Cationic Amino Acid Transporter Research Articles

Involvement of the carrier-mediated process in the retina-to-blood transport of spermine at the inner blood-retinal barrier

The elimination of spermine, the end product of cellular polyamine, from the retina to the blood across the blood-retinal barrier (BRB) was investigated. The in vivo microdialysis study revealed that the elimination of [3H]spermine from vitreous humor after vitreous bolus injection was in a biexponential manner. The rate constant for the elimination of [3H]spermine during the terminal phase was estimated to be 1.67-fold greater than that of [14C]d-mannitol, a bulk flow marker, and the difference in the terminal elimination rate constant between [3H]spermine and [14C]d-mannitol was reduced in the presence of 50 mM spermine, suggesting a retina-to-blood transport system for [3H]spermine across the BRB. The retina-to-blood transport of [3H]spermine was also supported by a study of the retinal uptake index (RUI). The in vitro transport study with TR-iBRB2 cells, a model cell line of the inner BRB, revealed time-, concentration- and temperature-dependent transport of [3H]spermine, suggesting the involvement of carrier-mediated processes in spermine transport across the inner BRB. The in vitro study also suggested that the transport of spermine at the inner BRB is pH-, membrane potential- and Cl−-sensitive and Na+-insensitive, and these functional properties of spermine transport suggest only a minor contribution of spermine transporters, such as CCC9 (SLC12A8), the expression of which was suggested at the inner BRB. In the inhibition study, [3H]spermine transport was markedly inhibited by putrescine, spermidine, spermine and agmatine while substrates of a well-characterized organic cation transporter (OCTs/SLC22A) and a cationic amino acid transporter (CATs/SLC7A) had no effect, suggesting the involvement of unknown transporters in spermine elimination from the retina.

Transcriptome-based analysis of kidney gene expression changes associated with diabetes in OVE26 mice, in the presence and absence of losartan treatment.

Diabetes is among the most common causes of end-stage renal disease, although its pathophysiology is incompletely understood. We performed next-generation sequencing-based transcriptome analysis of renal gene expression changes in the OVE26 murine model of diabetes (age 15 weeks), relative to non-diabetic control, in the presence and absence of short-term (seven-day) treatment with the angiotensin receptor blocker, losartan (n = 3–6 biological replicates per condition). We detected 1438 statistically significant changes in gene expression across conditions. Of the 638 genes dysregulated in diabetes relative to the non-diabetic state, >70% were downregulation events. Unbiased functional annotation of genes up- and down-regulated by diabetes strongly associated (p<1×10−8) with terms for oxidative stress and for endoplasmic reticulum stress/protein folding. Most of the individual gene products up- or down-regulated with diabetes were unaffected by losartan treatment; however, of the gene products dysregulated in diabetes and influenced by losartan treatment, the vast majority of changes were in the direction of amelioration rather than exacerbation of the diabetic dysregulation. This group of losartan-protected genes associated strongly with annotation terms for endoplasmic reticulum stress, heat shock proteins, and chaperone function, but not oxidative stress; therefore, the losartan-unaffected genes suggest avenues for additional therapeutic opportunity in diabetes. Interestingly, the gene product most highly upregulated by diabetes (>52-fold), encoded by the cationic amino acid transporter Slc7a12, and the gene product most highly downregulated by diabetes (>99%) – encoded by the “pseudogene” Gm6300 – are adjacent in the murine genome, are members of the SLC7 gene family, and are likely paralogous. Therefore, diabetes activates a near-total genetic switch between these two paralogs. Other individual-level changes in gene expression are potentially relevant to diabetic pathophysiology, and novel pathways are suggested. Genes unaffected by diabetes alone but exhibiting increased renal expression with losartan produced a signature consistent with malignant potential.

