1 and CAT2) and nitric oxide synthases (iNOS, eNOS, and bNOS) were determined by RT-PCR) in both glomeruli and tubuli preparations.The STZ diabetic rats compared with the non diabetic normal rats have a higher glomerular L-arginine uptake, higher iNOS mRNA, lower eNOS mRNA, and lower tubular CAT1 mRNA, eNOS mRNA, and bNOS mRNA. The diabetic iARF after one hour of reperfusion had lower glomerular L-arginine uptake, lower CAT1 mRNA, lower eNOS mRNA, lower bNOS, and higher tubular iNOS mRNA compared with iARF in normal rats.Our findings suggest a prolonged and more severe post-glomerular vasoconstriction very early after the reflow in the iARF of STZ diabetic rats compared with the iARF in the normal control rats. That may be a plausible explanation to the very significant decline in GFR and tubular necrosis that characterize the iARF in diabetic rats.

Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature cells that expand during benign and cancerassociated inflammation and are characterized by their ability to inhibit T cell immunity. Increased metabolism of L-Arginine (L-Arg), through the enzymes arginase 1 and NO synthase 2 (NOS2), is well documented as a major MDSC suppressive mechanism. Therefore, we hypothesized that restricting MDSC uptake of L-Arg is a critical control point to modulate their suppressor activity. Using murine models of prostate-specific inflammation and cancer, we have identified the mechanisms by which extracellular L-Arg is transported into MDSCs. We have shown that MDSCs recruited to localized inflammation and tumor sites upregulate cationic amino acid transporter 2 (Cat2), coordinately with Arg1 and Nos2. Cat2 expression is not induced in MDSCs in peripheral organs. CAT2 contributes to the transport of L-Arg in MDSCs and is an important regulator of MDSC suppressive function. MDSCs that lack CAT2 have significantly reduced suppressive ability ex vivo and display impaired capacity for regulating T cell responses in vivo as evidenced by increased T cell expansion and decreased tumor growth in Cat22/2 mice. The abrogation of suppressive function is due to low intracellular L-Arg levels, which leads to the impaired ability of NOS2 to catalyze L-Arg-dependent metabolic processes. Together, these findings demonstrate that CAT2 modulates MDSC function. In the absence of CAT2, MDSCs display diminished capacity for controlling T cell immunity in prostate inflammation and cancer models, where the loss of CAT2 results in enhanced antitumor activity.

Background L-Arginine transport mediated by type 2 cationic amino acid transporter (CAT-2) is one crucial mechanism that regulates nitric oxide production mediated by inducible nitric oxide synthase. Heme oxygenase (HO)-1 induction has been reported to significantly attenuate inducible nitric oxide synthase expression and nitric oxide production. The authors sought to explore the effects of HO-1 induction on CAT-2 expression and L-arginine transport. The effects of HO-1 induction on nuclear factor E2-related factor 2 (Nrf2) and nuclear factor kappaB (NF-kappaB) were also investigated. Methods Murine macrophages (RAW264.7 cells) were randomized to receive lipopolysaccharide, lipopolysaccharide plus hemin (an HO-1 inducer; 5, 50, or 500 microm), lipopolysaccharide plus hemin (5, 50, or 500 microm) plus tin protoporphyrin (an HO-1 inhibitor), or lipopolysaccharide plus hemin (5, 50, or 500 microm) plus hemoglobin (a carbon monoxide scavenger). Then, cell cultures were harvested and analyzed. Results Lipopolysaccharide significantly induced Nrf2 activation and HO-1 expression. Lipopolysaccharide also significantly induced NF-kappaB activation, CAT-2 expression, and L-arginine transport. In a dose-dependent manner, hemin enhanced the lipopolysaccharide-induced Nrf2 activation and HO-1 expression. In contrast, hemin, also in a dose-dependent manner, significantly attenuated the lipopolysaccharide-induced NF-kappaB activation, CAT-2 expression, and L-arginine transport. Furthermore, the effects of hemin were significantly reversed by both tin protoporphyrin and hemoglobin. Conclusions HO-1 induction significantly inhibited CAT-2 expression and L-arginine transport in lipopolysaccharide-stimulated macrophages, possibly through mechanisms involved activation of Nrf2 and inhibition of NF-kappaB. In addition, carbon monoxide mediated, at least in part, the effects of HO-1 induction on CAT-2 expression and L-arginine transport.

It has become evident that increased nitric oxide (NO) generation may be associated with production of reactive oxygen species, such as peroxynitrite (ONOO-). Peroxynitrite has been postulated to be responsible for several of the cytotoxic effects previously ascribed to NO. Since cellular arginine uptake has been shown to modulate nitric oxide synthase activity, we were intrigued to study the effect of ONOO- on arginine traffic in renal mesangial cells.Arginine uptake, CAT-1 and CAT-2 mRNA expression by northern blotting analysis, and CAT-1 protein content using western blotting were determined in mesangial cells pre-treated with peroxynitrite (0.1 and 0.5 mM) for 2 h.Peroxynitrite induced a significant increase in arginine uptake and CAT-2 mRNA expression compared with untreated cells. In contrast, CAT-1 mRNA expression and protein abundance were diminished.In rat mesangial cells, peroxynitrite augments arginine uptake via augmentation of CAT-2 while decreasing CAT-1 expression.

In murine macrophages, as a result of arginine catabolism during activation, citruline is produced under the effect of IFN-gamma and LPS, and ornithine and polyamines by IL-4 and IL-10. For proliferation, arginine is required from the extracellular medium and is used for protein synthesis. During activation, most arginine (95% in 6 h) was metabolized, while under proliferation only half was incorporated into proteins. Under basal conditions, this amino acid was preferentially transported by y(+)L activity. During activation, arginine transport increased drastically (4-5-fold) through y(+) cationic amino acid transporter (CAT) activity. By contrast, M-CSF induced only a modest increase in uptake (0.5-fold). The increase in arginine transport during activation, but not proliferation, was mediated by the SLC7A2/Cat2 gene. SLC7A1/Cat1 is constitutively expressed, and is not modified by proliferating or activating agents. M-CSF-dependent proliferation was not affected in the macrophages of SLC7A2 knockout mice; however, these cells showed a drastic reduction in the production of citruline or ornithine and polyamines during activation. The data show that a large increase in a specific transport system (CAT2) is necessary for activation-induced arginine metabolism, while arginine is in excess for the requirements of proliferation and a modest increase in transport occurs.

