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

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

Molecular Determinants for Nitric Oxide Regulation of the Murine Cationic Amino Acid Transporter CAT-2A.

Cationic amino acid transporters (CATs) supply cells with essential and semiessential dibasic amino acids. Among them, l-arginine is the substrate for nitric oxide synthases (NOS) to produce nitric oxide (NO), a key signaling molecule and second messenger. In cardiac preparations, we showed that NO acutely and directly modulates transport activity by noncompetitively inhibiting these CATs. We hypothesize that this NO regulation occurs through modification of cysteine residues in CAT proteins. Homology modeling and a computational chemistry approach identified Cys347 as one of two putative targets for NO binding, of 15 Cys residues present in the low-affinity mouse CAT-2A (mCAT-2A). To test this prediction, mammalian cell lines overexpressing mCAT-2A were used for site-directed mutagenesis and uptake studies. When Cys347 was replaced with alanine (Cys347Ala), mCAT-2A became insensitive to inhibition by NO donors. In addition, the transport capacity of this variant decreased by >50% compared to that of the control, without affecting membrane expression levels or apparent affinities for the transported amino acids. Interestingly, replacing Cys347 with serine (Cys347Ser) restored uptake levels to those of the control while retaining NO insensitivity. Other Cys residues, when replaced with Ala, still produced a NO-sensitive CAT-2A. In cells co-expressing NOS and mCAT-2A, exposure to extracellular l-arginine inhibited the uptake activity of control mCAT-2A, via NO production, but not that of the Cys347Ser variant. Thus, the -SH moiety of Cys347 is largely responsible for mCAT-2A inhibition by NO. Because of the endogenous NO effect, this modulation is likely to be physiologically relevant and a potential intervention point for therapeutics.

Modulation of CAT-2B-Mediated l-Arginine Uptake and Nitric Oxide Biosynthesis in HCT116 Cell Line Through Biological Activity of 4'-Geranyloxyferulic Acid Extract from Quinoa Seeds.

Chenopodium quinoa Wild is a "pseudocereal" 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'-Geranyloxyferulic acid (GOFA) was the only phytochemical product found that belongs to quinoa'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-α), and the products of intermediary metabolism (ONOO-, O2-). 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.

Sexually Dimorphic Regulation of Behavioral States by Dopamine in Caenorhabditis elegans.

Sex differences in behavior allow animals to effectively mate and reproduce. However, the mechanism by which biological sex regulates behavioral states, which underlie the regulation of sex-shared behaviors, such as locomotion, is largely unknown. In this study, we studied sex differences in the behavioral states of Caenorhabditis elegans and found that males spend less time in a low locomotor activity state than hermaphrodites and that dopamine generates this sex difference. In males, dopamine reduces the low activity state by acting in the same pathway as polycystic kidney disease-related genes that function in male-specific neurons. In hermaphrodites, dopamine increases the low activity state by suppression of octopamine signaling in the sex-shared SIA neurons, which have reduced responsiveness to octopamine in males. Furthermore, dopamine promotes exploration both inside and outside of bacterial lawn (the food source) in males and suppresses it in hermaphrodites. These results demonstrate that sexually dimorphic signaling allows the same neuromodulator to promote adaptive behavior for each sex.SIGNIFICANCE STATEMENT The mechanisms that generate sex differences in sex-shared behaviors, including locomotion, are not well understood. We show that there are sex differences in the regulation of behavioral states in the model animal Caenorhabditis elegans Dopamine promotes the high locomotor activity state in males, which must search for mates to reproduce, and suppresses it in self-fertilizing hermaphrodites through distinct molecular mechanisms. This study demonstrates that sex-specific signaling generates sex differences in the regulation of behavioral states, which in turn modulates the locomotor activity to suit reproduction for each sex.

Regulatory Aspects of the Vacuolar CAT2 Arginine Transporter of S. lycopersicum: Role of Osmotic Pressure and Cations.

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.

Lysine Restriction Affects Feed Intake and Amino Acid Metabolism via Gut Microbiome in Piglets.

BACKGROUND/AIMS: Our previous reports suggested that dietary supplementation with lysine influenced intestinal absorption and metabolism of amino acids. In this study, we further investigated the effect of lysine restriction (30%) on feed intake and we also tested the hypothesis that gut microbiome contributed to the potential mechanism of lysine restriction-mediated feeding behavior. Here, we profiled gut microbial communities by sequencing 16S ribosomal ribonucleic acid (rRNA) genes from gut samples as well as growth performance, serum hormones, and intestinal lysine transport in a piglet model. RESULTS: Piglets preferred to the lysine restricted diet when giving three diets and the feed intake was markedly higher in the lysine-restricted group than that in the control group. Altered hormones (leptin, CCK, and ghrelin) might contribute to the feeding behavior caused by lysine restriction. Meanwhile, lysine transporting ability (SLC7A1 and SLC7A2 expression, intestinal electrophysiological changes, and amino acid pool in mesenteric vein) was decreased in response to lysine restriction. Through deep sequencing of bacterial rRNA markers, we observed that bacterial diversity was enhanced in the lysine-restricted group (Shannon H, PD, and Chao1). At the phylum level, lysine restriction enhanced gut Actinobacteria, Saccharibacteria, and Synergistetes abundances. At the family level, Moraxellaceae, Halomonadaceae, Shewanellaceae, Corynebacteriaceae, Bacillaceae, Comamonadaceae, Microbacteriaceae, Caulobacteraceae, and Synergistaceae abundances were increased in response to lysine restriction. Predictive functional profiling of microbial communities by PICRUSt also confirmed that dietary lysine restriction affected gut microbiome, which might further mediate amino acid metabolism, membrane transport, and endocrine system. CONCLUSION: Our results indicated that lysine restriction inhibited intestinal lysine transport and promoted feed intake, which might be associated with gut microbiome.