Sa1746 Cationic Amino Acid Transporter 2 and L-Arginine Availability Are Required for Talin-1-Dependent Adherence of the Colonic Pathogen Citrobacter Rodentium and Subsequent Pro-Inflammatory Responses

Background: L-arginine (L-Arg) uptake in cells is mediated by cationic amino acid transporter (CAT) proteins, and CAT2 is the inducible form. We have found that CAT2-/mice exhibit less colitis in the IBD model induced by infection with Citrobacter rodentium, a rodent pathogen similar to EPEC that acts by forming attaching and effacing lesions on epithelial cells. The translocated intimin receptor is injected into host cells by C. rodentium and forms a complex with talin-1 that mediates bacterial binding and downstream signaling. Our aim was to investigate the role of talin-1 in C. rodentium infection. Methods: Male C57BL/6 wild-type (WT) or CAT2-/mice were infected with C. rodentium for 14 days. Talin-1 and the C. rodentium protein espB were assesed by confocal microscopy and Western blotting. mRNA levels of talin-1 and the proinflammatory CXC chemokines, KC and MIP-2, were determined by qPCR. Young adult mouse colon (YAMC) cells were transduced with control or talin-1 shRNA, and activated with C. rodentium at an MOI of 200. Bacterial adherence was assessed by culture after saponin treatment of cells. Levels of the NF-κB subunit p65 were assessed by immunofluorescence at 30 min, and of talin-1, KC and MIP-2 by qPCR at 4 h. Talin-1 mRNA stability was assessed in YAMC cells using the transcriptional inhibitor actinomycin D followed by serial assessment of mRNA levels. Results: C. rodentium infection in vivo induced a 4-fold increase in talin-1 mRNA and protein expression in whole tissues and a 236-fold increase in mRNA levels in colonic epithelial cells (CECs). Talin-1 and espB protein levels were markedly decreased in colon tissues from infected CAT2-/vs. WT mice; this was confirmed by confocal microscopy and there was also less colocalization of these proteins. Talin-1, KC and MIP-2 mRNA levels were each decreased by >92% in CAT2-/vs. WT mice (p<0.01). L-lysine, a competitive inhibitor of L-Arg uptake, reduced induction of talin-1, KC, and MIP-2 mRNA expression by 60.4 ± 18.3%, 91.7 ± 2.6%, and 68.9 ± 12.2%, respectively, in YAMC cells (p<0.01). In talin-1 knockdown cells, levels of adherent C. rodentium decreased from 4.9 ± 0.9 to 0.8 ± 0.2 bacteria/cell (p<0.05), and there was less nuclear translocation of p65. Similarly, KC and MIP-2 levels were decreased by 90.6 ± 0.3% and 92.5 ± 3.1%, respectively, in knockdown cells (p<0.01). There was a 4.2 ± 0.5-fold increase in talin-1 mRNA levels when cells starved of L-Arg were repleted with L-Arg for 4 h. Talin-1 mRNA was less stable in the absence of L-Arg; the mRNA half-life was 5.2fold (p<0.01) greater in cells repleted with L-Arg. Conclusions: Talin-1 is essential for C. rodentium adherence to colonic epithelial cells and induction of host inflammatory responses. L-Arg availability is needed for talin-1 mRNA stability, and thus mediates the immunopathogenesis of C. rodentium infection.

Expression of digestive enzymes and nutrient transporters in Eimeria acervulina-challenged layers and broilers

Avian coccidiosis is a disease caused by intestinal protozoa in the genus Eimeria. Clinical signs of coccidiosis include intestinal lesions and reduced feed efficiency and BW gain. This growth reduction may be due to changes in expression of digestive enzymes and nutrient transporters in the intestine. The objective of this study was to examine the differential expression of digestive enzymes, transporters of amino acids, peptides, sugars, and minerals, and an antimicrobial peptide in the small intestine of Eimeria acervulina-infected broilers and layers. Uninfected broilers and layers, in general, expressed these genes at comparable levels. Some differences included 3-fold and 2-fold greater expression of the peptide transporter PepT1 and the antimicrobial peptide LEAP2 (liver expressed antimicrobial peptide 2), respectively, in the jejunum of layers compared with broilers and 17-fold greater expression of LEAP2 in the duodenum of broilers compared with layers. In the duodenum of Eimeria-infected broilers and layers, there was downregulation of aminopeptidase N; sucrase-isomaltase; the neutral, cationic, and anionic amino acid transporters bo,+ AT/rBAT, Bo AT, CAT2, and EAAT3; the sugar transporter GLUT2; the zinc transporter ZnT1; and LEAP2. In the jejunum of infected layers there was downregulation of many of the same genes as in the duodenum plus downregulation of PepT1, bo,+ AT/rBAT, and the y+ L system amino acid transporters y+ LAT1 and y+ LAT2. In the ileum of infected layers there was downregulation of CAT2, y+ LAT1, the L type amino acid transporter LAT1, and the sugar transporter GLUT1, and upregulation of APN, PepT1, the sodium glucose transporter SGLT4, and LEAP2. In E. acervulina-infected broilers, there were no gene expression changes in the jejunum and ileum. These changes in intestinal digestive enzyme and nutrient transporter gene expression may result in a decrease in the efficiency of protein digestion, uptake of important amino acids and sugars, and disruption of mineral balance that may affect intestinal cell metabolism and Eimeria replication.