Endothelial cell dysfunction (ECD) is a common feature of chronic renal failure (CRF). Defective nitric oxide (NO) generation due to decreased endothelial NO synthase (eNOS) activity is a crucial parameter characterizing ECD. L-arginine is the sole precursor for NO biosynthesis. Among several transporters that mediate L-arginine uptake, cationic amino-acid transporter-1 (CAT-1) acts as the specific arginine transporter for eNOS. Our hypothesis implies that CAT-1 is a major determinant of eNOS activity in CRF. We studied glomerular and aortic arginine uptake, CAT-1, and CAT-2 messenger ribonucleic acid (mRNA) expression, and CAT-1 protein in: (a) rats 6 weeks following 5/6 nephrectomy (CRF), (b) sham-operated animals, and (c) rats with CRF treated orally with either atorvastatin or arginine in drinking water (modalities which have been shown to enhance eNOS activity and improve endothelial function). Both glomerular and aortic arginine transport were significantly decreased in CRF. Treatment with either arginine or atorvastatin abolished the decrease in arginine uptake in CRF rats. Using reverse transcriptase-polymerase chain reaction and Northern blotting, we found a significant increase in glomerular and aortic CAT-1 mRNA expression in CRF. Western blotting revealed that CAT-1 protein was decreased in CRF, but remained intact following arginine and atorvastatin administration. Renal and systemic arginine uptake is attenuated in CRF, through modulation of CAT-1 protein. These findings provide a possible novel mechanism to eNOS inactivation and endothelial dysfunction in uremia.

This study was designed to investigate bactericidal activity of and nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated peritoneal exudate macrophages (Mvarphi) from metallothionein (MT)-null mice. Control Mvarphi had a bactericidal effect on Staphylococcus aureus, but MT-null Mvarphi had significantly lower activity. NO is an important factor in the bactericidal function of Mvarphi. LPS-stimulated MT-null Mvarphi produced less NO than those of control mice. LPS-stimulated Mvarphi produce cytokines such as tumor necrosis factor (TNF)-alpha. TNF-alpha activate Mvarphi and stimulates NO production. We evaluated NO production by TNF-alpha-stimulated Mvarphi. MT-null Mvarphi produced less NO in response to TNF-alpha stimulation. Levels of expression of inducible NO synthase (iNOS) mRNA and production of iNOS protein in response to LPS stimulation were similar in MT-null and control cells, as were levels of expression of arginase, which competes in arginine metabolism with iNOS. No notable changes were found in arginine uptake or in expression of cationic amino acid transporter 2 (a major arginine transporter in Mvarphi) between control and MT-null Mvarphi. The rate of conversion of [(14)C]-l-arginine to citrulline, which is formed with NO by the action of iNOS, was much lower in MT-null Mvarphi than in control cells. These results indicate that the reduced production of NO in MT-deficient Mvarphi is due mainly to reduced activity of iNOS. Thus, MT plays important roles in bactericidal activity, NO production, and arginine metabolism in activated Mvarphi.

Catecholamines enhance inducible nitric oxide synthase (iNOS) expression that results in nitric oxide (NO) overproduction in lipopolysaccharide (LPS)-stimulated macrophages. L-arginine transport mediated by cationic amino acid transporters (including CAT-1, CAT-2, CAT-2A, and CAT-2B) is crucial in regulating iNOS activity. We sought to assess the effects of catecholamines on L-arginine transport and CAT isozyme expression in stimulated macrophages. Confluent RAW264.7 cells were cultured with LPS with or without catecholamines (epinephrine or norepinephrine, 5 x 10(-6) M) for 18 h. NO production, L-arginine transport, and enzyme expression were determined. Our data revealed that LPS co-induced iNOS, CAT-2, and CAT-2B expression, whereas CAT-1 and CAT-2A expression remained unaffected. Significant increases in NO production and L-arginine transport (approximately eight-fold and three-fold increases, respectively) were found in activated macrophages. Catecholamines significantly enhanced NO production and L-arginine transport (approximately 30% and 20% increases, respectively) in activated macrophages. Catecholamines also enhanced the expression of iNOS, CAT-1, and CAT-2A but not CAT-2 or CAT-2B in LPS-stimulated macrophages. Furthermore, the enhancement effects of catecholamines were inhibited by either dexamethasone or propranolol. We provide the first evidence to indicate that L-arginine transport in activated macrophages could be enhanced by catecholamines. Furthermore, this catecholamine-enhanced L-arginine transport might involve CAT-1 and CAT-2A but not CAT-2 or CAT-2B.

Expression of inducible nitric oxide synthase (iNOS) is generally accompanied by a parallel upregulation in L-arginine transport which is dependent, at least in part, on the synthesis of new carrier proteins. It is not clear however whether the induction of iNOS and its subsequent utilisation of L-arginine for NO synthesis contribute to the enhancement in L-arginine transport rates observed following induction of cells with pro-inXammatory mediators. To address this issue, we have transfected an iNOS construct in a pEGFP-N1 vector into HEK-293 cells and investigated the eVects this has on L-arginine transport. The expression of iNOS through transfection resulted in the production of signiWcant quantities of NO as detected by the standard Griess assay. Under these conditions, the transport of L-arginine was found to be unaltered, with rate of uptake being comparable in both transfected and non-transfected cells. Characterisation of the transporter(s) involved with uptake of L-arginine revealed features characteristic of the classical cationic amino acid transport system y + . Further analysis of the expression proWle of the cationic amino acid transporter (CAT) involved revealed the presence of transcripts for CAT-1 and CAT-2B. These data demonstrate that iNOS activity does not drive or enhance L-arginine transport despite the fact that HEK-293 cells transport L-arginine via the CATs, including CAT-2B which is thought to be critical for supply of substrate to iNOS.  2004 Published by Elsevier Inc.

In human saphenous vein endothelial cells (HSVECs), tumor necrosis factor-alpha (TNFalpha) and bacterial lipopolysaccharide (LPS), but neither interferon gamma (IFNgamma) nor interleukin 1beta (IL-1beta), stimulate arginine transport. The effects of TNFalpha and LPS are due solely to the enhancement of system y+ activity, whereas system y+L is substantially unaffected. TNFalpha causes an increased expression of SLC7A2/CAT-2B gene while SLC7A1/CAT-1 expression is not altered by the cytokine. The suppression of PKC-dependent transduction pathways, obtained with the inhibitor chelerytrhine, the inhibitor peptide of PKCzeta isoform, or chronic exposure to phorbol esters, does not prevent TNFalpha effect on arginine transport. Likewise, ERK, JNK, and p38 MAP kinases are not involved in the cytokine effect, since arginine transport stimulation is unaffected by their specific inhibitors. On the contrary, inhibitors of NF-kappaB pathway hinder the increase in CAT2B mRNA and the stimulation of arginine uptake. These results indicate that in human endothelial cells the activation of NF-kappaB pathway mediates the TNFalpha effects on arginine transport.