Expression of cationic amino acid transporters in pig skeletal muscles during postnatal development.

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.

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.

ADAR2 regulates RNA stability by modifying access of decay-promoting RNA-binding proteins.

Adenosine deaminases acting on RNA (ADARs) catalyze the editing of adenosine residues to inosine (A-to-I) within RNA sequences, mostly in the introns and UTRs (un-translated regions). The significance of editing within non-coding regions of RNA is poorly understood. Here, we demonstrate that association of ADAR2 with RNA stabilizes a subset of transcripts. ADAR2 interacts with and edits the 3΄UTR of nuclear-retained Cat2 transcribed nuclear RNA (Ctn RNA). In absence of ADAR2, the abundance and half-life of Ctn RNA are significantly reduced. Furthermore, ADAR2-mediated stabilization of Ctn RNA occurred in an editing-independent manner. Unedited Ctn RNA shows enhanced interaction with the RNA-binding proteins HuR and PARN [Poly(A) specific ribonuclease deadenylase]. HuR and PARN destabilize Ctn RNA in absence of ADAR2, indicating that ADAR2 stabilizes Ctn RNA by antagonizing its degradation by PARN and HuR. Transcriptomic analysis identified other RNAs that are regulated by a similar mechanism. In summary, we identify a regulatory mechanism whereby ADAR2 enhances target RNA stability by limiting the interaction of RNA-destabilizing proteins with their cognate substrates.

The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria.

Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2-/-mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2-/-mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1ß, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.

Expression Profile of Cationic Amino Acid Transporters in Rats with Endotoxin-Induced Uveitis.

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 the in vivo findings. 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.

Neuropeptidergic Signaling and Active Feeding State Inhibit Nociception in Caenorhabditis elegans.

Food availability and nutritional status are important cues affecting behavioral states. Here we report that, in Caenorhabditis elegans, a cascade of dopamine and neuropeptide signaling acts to inhibit nociception in food-poor environments. In the absence of food, animals show decreased sensitivity and increased adaptation to soluble repellents sensed by the polymodal ASH nociceptors. The effects of food on adaptation are affected by dopamine and neuropeptide signaling; dopamine acts via the DOP-1 receptor to decrease adaptation on food, whereas the neuropeptide receptors NPR-1 and NPR-2 act to increase adaptation off food. NPR-1 and NPR-2 function cell autonomously in the ASH neurons to increase adaptation off food, whereas the DOP-1 receptor controls neuropeptide release from interneurons that modulate ASH activity indirectly. These results indicate that feeding state modulates nociception through the interaction of monoamine and neuropeptide signaling pathways.

Changes in ADMA/DDAH pathway after hepatic ischemia/reperfusion injury in rats: the role of bile.

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.

Campylobacter jejuni influences the expression of nutrient transporter genes in the intestine of chickens.

The gastrointestinal tract represents the first barrier against pathogens. However, the interaction of Campylobacter with intestinal epithelial cells and its effects on the intestinal function of chickens are poorly studied. Therefore, the goal of the present study was to characterize the effects of C. jejuni oral infection on the mRNA expression of nutrient transporters in the intestine. Newly hatched specific pathogen-free (SPF) chickens were orally infected with C. jejuni (NCTC 12744; 1 × 10(8)CFU/bird) at 14 days of age. Quantitative RT-PCR analyses at 14 days-post infection (dpi) revealed that the relative gene expression of the sodium/glucose cotransporter (SGLT-1) and the peptide transporter (PepT-1) was down-regulated (P<0.05) in all investigated segments (duodenum, jejunum and cecum) of Campylobacter-infected birds, while the facilitated glucose transporter (GLUT-2) was down-regulated (P<0.05) in jejunal and cecal tissues only. Furthermore, down-regulation (P<0.05) of the cationic amino acid transporter (CAT-2) and the excitatory amino acid transporter (EAAT-3) was seen in the jejunum, and down-regulation (P<0.05) of the l-type amino acid transporter (y(+)LAT-2) was noticed in the duodenum of infected birds. The decreased expression of intestinal nutrient transporters coincided with a decrease (P<0.05) in body weight and body weight gain during a 2-week post infection period. For the first time, it can be concluded that nutrient transporter expression is compromised in the small and large intestine of Campylobacter-infected birds with negative consequences on growth performance. Furthermore, the down-regulation of mRNA expression of glucose and amino acid transporters may result in accumulation of nutrients in the intestinal lumen, which may favor C. jejuni replication and colonization.