Anti-viral effects of CGRP in mixed glial cells infected with LP-BM5 virus (VIR5P.1022)

Abstract LP-BM5 retrovirus infection is an established murine model used to study HIV infection in humans due to that susceptible strains of mice (including C57BL/6(B6) mice) develop immunodeficiency syndrome (termed as MAIDS) following LP-BM5 infection. Previously, we observed that calcitonin gene related protein (CGRP), a neuropeptide produced primarily by sensory neurons, reduced LP-BM5 viral loads in infected mixed glial cells (primarily astrocytes and microglia). The mechanism through which CGRP decreases the viral loads is still not known. To elucidate the role of CGRP in glial LP-BM5 infection, we first determined that CGRP did not induce an increased cell death post-infection or directly decrease the viral infectivity. Then, we investigated whether CGRP could affect viral infectivity by enhancing the glycosylation of the cell receptor for viral attachment, murine cationic amino acid transporter 1 (mCAT-1). Our data indicate that blocking glycosylation by Tunicamycin did not affect CGRP’s anti-viral effects. Further, we tested the involvement of p38 MAPK pathway in CGRP-induced inhibitory effects. Preliminary experiment using a p38 inhibitor, SB203580, suggests a p38 mediated mechanism. Future studies will further elucidate the role of p38 and examine the role of other CGRP downstream pathways on LPBM5 infection.

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; 1×108CFU/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.

T-cell receptor transfer into human T cells with ecotropic retroviral vectors

Adoptive T-cell transfer for cancer immunotherapy requires genetic modification of T cells with recombinant T-cell receptors (TCRs). Amphotropic retroviral vectors(RVs) used for TCR transduction for this purpose are considered safe in principle. Despite this, TCR-coding and packaging vectors could theoretically recombine to produce replication competent vectors (RCVs), and transduced T-cell preparations must be proven free of RCV. To eliminate the need for RCV testing, we transduced human T cells with ecotropic RVs so potential RCV would be non-infectious for human cells. We show that transfection of synthetic messenger RNA encoding murine cationic amino-acid transporter 1 (mCAT-1), the receptor for murine retroviruses, enables efficient transient ecotropic transduction of human T cells. mCAT-1-dependent transduction was more efficient than amphotropic transduction performed in parallel, and preferentially targeted naive T cells. Moreover, we demonstrate that ecotropic TCR transduction results in antigen-specific restimulation of primary human T cells. Thus, ecotropic RVs represent a versatile, safe and potent tool to prepare T cells for the adoptive transfer.

Characterization of the putative amino acid transporter genes AtCAT2, 3 &4: The tonoplast localized AtCAT2 regulates soluble leaf amino acids

The plant vacuole constitutes a large transient storage compartment for nutrients, proteins and metabolites, and is a major cellular sink for toxic waste compounds. Amino acids can cross the vacuolar membrane via specific transport proteins, which are molecularly not well characterized. Two members of a small subfamily of the cationic amino acid transporters, AtCAT2 and AtCAT4, were primarily localized at the tonoplast when tagged with GFP. The closely related AtCAT3, by contrast, was detected in the endoplasmic reticulum membrane. The exchange of a di-acidic motif at the carboxy-tail affected their sub-cellular localization, with larger effects visible in transiently transformed protoplasts compared to stably expressing plant lines. The genes have broad, partially overlapping tissue expression, with CAT2 dominating in most tissues. Loss-of-function mutants of individual CATs showed no visible phenotype under various conditions, but the overall tissue concentration of amino acids was increased in soil-grown cat2 mutants. The data suggest that CAT2 is a critical target of leaf amino acid concentrations and manipulation of this tonoplast transporter can significantly alter total tissue amino acid concentrations.