Augmented arginine uptake, through modulation of cationic amino acid transporter-1, increases GFR in diabetic rats. Background It is suggested that either arginine or its metabolites, nitric oxide and polyamines play a role in the renal hemodynamic alterations observed in the early stages of diabetes. Yet, the regulation of arginine transport in diabetic kidneys has never been studied. Methods Arginine uptake was determined in glomeruli harvested from control rats; diabetic rats (2weeks following an intraperitoneal injection of streptozotocin, 60mg/kg body weight); rats, 4days following left nephrectomy (a nondiabetic model of hyperfiltration); diabetes + lysine (0.5% in the drinking water to attenuate arginine uptake); and control + lysine. Results Glomerular arginine transport was significantly increased in diabetic rats, but remained unchanged following uninephrectomy. Lysine abolished the increase in arginine uptake in diabetic rats but had no effect in controls. The increase in creatinine clearance observed in diabetes was completely abolished by lysine. Using reverse transcription-polymerase chain reaction (RT-PCR), Northern blotting, and immunohistochemistry, we found a significant increase in glomerular cationic amino acid transporter-1 (CAT-1) expression in diabetic animals, which was unaffected by lysine. When human endothelial cells were incubated with arginine end products no effect on arginine transport was observed. However, only in the presence of 0.5mmol/L sodium nitroprusside (SNP) an augmented steady-state CAT-1 mRNA was demonstrated by RT-PCR. Conclusion In a rat model of early diabetes, glomerular arginine uptake is elevated through modulation of CAT-1 expression, thus, contributing to the pathogenesis of hyperfiltration. Increased nitric oxide formation may play a role in this process.

Cationic amino acid transporters play an important role in the intracellular supply of l-Arg and the generation of nitric oxide. Since the transport of l-Arg is voltage-dependent, we aimed at determining the intracellular l-Arg concentration and describing the transport of l-Arg in terms of Michaelis–Menten kinetics, taking into account membrane voltage. The human isoforms of the cationic amino acid transporters, hCAT-1, hCAT-2A, and hCAT-2B, were expressed in oocytes from Xenopus laevis and studied with the voltage clamp technique and in tracer experiments. We found that l-Arg was concentrated intracellularly by all hCAT isoforms and that influx and efflux, in the steady state of exchange, were nearly mirror images. Conductance measurements at symmetric concentrations of l-Arg (inside/outside) allowed us to determine KM and Vmax. The empty transporter of hCAT-2B featured an unexpected potassium conductance, which was inhibited by l-Arg.

The in vivo mechanisms by which glucocorticoids inhibit nitric oxide expression await detailed investigation. In cell culture experiments, glucocorticoids have been shown to inhibit inducible nitric oxide synthase (iNOS) formation and activity. Glucocorticoids can inhibit iNOS activity in cultured cells by blocking arginine transport and inhibiting tetrahydrobiopterin biosynthesis. We recently reported that changes in intrapulmonary formation of nitric oxide in endotoxemic rats correspond with changes in transcription of the predominant arginine transporter cationic amino acid transporter (CAT)-2. Realizing that hemorrhagic shock induces nitric oxide overproduction in intact animals, we sought to explore whether glucocorticoids attenuate hemorrhagic shock-induced increases in intrapulmonary nitric oxide formation and whether they might do so by inhibiting the formation of tetrahydrobiopterin, iNOS protein, and CAT-2. We randomly assigned 10 male Sprague-Dawley rats to receive dexamethasone or normal saline. Bleeding the animals to a mean systemic blood pressure of between 40 and 45 mmHg created the hemorrhagic shock. Dexamethasone abrogated the increase in exhaled nitric oxide concentrations caused by hemorrhagic shock. At the end of the experiment, plasma nitrate/nitrite values were lower in the dexamethasone group than in the control group. The iNOS protein concentrations were also lower in the dexamethasone group than in the control group. Dexamethasone decreased the intrapulmonary iNOS mRNA concentrations yet increased both guanosine triphosphate cyclohydrolase I mRNA and CAT-2 mRNA. Our results support the idea that dexamethasone inhibits nitric oxide formation in a manner that is independent of tetrahydrobiopterin and arginine transport yet dependent on downregulation of iNOS mRNA expression.

l-Arginine plays a central role in the normal function of several organs including the immune system. It is metabolized in macrophages by inducible nitric oxide synthase to produce nitric oxide, important in the cytotoxic mechanisms, and by arginase I (ASE I) and arginase II (ASE II) to synthesize l-ornithine and urea, the first being the precursor for the production of polyamines needed for cell proliferation. l-Arginine availability can modulate T cell function. Human T cells stimulated and cultured in the absence of l-arginine lose the expression of the TCR ζ-chain (CD3ζ) and have an impaired proliferation and a decreased cytokine production. The aim of this work was to test whether activated macrophages could modulate extracellular levels of l-arginine and alter T cell function, and to determine which metabolic pathway was responsible for this event. The results show that macrophages stimulated with IL-4 + IL-13 up-regulate ASE I and cationic amino acid transporter 2B, causing a rapid reduction of extracellular levels of l-arginine and inducing decreased expression of CD3ζ and diminished proliferation in normal T lymphocytes. Competitive inhibitors of ASE I or the addition of excess l-arginine lead to the re-expression of CD3ζ and recovery of T cell proliferation. In contrast, inducible nitric oxide synthase or ASE II failed to significantly reduce the extracellular levels of l-arginine and modulate CD3ζ expression. These results may provide new insights into the mechanisms leading to T cell dysfunction and the down-regulation of CD3ζ in cancer and chronic infectious diseases.