Glycinergic feedback enhances synaptic gain in the distal retina.

Glycine input originates with interplexiform cells, a group of neurons situated within the inner retina that transmit signals centrifugally to the distal retina. The effect on visual function of this novel mechanism is largely unknown. Using gramicidin-perforated patch whole cell recordings, intracellular recordings and specific antibody labelling techniques, we examined the effects of the synaptic connections between glycinergic interplexiform cells, photoreceptors and bipolar cells. To confirm that interplexiform cells make centrifugal feedback on bipolar cell dendrites, we recorded the postsynaptic glycine currents from axon-detached bipolar cells while stimulating presynaptic interplexiform cells. The results show that glycinergic interplexiform cells activate bipolar cell dendrites that express the α3 subunit of the glycine receptor, as well as a subclass of unidentified receptors on photoreceptors. By virtue of their synaptic contacts, glycine centrifugal feedback increases glutamate release from photoreceptors and suppresses the uptake of glutamate by the type 2A excitatory amino acid transporter on photoreceptors. The net effect is a significant increase in synaptic gain between photoreceptors and their second-order neurons.

Maternal hypercholesterolemia in pregnancy associates with umbilical vein endothelial dysfunction: role of endothelial nitric oxide synthase and arginase II.

OBJECTIVE: Human pregnancy that courses with maternal supraphysiological hypercholesterolemia (MSPH) correlates with atherosclerotic lesions in fetal arteries. It is known that hypercholesterolemia associates with endothelial dysfunction in adults, a phenomenon where nitric oxide (NO) and arginase are involved. However, nothing is reported on potential alterations in the fetoplacental endothelial function in MSPH. The aim of this study was to determine whether MSPH alters fetal vascular reactivity via endothelial arginase/urea and L-arginine transport/NO signaling pathways. APPROACH AND RESULTS: Total cholesterol <280 mg/dL was considered as maternal physiological hypercholesterolemia (n=46 women) and ≥ 280 mg/dL as MSPH (n=28 women). Maternal but not fetal total cholesterol and low-density lipoprotein-cholesterol levels were elevated in MSPH. Umbilical veins were used for vascular reactivity assays (wire myography), and primary cultures of umbilical vein endothelial cells to determine arginase, endothelial NO synthase (eNOS), and human cationic amino acid transporter 1 and human cationic amino acid transporter 2A/B expression and activity. MSPH reduced calcitonine gene-related peptide-umbilical vein relaxation and increased intima/media ratio (histochemistry), as well as reduced eNOS activity (L-citrulline synthesis from L-arginine, eNOS phosphorylation/dephosphorylation), but increased arginase activity and arginase II protein abundance. Arginase inhibition increased eNOS activity and L-arginine transport capacity without altering human cationic amino acid transporter 1 or human cationic amino acid transporter 2A/B protein abundance in maternal physiological hypercholesterolemia and MSPH. CONCLUSIONS: MSPH is a pathophysiological condition altering umbilical vein reactivity because of fetal endothelial dysfunction associated with arginase and eNOS signaling imbalance. We speculate that elevated maternal circulating cholesterol is a factor leading to fetal endothelial dysfunction, which could have serious consequences to the growing fetus.

Dual effect of nickel on L-arginine/nitric oxide system in RAW 264.7 macrophages.

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.

Differential topochemistry of three cationic amino acid transporter proteins, hCAT1, hCAT2 and hCAT3, in the adult human brain.

The cellular uptake of L-arginine and other cationic amino acids (such as L-lysine and L-ornithine) is mainly mediated by cationic amino acid transporter (CAT) proteins. Despite the important roles of cationic amino acid transporters for normal brain functioning and various brain diseases there is currently only fragmentary knowledge about their cellular and regional distribution patterns in the human brain. We mapped the immunohistochemical localization of human cationic amino acid transporters 1, 2 and 3 (hCAT1, 2, and 3) throughout five adult human brains and found a wide but uneven distribution of these transporters. All three hCAT1s were mainly localized in neurons, but were also found in numerous astrocytes, oligodendrocytes, plexus choroideus epithelial cells, and small blood vessels. The highest density of hCAT expressing neurons was observed in the hypothalamus, in some areas of the cerebral cortex, the thalamic reticular nucleus and the caudate nucleus, whereas weak to moderate expression was detected in the hippocampus, the prefrontal cortex (hCAT1 only), pons, brain stem and cerebellum. In contrast to what has been found in rodent brain, we detected hCAT2 and hCAT3 also in astrocytes. Overall, each hCAT has its characteristic, individual cerebral expression patterns, which, however, overlap with the others.

LPS-stimulated RAW264.7 macrophage CAT-2-mediated l-arginine uptake and nitric oxide biosynthesis is inhibited by omega fatty acid lipid emulsion.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (P < 0.05) 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 (P < 0.05). CONCLUSIONS: 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.

L-arginine supplementation improves responses to injury and inflammation in dextran sulfate sodium colitis.

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.