Runx1t1 (Runt-Related Transcription Factor 1; Translocated to, 1) Epigenetically Regulates the Proliferation and Nitric Oxide Production of Microglia

BackgroundMicroglia, the resident immune cells of the brain, undergo rapid proliferation and produce several proinflammatory molecules and nitric oxide (NO) when activated in neuropathological conditions. Runx1t1 (Runt-related transcription factor 1, translocated to 1) has been implicated in recruiting histone deacetylases (HDACs) for transcriptional repression, thereby regulating cell proliferation. In the present study, Runx1t1 expression was shown to localize in amoeboid microglial cells of the postnatal rat brain, being hardly detectable in ramified microglia of the adult brain. Moreover, a marked expression of Runx1t1was induced and translocated to nuclei in activated microglia in vitro and in vivo. In view of these findings, it was hypothesized that Runx1t1 regulates microglial functions during development and in neuropathological conditions.Methods and FindingssiRNA-mediated knockdown of Runx1t1 significantly decreased the expression level of cell cycle-related gene, cyclin-dependent kinase 4 (Cdk4) and proliferation index in activated BV2 microglia. It was also shown that HDAC inhibitor (HDACi) treatment mimics the effects of Runx1t1 knockdown on microglial proliferation, confirming that microglial proliferation is associated with Runx1t1 expression and HDACs activity. Further, Runx1t1 and HDACs were shown to promote neurotoxic effect of microglia by repressing expression of LAT2, L-aminoacid transporter-2 (cationic amino acid transporter, y+ system), which normally inhibits NO production. This was confirmed by chromatin immunoprecipitation (ChIP) assay, which revealed that Runx1t1 binds to the promoter region of LAT2 and this binding increased upon microglial activation. However, the enhanced binding of Runx1t1 to the LAT2 promoter could not repress the LAT2 expression when the BV2 microglia cells were treated with HDACi, indicating that Runx1t1 requires HDACs to transcriptionally repress the expression of LAT2.Conclusion/InterpretationIn conclusion, it is suggested that Runx1t1 controls proliferation and the neurotoxic effect of microglia by epigenetically regulating Cdk4 and LAT2 via its interaction with HDACs.

Susceptibility of muridae cell lines to ecotropic murine leukemia virus and the cationic amino acid transporter 1 viral receptor sequences: implications for evolution of the viral receptor

Ecotropic murine leukemia viruses (Eco-MLVs) infect mouse and rat, but not other mammalian cells, and gain access for infection through binding the cationic amino acid transporter 1 (CAT1). Glycosylation of the rat and hamster CAT1s inhibits Eco-MLV infection, and treatment of rat and hamster cells with a glycosylation inhibitor, tunicamycin, enhances Eco-MLV infection. Although the mouse CAT1 is also glycosylated, it does not inhibit Eco-MLV infection. Comparison of amino acid sequences between the rat and mouse CAT1s shows amino acid insertions in the rat protein near the Eco-MLV-binding motif. In addition to the insertion present in the rat CAT1, the hamster CAT1 has additional amino acid insertions. In contrast, tunicamycin treatment of mink and human cells does not elevate the infection, because their CAT1s do not have the Eco-MLV-binding motif. To define the evolutionary pathway of the Eco-MLV receptor, we analyzed CAT1 sequences and susceptibility to Eco-MLV infection of other several murinae animals, including the southern vole (Microtus rossiaemeridionalis), large Japanese field mouse (Apodemus speciosus), and Eurasian harvest mouse (Micromys minutus). Eco-MLV infection was enhanced by tunicamycin in these cells, and their CAT1 sequences have the insertions like the hamster CAT1. Phylogenetic analysis of mammalian CAT1s suggested that the ancestral CAT1 does not have the Eco-MLV-binding motif, like the human CAT1, and the mouse CAT1 is thought to be generated by the amino acid deletions in the third extracellular loop of CAT1.