Inducible nitric oxide synthase and arginase activities are acknowledged as important players in human skin epidermal function. For proper enzyme function the substrate availability of L-arginine for both enzymes and thus its transport across the cell membrane via the y+-system (also named cationic amino acid transporters) is critical. Here, we examine the expression of cationic amino acid transporters and their functional role in modulating inducible nitric oxide synthase and arginase activities in human skin and primary keratinocytes, fibroblasts and endothelial cells as well as their impact on keratinocyte proliferation. Skin biopsies were found to express constitutively both cationic amino acid transporter-1 and cationic amino acid transporter-2 mRNA, an expression pattern known to occur in hepatocytes and muscle cells only. To determine the cellular components expressing cationic amino acid transporter, we analyzed the expression patterns in the different human skin cell types in vitro, i.e., in fibroblasts, dermal endothelial cells, and keratinocytes as well as in the HaCaT cell line. An ubiquitous cationic amino acid transporter-1 mRNA expression was found in all cells, whereas constitutive cationic amino acid transporter-2 mRNA expression occurs in resident keratinocytes and dermal endothelial cells only. De novo induction of cationic amino acid transporter-2 and inducible nitric oxide synthase by proinflammatory cytokines was seen in fibroblasts and HaCaT. Competitive inhibition of the cationic amino acid transporter-mediated L-arginine transport by culturing primary human keratinocytes in the presence of increased L-lysine concentration led to decreased inducible nitric oxide synthase and arginase activities with a concomitant significant decrease in keratinocyte proliferation. In summary, our results demonstrate that human keratinocytes constitutively express cationic amino acid transporters 1 and 2 and that cationic amino acid transporter mediated L-arginine influx, is essential for both inducible nitric oxide synthase and arginase enzyme activities, which in turn modulate proliferation and differentiation of human epidermal skin cells.

l-Arginine transport is augmented through up-regulation of tubular CAT-2 mRNA in ischemic acute renal failure in rats. Background Ischemic acute renal failure (iARF) is associated with increased nitric oxide (NO) production during the reperfusion period, as endothelial nitric oxide synthase (eNOS) is maximally activated, and renal tubular inducible NOS (iNOS) is stimulated. Increased NO production leads to augmented tubular injury, probably through the formation of peroxynitrite. l-Arginine (l-Arg), the only precursor for NO, is transported into cells by cationic amino acid transporters, CAT-1 and CAT-2. We hypothesized that the increased NO production observed in iARF may result from increased l-Arg uptake, which would be reflected in the augmented expression of l-Arg transporter(s). Methods Ischemic acute renal failure was induced in rats by right nephrectomy + left renal artery clamping for 60 minutes. l-Arg uptake was examined in freshly harvested glomeruli and tubuli from control, sham operated, and animals subjected to 15, 30, and 60 minutes, and 24 hours of reperfusion, following 60 minutes of ischemia. Using RT-PCR, renal tissues were examined further for the expression of iNOS, CAT-1, CAT-2, arginase I and arginase II. Results Tubular expression of iNOS mRNA was initiated by ischemia, continued to increase after 60 minutes of reperfusion, and decreased after 24 hours. l-Arg transport into glomeruli was similar in all experimental groups. l-Arg uptake into tubuli was markedly augmented following the 60-minute reperfusion, while it moderately increased after 24 hours of reperfusion. This was accompanied by a parallel, preferential increase in tubular CAT-2 mRNA expression at 60 minutes of reperfusion. CAT-1 mRNA expression was unchanged, as detected by RT-PCR. In addition, the expression of arginase II and arginase I mRNA was attenuated by 30 minutes and one hour of reperfusion, and returned to baseline values after 24 hours of reperfusion. Conclusions Ischemic ARF is associated with augmented tubular CAT-2 mRNA expression, which leads to enhanced l-Arg transport and increased NO production. This may contribute to the renal injury exhibited in iARF.

Pulmonary inflammation increases nitric oxide (NO) production via inducible nitric oxide synthase (iNOS). This study was performed to determine some of the factors that affect the availability of the NOS substrate, L-arginine (L-arg), in the intact lung subjected to silica-induced inflammation. Nitrate production, as an index of NO production, was significantly greater in silica-exposed lungs (53.5 +/- 12.1 nmol/90 min) compared with controls (22.5 +/- 5.1 nmol/90 min, P0.05). This was accompanied by greater (P0.0001) 90-min [(3)H]L-arg uptake (62 +/- 3% control, 82 +/- 1% silica), a significantly (P0.005) increased permeability-surface area product for L-arg (0.28 +/- 0.05 ml/min control, 0.63 +/- 0.07 ml/min silica), and a significantly (P0.001) increased urea production (1.16 +/- 0.08 micromol/90 min control, 1.77 +/- 0.06 micromol/90 min silica). There was no difference in eNOS protein between groups and eNOS mRNA was not detectable in either group, whereas silica exposure resulted in the appearance of both iNOS protein and mRNA. Silica exposure increased CAT-1 and CAT-2 mRNA approximately 8-fold compared with controls. We conclude that the increase in NO production in silica-exposed lungs was associated with increased L-arg uptake from the vasculature, presumably resulting from increased CAT-1 and CAT-2, and by increased L-arg metabolism via arginase.

One of the limiting steps in the regulation of nitric oxide (NO) synthesis is the availability of its precursor, L-arginine, which depends on the presence of a specific uptake system. A characterization of the L-arginine uptake mechanism in the golden hamster retina was performed. This mechanism was stereospecific, saturable, and monophasic, with an apparent of 56.1 +/- 2.0 microM and a maximum velocity of 36.0 +/- 2.8 pmol/mg prot/min. The basic amino acids L-lysine and L-ornithine but not D-arginine or the nitric oxide synthase inhibitors, N(omega)-nitro-L-arginine methyl ester and N(omega)-nitro-L-arginine impaired L-arginine influx. Preincubation with L-lysine for 1 h prior to the transport assay significantly stimulated L-arginine uptake. Saturation studies of L-arginine uptake performed at 12.00 and 24.00 h indicated a higher value of Vmax at midnight than at midday. When the hamsters were placed under constant darkness or constant light for 48 h and killed at equivalent time points, representing subjective day and subjective night, the differences in L-arginine influx disappeared. Semiquantitative RT-PCR analysis showed that the levels of mRNAs for both CAT-1 and CAT-2B were significantly higher at midnight than at midday. L-Arginine significantly increased cGMP accumulation in a time-dependent manner, with maximal effects during the night. Based on these results, it might be presumed that hamster retinal L-arginine uptake is regulated by the photic stimulus.