Amino acid prodrug of quinidine: An approach to circumvent P-glycoprotein mediated cellular efflux

In the present study, we investigated the effect of large neutral amino acid modification in overcoming P-gp mediated cellular efflux of quinidine. L-isoleucine ester prodrug of quinidine (Ile-quinidine) was synthesized in our laboratory. [14C]-erythromycin was selected as a model substrate to study interaction of quinidine and Ile-quinidine with P-gp. Transport studies were conducted to study translocation kinetics of quinidine and Ile-quinidine in MDCK-MDR1 cells. In cellular accumulation study, uptake rate of [14C]-erythromycin elevated drastically in presence of cyclosporine A and GF 120918. This result indicates that [14C]-erythromycin is an excellent substrate of P-gp. Similarly, uptake rate of [14C]-erythromycin was enhanced significantly in presence of quinidine (25 and 50 μM). However, [14C]-erythromycin uptake rate remained fairly constant in presence of Ile-quinidine (25 μM). Apparent A-B and B-A permeability of quinidine observed in MDCK-MDR1 cells were 1.6 ± 0.2 × 10(-6) and 7.0 ± 0.4 × 10(-6)cm/s, a 4.4-fold difference. Moreover, A-B permeability of quinidine increased dramatically in the presence of cyclosporine A and GF 120918. Apparent permeability values of Ile-quinidine observed from A-B and B-A direction were 4.3 ± 0.9 × 10(-6) and 5.5 ± 0.4 × 10(-6)cm/s, a 1.3-fold difference. Importantly, A-B transport of Ile-quinidine did not change dramatically in the presence of cyclosporine and GF 120918. Based on these results, it was apparent that quinidine displayed higher substrate affinity toward P-gp relative to Ile-quinidine. Chemical or enzymatic hydrolysis of Ile-quinidine resulted in regeneration of low quantities of quinidine during transport studies. Competitive inhibition studies demonstrated that Ile-quinidine was recognized by multiple amino acid transporters such as LAT1, LAT2 and cationic amino acid transporter. In conclusion, chemical modification of quinidine with neutral amino acids results in circumvention of P-gp mediated drug efflux. Hence, amino acid transporter targeted prodrug delivery seems to be a viable strategy for improving drug accumulation in P-gp overexpressing cells.

Endothelial cationic amino acid transporter-1 overexpression can prevent oxidative stress and increases in arterial pressure in response to superoxide dismutase inhibition in mice

Oxidative stress may play an important role in the pathogenesis of hypertension. The aim of our study is to examine whether increased expression of the predominant endothelial l-arginine transporter, cationic amino acid transporter-1 (CAT1), can prevent oxidative stress-induced hypertension. Wild-type mice (WT; n = 9) and endothelial CAT1 overexpressing (CAT+) mice (n = 6) had telemetry probes implanted for the measurement of mean arterial pressure (MAP), heart rate (HR) and locomotor activity. Minipumps were implanted for infusion of the superoxide dismutase inhibitor diethyldithiocarbamic acid (DETCA; 30 mg kg(-1) day(-1) ; 14 days) or its saline vehicle. Baseline levels of MAP, HR and locomotor activity were determined before and during chronic DETCA administration. Mice were then killed, and their plasma and kidneys collected for analysis of F2 -isoprostane levels. Basal MAP was less in CAT+ (92 ± 2 mmHg; n = 6) than in WT (98 ± 2 mmHg; n = 9; P < 0.001). During DETCA infusion, MAP was increased in WT (by 4.2 ± 0.5%; P < 0.001) but not in CAT+, when compared to appropriate controls (PDETCA*genotype = 0.006). DETCA infusion increased total plasma F2 -isoprostane levels (by 67 ± 11%; P = 0.05) in WT but not in CAT+. Total renal F2 -isoprostane levels were greater during DETCA infusion in WT (by 72%; P < 0.001), but not in CAT+, compared to appropriate controls. Augmented endothelial l-arginine transport attenuated the prohypertensive effects of systemic and renal oxidative stress, suggesting that manipulation of endothelial CAT1 may provide a new therapeutic approach for the treatment of cardiovascular disease associated with oxidative stress.

Performance, serum amino acid concentrations and expression of selected genes in pair‐fed growing pigs exposed to high ambient temperatures

Heat stress (HS) depresses pig performance mainly because of appetite reduction, although other factors involved in the cellular availability of nutrients may also contribute to that depression. An experiment was conducted with twelve pair-fed pigs (30.3±2.7kg BW) to examine the effect of severe HS (up to 45°C) on the expression of genes coding for two cationic amino acid (AA) transporters (b(0,+) AT and CAT-1), leptin, heat-shock protein (Hsp-90) and myosin in several tissues; serum concentrations (SC) of AA; and performance. There were two treatments: Comfort, pigs housed at an average temperature of 22 (±2)°C; and HS, pigs housed in a similar room with no climate control, where temperature was raised up to 45°C. All pigs received the same wheat-soybean meal diet and had similar daily feed intake. Comfort pigs had a higher daily gain and better gain/feed ratio than HS pigs (p<0.05). The expression of b(0,+) AT in jejunum and liver, that of myosin in the Semitendinosus muscle, and leptin in adipose tissue was lower, but CAT-1 in jejunum and liver, and Hsp-90 in liver was higher in HS pigs. The SC of Lys and Met in HS pigs were around 55% and 20%, respectively, of that in Comfort pigs (p<0.05). In conclusion, HS affects the expression of cationic AA transporters, myosin, Hsp-90, leptin; the SC of Lys and Met; and the performance of pair-fed pigs. These results suggest that HS-related changes in gene expression affect the performance of pigs beyond the effect caused by the reduction in voluntary feed intake.

Agmatinase and human cationic amino acid transporter 1 in mood disorder: what&amp;#180;s under the microscope?

Agmatinase and human cationic amino acid transporter 1 in mood disorder: what&amp;#180;s under the microscope?

Altered growth and improved resistance of Arabidopsis against Pseudomonas syringae by overexpression of the basic amino acid transporter AtCAT1

Amino acid transporters in plants are crucial for distributing amino acids between plant organs and cellular compartments. The H(+)-coupled plasma membrane transporter CAT1 (cationic amino acid transporter 1) facilitates the high-affinity uptake of basic amino acids. The uptake of lysine (Lys) via the roots was not altered in loss-of-function mutants, in accordance with the minor expression of CAT1 in roots, but plants ectopically overexpressing CAT1 incorporated Lys at higher rates. Exogenous Lys inhibited the primary root of Arabidopsis, whereas lateral roots were stimulated. These effects were augmented by the presence or absence of CAT1. Furthermore, the total biomass of soil-grown plants ectopically overexpressing CAT1 was reduced and the time to flowering was accelerated. These effects were accompanied by only minor changes in the overall amino acid profile. Interestingly, CAT1 belongs to a specific small cluster of nitrogen-containing metabolite transporter genes that are rapidly up-regulated upon infection with Pseudomonas syringae and that may participate in the systemic response of plants to pathogen attack. The overexpression of CAT1 indeed enhanced the resistance to the hemibiotrophic bacterial pathogen P. syringae via a constitutively activated salicylic acid (SA) pathway, which is consistent with the developmental defects and the resistance phenotype.

Alteration of the microRNA-122 regulatory network in rat models of hepatotoxicity

MicroRNAs are small RNA molecules that post-transcriptionally regulate gene expression. MicroRNA-122 is the most abundant and specific liver microRNA. Hepatotoxicity involves a significant alteration of liver gene expression. The aim of this work was to evaluate the microRNA-122 regulatory network in models of hepatotoxicity induced by thioacetamide or carbon tetrachloride. We report that the toxins decreased the expression of microRNA-122, which corresponded with an increase in two target genes: Cyclin G1 and the cationic amino acid transporter CAT-1. We found a decreased expression of its precursor, pri-microRNA-122, and of the transcription factors that specifically bind its promoter: CCAAT/enhancer-binding protein alpha, and members of the hepatocyte nuclear factor family. Therefore, microRNA-122 expression levels are under transcriptional control during hepatotoxicity. We propose that the changes observed are associated with the liver response to cope with the injury caused by the hepatotoxins, likely through a cell proliferation process to repair the damaged tissue.