l-Arginine (l-Arg) is a semiessential amino acid that has altered availability in human ulcerative colitis (UC), a form of inflammatory bowel disease, and is beneficial in murine colitis induced by dextran sulfate sodium (DSS), a model with similarity to UC. We assessed the role of cationic amino acid transporter 2 (CAT2), the inducible transporter of l-Arg, in DSS colitis. Expression of CAT2 was upregulated in tissues from colitic mice and localized predominantly to colonic macrophages. CAT2-deficient (CAT2−/−) mice exposed to DSS exhibited worsening of survival, body weight loss, colon weight, and histological injury. These effects were associated with increased serum l-Arg and decreased tissue l-Arg uptake and inducible nitric oxide synthase protein expression. Clinical benefits of l-Arg supplementation in wild-type mice were lost in CAT2−/−mice. There was increased infiltration of macrophages, dendritic cells, granulocytes, and T cells in colitic CAT2−/−compared with wild-type mice. Cytokine profiling revealed increases in proinflammatory granulocyte colony-stimulating factor, macrophage inflammatory protein-1α, IL-15, and regulated and normal T cell-expressed and -secreted and a shift from an IFN-γ- to an IL-17-predominant T cell response, as well as an increase in IL-13, in tissues from colitic CAT2−/−mice. However, there were no increases in other T helper cell type 2 cytokines, nor was there a global increase in macrophage-derived proinflammatory cytokines. The increase in IL-17 derived from both CD4 and γδ T cells and was associated with colonic IL-6 expression. Thus CAT2 plays an important role in controlling inflammation and IL-17 activation in an injury model of colitis, and impaired l-Arg availability may contribute to UC pathogenesis.

The immunogenic mechanisms of the potent contact allergen nickel are not completely clear. Nitric oxide (NO) serves as a fundamental signalling and effector molecule in the immune system, but little is known about its possible role in immune reactions elicited by nickel. We investigated the effects of nickel on the L-arginine/inducible NO synthase (iNOS) system in a murine macrophage cell line, RAW 264.7. Both LPS-stimulated and non-stimulated RAW 264.7 cells were incubated in the presence of 0-100 μM nickel sulphate for 24 h. Subsequently, NO production, iNOS protein expression, L-arginine uptake and gene expression of iNOS and cationic amino acid transporter systems (CAT) were measured. We found that 100 μM NiSO4 increased LPS-induced nitrite production as well as the formation of [(3)H]-L-citrulline from [(3)H]-L-arginine in the RAW 264.7 cells. Correspondingly, the expression of iNOS gene and protein was also remarkably enhanced. Nevertheless, nickel had an inhibitory effect on L-arginine transport which disappeared gradually upon LPS-stimulation in parallel with an increase in NO output. LPS was found to significantly amplify CAT-3 as well as CAT-2 mRNA expression, mirroring the increase in L-arginine transport. In the range of 1-10 μM, NiSO4 did not have any additional effect on CAT mRNA expression, but at 100 μM it was able to enhance CAT-1 and CAT-3 mRNA expression upon LPS stimulation. Our data indicate that nickel interferes with macrophages' L-arginine/NOS system on multiple levels. Considering the potent biological effects of NO, these influences may contribute to nickel toxicity.

Restoration of the colonic epithelial barrier is an important response during colitis. L-arginine (L-Arg) is a semiessential amino acid that reduces murine colitis induced by Citrobacter rodentium . Cationic amino acid transporter (CAT) proteins increase L-Arg uptake into cells. L-Arg is utilized to produce nitric oxide (NO), by inducible NO synthase (iNOS), or L-ornithine (L-Orn) by arginase (Arg) enzymes. The latter is followed by generation of polyamines by ornithine decarboxylase (ODC) and L-proline (L-Pro) by ornithine aminotransferase (OAT). We show that L-Arg enhanced epithelial restitution in conditionally immortalized young adult mouse colon (YAMC) cells in a wound repair model, and in isolated mouse colonic epithelial cells (CECs), using a cell migration assay. Restitution was impaired by C. rodentium . Wounding induced CAT2, and inhibition of L-Arg uptake by the competitive inhibitor L-lysine (L-Lys) or by CAT2 shRNA, but not CAT1 shRNA, decreased restitution. Migration was impaired in CECs treated with L-Lys or from CAT2−/− mice. Wounding increased Arg1 expression, and inhibition of arginase with S-(2-boronoethyl)-L-cysteine (BEC) or Arg1 shRNA inhibited restitution in YAMC cells; cell migration in CECs was also impaired by BEC. Inhibition of ODC or iNOS did not alter restitution. L-Orn or L-Pro restored restitution in cells treated with BEC or Arg1 shRNA, whereas the polyamine putrescine had no benefit. Wounding increased OAT levels, OAT shRNA inhibited restitution, and L-Pro restored restitution in cells with OAT knockdown. Uptake of L-Arg, and its metabolism by Arg1 to L-Orn and conversion to L-Pro by OAT is essential for colonic epithelial wound repair.

Inflammatory bowel disease (IBD), consisting of Crohn's disease and ulcerative colitis (UC), results in substantial morbidity and is difficult to treat. New strategies for adjunct therapies are needed. One candidate is the semi-essential amino acid, L-arginine (L-Arg), a complementary medicine purported to be an enhancer of immunity and vitality in the lay media. Using dextran sulfate sodium (DSS) as a murine colonic injury and repair model with similarities to human UC, we assessed the effect of L-Arg, as DSS induced increases in colonic expression of the y(+) cationic amino acid transporter 2 (CAT2) and L-Arg uptake. L-Arg supplementation improved the clinical parameters of survival, body weight loss, and colon weight, and reduced colonic permeability and the number of myeloperoxidase-positive neutrophils in DSS colitis. Luminex-based multi-analyte profiling demonstrated that there was a marked reduction in proinflammatory cytokine and chemokine expression with L-Arg treatment. Genomic analysis by microarray demonstrated that DSS-treated mice supplemented with L-Arg clustered more closely with mice not exposed to DSS than to those receiving DSS alone, and revealed that multiple genes that were upregulated or downregulated with DSS alone exhibited normalization of expression with L-Arg supplementation. Additionally, L-Arg treatment of mice with DSS colitis resulted in increased ex vivo migration of colonic epithelial cells, suggestive of increased capacity for wound repair. Because CAT2 induction was sustained during L-Arg treatment and inducible nitric oxide (NO) synthase (iNOS) requires uptake of L-Arg for generation of NO, we tested the effect of L-Arg in iNOS(-/-) mice and found that its benefits in DSS colitis were eliminated. These preclinical studies indicate that L-Arg supplementation could be a potential therapy for IBD, and that one mechanism of action may be functional enhancement of iNOS activity.