L-Arginine improves endothelial function, independently of arginine uptake, in aortas from chronic renal failure female rats

Endothelial cell dysfunction (ECD) is a common feature of chronic renal failure (CRF). Defective nitric oxide (NO) generation due to decreased endothelial nitric oxide synthase (eNOS) activity is a crucial parameter characterizing ECD. Decreased activity of cationic amino acid transporter-1 (CAT-1), the selective arginine transporter of eNOS, has been shown to inhibit eNOS in uremia. Recently, we failed to demonstrate a decrease in glomerular arginine transport in uremic female rats (Schwartz IF, Grupper A, Soetendorp H, Hillel O, Laron I, Chernichovski T, Ingbir M, Shtabski A, Weinstein T, Chernin G, Shashar M, Hershkoviz R, Schwartz D. Am J Physiol Renal Physiol 303: F396-F404, 2012). The current experiments were designed to determine whether sexual dimorphism which characterizes glomerular arginine transport system in uremia involves the systemic vasculature as well and to assess the effect of L-arginine in such conditions. Contractile and vasodilatory responses, ultrastructural changes, and measures of the L-arginine-NO system were performed in thoracic aortas of female rats subjected to 5/6 nephrectomy. The contractile response to KCl was significantly reduced, and acetylcholine-induced vasodilation was significantly impaired in aortas from CRF dames compared with healthy rats. Both of these findings were prevented by the administration of arginine in the drinking water. The decrease in both cGMP generation, a measure of eNOS activity, and aortic eNOS and phosphorylated eNOS abundance observed in CRF rats was completely abolished by l-arginine, while arginine transport and CAT-1 protein were unchanged in all experimental groups. Arginine decreased both serum levels of advanced glycation end products and the asymmetrical dimethylarginine/arginine ratio and restored the endothelial ultrastructure in CRF rats. In conclusion. arginine administration has a profound beneficial effect on ECD, independently of cellular arginine uptake, in CRF female rats.

Measurement of Arginine Metabolites: Regulators of Nitric Oxide Metabolism

Arginine is the substrate for nitric oxide synthases (NOS), and arginine availability regulates the production of nitric oxide. Through the activity of methyltransferases, arginine can be methylated to form monomethylarginine (NMMA), asymmetrical dimethylarginine (ADMA), and symmetrical dimethylarginine (SDMA). NMMA and ADMA directly inhibit NOS, whereas SDMA inhibits the cellular import of arginine through the cationic amino acid transporter. Increased levels of methylarginine compounds have been associated with many diseases including atherosclerosis, renal failure, pulmonary hypertension, and preeclampsia. Previous HPLC methods to measure these molecules rely on derivatization with ortho-phthalaldehyde, which is unstable and requires immediate pre- or post-column reactions. We have identified a new fluorometric agent that is stable for at least 1 week and provides chromatographic properties that facilitate separation of these chemically similar compounds by reverse phase chromatography.

Maternal Hypercholesterolemia in Pregnancy Associates With Umbilical Vein Endothelial Dysfunction

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 &lt;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.

Identification of Cysteine Residues in Human Cationic Amino Acid Transporter hCAT-2A That Are Targets for Inhibition by N-Ethylmaleimide

In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y(+)L (y(+)LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y(+) (most likely CAT-1), but not system y(+)L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753-766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y(+)LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y(+)LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced transporter expression in the plasma membrane, indicating that NEM reduces the intrinsic transporter activity. Individual mutation of each of the seven cysteine residues conserved in all CAT isoforms did not lead to NEM insensitivity of hCAT-2A. However, a cysteine-less mutant was no longer inhibited by NEM, suggesting that inhibition occurs through modification of more than one cysteine in hCAT-2A. Indeed, also the double mutant C33A/C273A was insensitive to NEM inhibition, whereas reintroduction of a cysteine at either position 33 or 273 in the cysteine-less mutant led to NEM sensitivity. We thus identified Cys-33 and Cys-273 in hCAT-2A as the targets of NEM inhibition. In addition, all proteins with Cys-33 mutations showed a pronounced reduction in transport activity, suggesting that, surprisingly, this residue, located in the cytoplasmic N terminus, is important for transporter function.