Background Sexual dimorphism plays a role in the liver and in renal injuries. However, whether sex is a risk factor in bile duct ligation (BDL) in young rats has never been examined. Methods Six male and six female rats treated with BDL were sacrificed 2 weeks after surgery and were designated as BDL-M and BDL-F groups. The other six male and six female rats that received sham ligation were designated as sham-M and sham-F groups. Plasma biochemistry and liver and kidney asymmetric dimethylarginine (ADMA)-related molecules were examined. Results Both BDL-M and BDL-F groups had elevated plasma aspartate transaminase (AST), alanine transaminase (ALT), bilirubin, and transforming growth factor-β1 levels. The BDL-F group had lower plasma AST and ALT levels than the BDL-M group. The BDL-M and BDL-F groups had elevated plasma ADMA levels. The cationic amino acid transporter 1 (CAT1) level was increased in the BDL-F group as compared to the sham-F group, whereas the CAT2 level was reduced in the both BDL-M and BDL-F groups. Conclusion We found that young male rats were prone to higher degrees of biochemical liver and kidney injury to cholestasis. Sex differences in modulation of oxidative stress markers, such as ADMA, may play a role. Our results support careful monitoring and optimal treatment of cholestatic disease, especially in young male patients.

Omega-3 fatty acid (ω-3 FA) lipid emulsion has been reported to inhibit nitric oxide (NO) production and alter inducible nitric oxide synthase (iNOS) protein expression in lipopolysaccharide (LPS)-stimulated murine macrophages. However, the role of cellular uptake of l-arginine and iNOS transcription in ω-3 FA emulsion-induced inhibition of NO has not been explored. In addition, cationic amino acid transporter-2 (CAT-2) can regulate iNOS activity. The effect of ω-3 FA emulsion on CAT-2 expression is unknown. In the present study, we hypothesized that ω-3 FA emulsion pretreatment would decrease the production of NO in LPS-stimulated macrophages and that this effect would occur through alterations in the cellular uptake of l-arginine and CAT-2 expression, in addition to iNOS expression.Confluent immortalized murine macrophages (RAW264.7cells) were incubated with Dulbecco's modified Eagle's medium, ω-3 FA emulsion, or an isoenergetic ω-6 lipid emulsion for 4 h. The cells were washed and then stimulated with LPS (1 μg/mL) or media alone for 12 or 24 h before harvesting. Greiss reagent was used to assess NO production of plate well supernatants. Cellular uptake of l-arginine was assessed through [(3)H]-l-arginine. The expression of iNOS and CAT-2 mRNA in harvested RAW264.7 was quantified by reverse transcriptase-polymerase chain reaction.NO production of unstimulated RAW264.7 cells was similar in all groups. After LPS stimulation, ω-3 FA pretreatment at 12 and 24 h produced significantly less NO (P0.05) compared with ω-6 FA or media only. ω-3 FA pretreatment at 12 and 24 h resulted in less l-arginine uptake. iNOS and CAT-2 mRNA was significantly decreased with ω-3 FA pretreatment compared with ω-6 FA or media-only treatment (P0.05).These experiments demonstrated that, in addition to other anti-inflammatory effects, ω-3 FA lipid emulsion also significantly lowers NO production and l-arginine transport through altered expression of iNOS and CAT-2 in LPS-stimulated RAW264.7 macrophage cells.

Growth of the human MCF-7 breast cancer cell line is highly dependent on L-arginine. We have reported that L-arginine, released from extracellular substrates by prolactin (PRL)- and 17β-estradiol (E2)-induced carboxypeptidase-D in the cell membrane, promotes nitric oxide (NO) production for MCF-7 cell survival. Arginine uptake is mediated by members of the cationic amino acid transporter (CAT) family and may coincide with induction of nitric oxide synthase (NOS) for the production of NO. The present study investigated the CAT isoforms and PRL/E2 regulation of CAT and NOS in breast cancer cell lines. Using RT-PCR analysis, CAT-1, CAT-2A, and CAT-2B transcripts were detected in MCF-7, T47D, and MDA-MB-231 cells. The CAT-4 transcript was detected in MDA-MB-231 only. CAT-3 was not detected in any of these cells. PRL and E2 did not significantly alter levels of CAT-1 mRNA and protein, nor CAT-2A and CAT-2B mRNAs in MCF-7 and T47D cells. PRL and E2 also had no effect on the overall uptake of L-[2,3,4,5-H(3)] arginine into these cells. However, confocal immunofluorescent microscopy showed that PRL and E2 upregulated eNOS and iNOS proteins, which distributed in the cytoplasm and/or nucleus of MCF-7 cells. Knockdown of CAT-1 gene expression using small interfering RNA significantly decreased L-[2,3,4,5-H(3)]-arginine uptake, decreased viability and increased apoptosis of MCF-7 and T47D cells. In summary, several CAT isoforms are expressed in breast cancer cells. The CAT-1 isoform plays a role in arginine uptake and, together with PRL/E2-induced NOS, contribute to NO production for the survival of MCF-7 and T47D cells.

International audience; BACKGROUND & AIMS: Helicobacter pylori-induced immune responses fail to eradicate the bacterium. Nitric oxide (NO) can kill H pylori. However, translation of inducible NO synthase (iNOS) and NO generation by H pylori-stimulated macrophages is inhibited by the polyamine spermine derived from ornithine decarboxylase (ODC), and is dependent on availability of the iNOS substrate L-arginine (L-Arg). We determined if spermine inhibits iNOS-mediated immunity by reducing L-Arg uptake into macrophages. METHODS: Levels of the inducible cationic amino acid transporter (CAT) 2, ODC, and iNOS were measured in macrophages and H pylori gastritis tissues. L-Arg uptake, iNOS expression, and NO levels were assessed in cells with small interfering RNA knockdown of CAT2 or ODC, and in gastric macrophages. The ODC inhibitor, alpha-difluoromethylornithine, was administered to H pylori-infected mice for 4 months after inoculation. RESULTS: H pylori induced CAT2 and uptake of L-Arg in RAW 264.7 or primary macrophages. Addition of spermine or knockdown of CAT2 inhibited L-Arg uptake, NO production, and iNOS protein levels, whereas knockdown of ODC had the opposite effect. CAT2 and ODC were increased in mouse and human H pylori gastritis tissues and localized to macrophages. Gastric macrophages from H pylori-infected mice showed increased ODC expression, and attenuated iNOS and NO levels upon ex vivo H pylori stimulation versus cells from uninfected mice. alpha-Difluoromethylornithine treatment of infected mice restored L-Arg uptake, iNOS protein expression, and NO production in gastric macrophages, and significantly reduced both H pylori colonization levels and gastritis severity. CONCLUSIONS: Up-regulation of ODC in gastric macrophages impairs host defense against H pylori by suppressing iNOS-derived NO production.

Utilization of lysine and arginine by mammalian skeletal muscle is due in large part to the system y+ cationic amino acid transporters (CAT), particularly CAT-2A. The chicken CAT-2 (cCAT-2) gene is alternatively spliced to produce three isoforms (cCAT-2A/B/C). Chicken (Gallus gallus) CAT-2 isoforms were transiently and stably expressed in mammalian cell lines to determine cCAT-2 isoform cellular localization and transport properties. The cCAT-2A protein localized to the plasma membrane and the cCAT-2B protein localized to the cytoplasm juxtaposed to the plasma membrane. The cCAT-2C protein localized non-specifically throughout the cytoplasm. The cCAT-2A protein exhibited saturable transport. The Kt of cCAT-2A for lysine using transient transfection was 2.6 mM and the Vmax was 11.9 pmol/mg protein/min. The Kt of cCAT-2A for lysine using stable transfection was 7.9 mM and the Vmax was 12.8 pmol/mg protein/min. Transient and stable transfections of cCAT-2B or cCAT-2C resulted in no lysine or arginine transport. These data indicate that cCAT-2A is a low affinity, high velocity transporter for lysine and arginine and is the cCAT-2 isoform responsible for lysine and arginine transport in avian skeletal muscle.

Arginine is an essential amino acid for conceptus (embryo/fetus and trophoblast/placenta) growth and development; however, the mechanisms for arginine transport into the uterine lumen and uptake by conceptuses are largely unknown. In this study, expression of System y(+) (SLC7A1, SLC7A2, and SLC7A3) cationic amino acid transporters in uteri of cyclic and pregnant ewes and conceptuses was studied, and effects of pregnancy, progesterone (P4), and interferon tau (IFNT) on their expression were investigated. SLC7A1 mRNA was most abundant in endometrial luminal (LE) and superficial glandular (sGE) epithelia on Day 16 of the estrous cycle and on Days 16-20 of pregnancy, whereas SLC7A2 mRNA was most abundant in LE and mid to deep glandular (GE) epithelia on Days 14-20 of gestation. Expression of SLC7A1 and SLC7A2 was enhanced in pregnant ewes in a cell-specific manner, but abundance of SLC7A3 was not affected by day of the estrous cycle or by pregnancy status. SLC7A1, SLC7A2, and SLC7A3 mRNAs were expressed in trophectoderm and endoderm of conceptuses. In ovariectomized ewes, short-term treatment of ewes with P4 and IFNT did not affect endometrial SLC7A1 mRNA, while long-term treatment with P4 stimulated SLC7A1 in LE and GE, and IFNT tended to increase SLC7A1 abundance in LE. SLC7A2 mRNA abundance increased 4.1-fold in response to short-term P4 treatment and an additional 1.7-fold by IFNT primarily in endometrial LE/sGE, and these effects were ablated by a P4 receptor antagonist. These results indicate that coordinate changes in SLC7A1, SLC7A2, and SLC7A3 expression in uterine endometria and conceptuses are likely important in transport of arginine that is critical to conceptus growth, development, and survival.

To determine the specific transport system activities and expression of transporter genes responsible for uptake of L-arginine from the lumen of normal and Cryptosporidium parvum-infected neonatal porcine ileum and the influence of L-arginine catabolic pathways on L-arginine uptake.Intact sheets of ileal mucosa from control and C parvum-infected neonatal piglets were mounted in Ussing chambers and the uptake of 14C-L-arginine was determined under initial rate conditions and in the presence of transport system-selective inhibitors. Epithelial expression of L-arginine transporter genes was quantified by real-time reverse transcription polymerase chain reaction. L-Arginine catabolic enzyme expression was examined by immunoblotting epithelial lysates for arginase I and II. The role of intracellular catabolism in promoting the uptake of L-arginine was determined by pharmacological inhibition of nitric oxide synthase and arginase activities.C parvum-infected ileum transported L-arginine at rates equivalent to uninfected epithelium despite profound villous atrophy. This was attributed to enhanced uptake of L-arginine by individual epithelial cells in the infection. There were no differences in L-arginine transport system activities (y(+) and B(0, +)) or level of transporter gene expression (CAT-1, CAT-2A, and ATB(0, +)) between uninfected and C parvum-infected epithelial cells. However, infected epithelia had induced expression of the L-arginine hydrolytic enzyme arginase II and lower concentrations of L-arginine. Furthermore, transport of L-arginine by the infected epithelium was significantly inhibited by pharmacological blockade of arginase.Intracellular catabolism by arginase II, the induction of which has not been described previously for intestinal epithelium, facilitates uptake of L-arginine by infected epithelium using transport systems that do not differ from those of uninfected cells. Induction of arginase II may limit nitric oxide synthesis by competing with nitric oxide synthase for utilization of L-arginine or promote use of L-arginine for the synthesis of reparative polyamines.

The system y+ cationic amino acid transporters (CATs) play an important role in the regulation of lysine and arginine utilization. We have characterized the genomic organization and tissue transcription of the chicken CAT-2 (cCAT-2) isoforms from pectoralis muscle. The primary cCAT-2 transcript is alternatively spliced within the same position of the mRNA to produce cCAT-2A and cCAT-2B isoforms that share high nucleotide homology with their mammalian counterparts. We also identified a novel third CAT-2 isoform, cCAT-2C. This isoform contains a premature termination codon and studies in the chicken LMH cell line show that cCAT-2C mRNA is degraded by the nonsense-mediated mRNA decay pathway. All three cCAT-2 isoforms are transcribed in liver, pectoralis, gastrocnemius and heart. Chicken cCAT-2A was the predominant cCAT-2 isoform in liver, pectoralis and gastrocnemius. Analysis of the 5′-untranslated region of cCAT-2 identified multiple cCAT-2 promoters. Chicken CAT-2 promoter usage was tissue dependent and was not responsible for cCAT-2 isoform production. Analysis of genomic sequence upstream cCAT-2 promoter regions revealed binding sites for transcription factors involved in amino acid sensing, hormone signaling and immune function. These results provide insight into the role of the cCAT-2 gene and its regulation of lysine and arginine utilization in aves.

In the present study, we characterized the distribution of human cationic amino acid transporters 1 (hCAT1) and 2 (hCAT2) in healthy skin and compared it to psoriatic skin lesions by means of immunohistochemistry. Moreover, we tested the hypothesis that L -arginine and L -ornithine influence the expression and synthesis of hCAT1 and hCAT2 in cell culture experiments by means of real-time-PCR and Western blot. Immunohistochemical comparison between healthy and psoriatic skin revealed a decreased amount of hCAT1, especially in the stratum granulosum of psoriatic skin; the distribution pattern of hCAT2 was not significantly affected in psoriatic skin. Cell culture experiments showed that supraphysiological concentrations of 15 mM L -arginine (72 h) lead to a significant increase of the hCAT1-mRNA and protein expression, whereas other concentrations had no significant influence. In contrast, L -arginine concentrations of 2 mM led to a significant increase of the hCAT2B mRNA-expression after 24 h. However, 48 and 72 h revealed no significant changes and high concentrations (15 mM L -arginine) led to a significant downregulation of the hCAT2B transporter over all time points analyzed. L -ornithine had no effect on the hCAT1 expression of mRNA and protein level. On the other hand the expression of hCAT2B was significantly up regulated at a 5-mM concentration of L -ornithine at all analyzed time points. Other concentrations had no effect. For the first time, the findings yield data about hCAT1 and hCAT2 on protein-level and suggest that L -arginine is a worthwhile object of studies, which investigated L -arginine as a possible therapeutic agent to reduce psoriatic symptoms.

Nitric oxide (NO) is a potent regulator of both vascular tone and oxygen utilization. Diabetes is commonly associated with both NO deficiency and reduced renomedullary oxygen availability. Arginine availability as regulator of NO production has gained growing interest. We hypothesized that arginine limitation causes diabetes-induced renomedullary NO deficiency, which directly influences renomedullary oxygen tension (Po2). Medullary NO, Po2, and blood flow were measured in control and streptozotocin-induced diabetic rats, which were treated or not treated with α-tocopherol, and administered l-arginine followed by Nω-nitro-l-arginine methyl ester. Major components of arginine metabolism were also investigated. Diabetic rats had reduced renomedullary NO levels compared with controls. Arginine selectively increased NO levels in diabetic rats and totally restored NO levels in α-tocopherol-treated animals. Tocopherol prevented the reduction in medullary Po2 in the diabetic animals. Although blood flow increased equally in all groups, arginine increased Po2 exclusively in the diabetic groups. Diabetes decreased plasma arginine and asymmetric dimethylarginine concentrations, but increased hepatic CAT-2A and plasma ornithine independently of α-tocopherol treatment. In conclusion, diabetic rats had reduced renomedullary NO due to decreased plasma arginine following increased hepatic arginine uptake and degradation. This was unrelated to oxidative stress. The diabetes-induced reduction in renomedullary Po2 was restored by either acute arginine administration, which also restored NO levels, or long-term antioxidant treatment. Arginine increased medullary NO and Po2 independently of altered hemodynamics in the diabetic groups. This reveals a direct regulatory function of NO for renomedullary Po2 especially during situations of elevated oxidative stress.

Leishmania spp. are obligate intracellular parasites, requiring a suitable microenvironment for their growth within host cells. We previously reported that the growth of Leishmania amazonensis amastigotes in murine macrophages (Ms) was enhanced in the presence of gamma interferon (IFN-), a Th1 cytokine normally associated with classical M activation and killing of intracellular pathogens. In this study, we provided several lines of evidence suggesting that IFN--mediated parasite growth enhancement was associated with L-arginine transport via mouse cationic amino acid transporter 2B (mCAT-2B). (i) mRNA expression of Slc7A2, the gene encoding for mCAT-2B, as well as L-arginine transport was increased in IFN--treated Ms. (ii) Supplementation of L-arginine in M cultures increased parasite growth. (iii) Parasite growth enhancement in wild-type Ms was inhibited in the presence of nonmetabolized L-arginine analogues. (iv) IFNmediated parasite growth was absent in Ms derived from mCAT-2B-deficient mice. Although we detected a clear upregulation of mCAT-2B and L-arginine transport, no measurable iNOS or arginase activities were observed in IFN--treated, infected Ms. Together, these data suggest an involvement of a novel L-arginine usage independent of iNOS and arginase activities during IFN--mediated parasite growth enhancement. A possible role of mCAT-2B in supplying L-arginine directly to the parasites for their proliferation is discussed. Protozoan parasites in the genus Leishmania are obligate intracellular pathogens that can cause a wide spectrum of diseases, ranging from benign cutaneous lesions to life-threatening visceral infections. Leishmania spp. exhibit a dimorphic life cycle, in which parasites are transmitted by a sand fly vector as flagellated promastigotes and reside within mammalian macrophages (Ms) as amastigotes. The fate of intracellular amastigotes depends largely on the activation status of Ms, which is greatly influenced by various cytokines and chemokines (19

SUMMARY 1 In recent studies, the vascular adventitia has been established as an important source of inducible nitric oxide synthase (iNOS) and subsequent nitric oxide (NO) production, even more powerful than the media in response to certain inflammatory factors, such as lipopolysaccharide (LPS). The adventitia has an independent l-arginine (l-Arg)/NOS/NO pathway and is involved in the regulation of vascular function. In the present study, we explored the changes in and the pathophysiological significance of the l-Arg/NOS/NO pathway in the adventitia of rats with sepsis. 2 Sepsis was induced by caecal ligation and puncture in order to observe changes in l-Arg transport, NOS gene expression and activity and NO generation in the vascular adventitia to determine the mechanism of activation of the l-Arg/NOS/NO pathway. 3 Severe sepsis resulted in severe disturbance of haemodynamic features, with decreased mean arterial blood pressure, brachycardia and inhibited cardiac function (decreased left ventricular ±dP/dtmax). Left ventricular end-diastolic pressure was elevated threefold (P 90%). The Vmax of l-Arg transport in the sepsis group was increased by 83.5% (P

We use a modeling and simulation approach to carry out an in silico analysis of the metabolic pathways involving arginine as a precursor of nitric oxide or polyamines in aorta endothelial cells. Our model predicts conditions of physiological steady state, as well as the response of the system to changes in the control parameter, external arginine concentration. Metabolic flux control analysis allowed us to predict the values of flux control coefficients for all the transporters and enzymes included in the model. This analysis fulfills the flux control coefficient summation theorem and shows that both the low affinity transporter and arginase share the control of the fluxes through these metabolic pathways.