Immune Alterations in a Patient With Hyperornithinemia-Hyperammonemia-Homocitrullinuria Syndrome: A Case Report.

The hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is a rare autosomal recessive inborn error of the urea cycle caused by mutations in the SLC25A15 gene. Besides the well-known metabolic complications, patients often present intercurrent infections associated with acute hyperammonemia and metabolic decompensation. However, it is currently unknown whether intercurrent infections are associated with immunological alterations besides the known metabolic imbalances. Herein, we describe the case of a 3-years-old girl affected by the HHH syndrome caused by two novel SLC25A15 gene mutations associated with immune phenotypic and functional alterations. She was admitted to the hospital with an episode of recurrent otitis, somnolence, confusion, and lethargy. Laboratory tests revealed severe hyperammonemia, elevated serum levels of liver transaminases, hemostasis alterations, hyperglutaminemia and strikingly increased orotic aciduria. Noteworthy, serum protein electrophoresis showed a reduction in the gamma globulin fraction. Direct sequencing of the SLC25A15 gene revealed two heterozygous non-conservative substitutions in the exon 5: c.649G>A (p.Gly217Arg) and c.706A>G (p.Arg236Gly). In silico analysis indicated that both mutations significantly impair protein structure and function and are consistent with the patient clinical status confirming the diagnosis of HHH syndrome. In addition, the immune analysis revealed reduced levels of serum IgG and striking phenotypic and functional alterations in the T and B cell immune compartments. Our study has identified two non-previously described mutations in the SLC25A15 gene underlying the HHH syndrome. Moreover, we are reporting for the first time functional and phenotypic immunologic alterations in this rare inborn error of metabolism that would render the patient immunocompromised and might be related to the high frequency of intercurrent infections observed in patients bearing urea cycle disorders. Our results point out the importance of a comprehensive analysis to gain further insights into the underlying pathophysiology of the disease that would allow better patient care and quality of life.

Effect of pH on weakly acidic and basic model drugs and determination of their ex vivo transdermal permeation routes

ABSTRACT The aim of the present study was to investigate the effect of donor pH on the transdermal permeability of the model drugs across rat skin and also to determine the major route of transport of the drugs. Weakly acidic drugs (partition coefficient) ibuprofen (3.6), aceclofenac (3.9), glipizide (1.9) and weakly basic drugs olanzapine (3.6), telmisartan (6.0), and sildenafil citrate (1.9) were selected for the study. The ex vivo permeation studies of these drugs at different donor pH (pH - 1.2, 4, 5, 6.8, 7.4, and 8) using Franz diffusion cell (area, 7.54 cm2) has shown a pH-dependent permeability. Among these drugs the weakly acidic drugs has shown higher permeation rates compared to the weakly basic drugs. The permeability coefficient and the distribution coefficient of the weakly basic drugs increased on increasing the pH whereas the weakly acidic drugs showed an inverse relation. The weakly basic drugs also showed an increase in permeation with increase in the fraction of unionized species indicating dominance of transcellular route of permeation. With an exception of sildenafil citrate, a weakly basic salt form of the drug which showed a high permeation value at pH 7.4 where 57% of the drug was unionized, indicating the involvement of both paracellular and transcellular route in its permeation.

Ornithine and Homocitrulline Impair Mitochondrial Function, Decrease Antioxidant Defenses and Induce Cell Death in Menadione-Stressed Rat Cortical Astrocytes: Potential Mechanisms of Neurological Dysfunction in HHH Syndrome.

Hyperornithinemia-hyperammonemia-homocitrullinuria (HHH) syndrome is caused by deficiency of ornithine translocase leading to predominant tissue accumulation and high urinary excretion of ornithine (Orn), homocitrulline (Hcit) and ammonia. Although affected patients commonly present neurological dysfunction manifested by cognitive deficit, spastic paraplegia, pyramidal and extrapyramidal signs, stroke-like episodes, hypotonia and ataxia, its pathogenesis is still poorly known. Although astrocytes are necessary for neuronal protection. Therefore, in the present study we investigated the effects of Orn and Hcit on cell viability (propidium iodide incorporation), mitochondrial function (thiazolyl blue tetrazolium bromide-MTT-reduction and mitochondrial membrane potential-ΔΨm), antioxidant defenses (GSH) and pro-inflammatory response (NFkB, IL-1ß, IL-6 and TNF-α) in unstimulated and menadione-stressed cortical astrocytes that were previously shown to be susceptible to damage by neurotoxins. We first observed that Orn decreased MTT reduction, whereas both amino acids decreased GSH levels, without altering cell viability and the pro-inflammatory factors in unstimulated astrocytes. Furthermore, Orn and Hcit decreased cell viability and ΔΨm in menadione-treated astrocytes. The present data indicate that the major compounds accumulating in HHH syndrome impair mitochondrial function and reduce cell viability and the antioxidant defenses in cultured astrocytes especially when stressed by menadione. It is presumed that these mechanisms may be involved in the neuropathology of this disease.

Melatonin attenuates Leishmania (L.) amazonensis infection by modulating arginine metabolism.

Acute inflammatory responses induced by bacteria or fungi block nocturnal melatonin synthesis by rodent pineal glands. Here, we show Leishmania infection does not impair daily melatonin rhythm in hamsters. Remarkably, the attenuated parasite burden and lesion progression in hamsters infected at nighttime was impaired by blockage of melatonin receptors with luzindole, whereas melatonin treatment during the light phase attenuated Leishmania infection. In vitro studies corroborated in vivo observations. Melatonin treatment reduced macrophage expression of Cat-2b, Cat1, and ArgI, genes involved in arginine uptake and polyamine synthesis. Indeed, melatonin reduced macrophage arginine uptake by 40%. Putrescine supplementation reverted the attenuation of infectivity by melatonin indicating that its effect was due to the arrest of parasite replication. This study shows that the Leishmania/host interaction varies in a circadian manner according to nocturnal melatonin pineal synthesis. Our results provide new data regarding Leishmania infectiveness and show new approaches for applying agonists of melatonin receptors in Leishmaniasis therapy.

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

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

Feline cystinuria caused by a missense mutation in the SLC3A1 gene.

BACKGROUND: Cystinuria is an inherited metabolic disease that is relatively common in dogs, but rare in cats and is characterized by defective amino acid reabsorption, leading to cystine urolithiasis. OBJECTIVES: The aim of this study was to report on a mutation in a cystinuric cat. ANIMALS: A male domestic shorthair (DSH) cat with cystine calculi, 11 control cats from Wyoming, and 54 DSH and purebred control cats from elsewhere in the United States. METHODS: Exons of the SLC3A1 gene were sequenced from genomic DNA of the cystinuric cat and a healthy cat. Genetic screening for the discovered polymorphisms was conducted on all cats. RESULTS: A DSH cat showed stranguria beginning at 2 months of age, and cystine calculi were removed at 4 months of age. The cat was euthanized at 6 months of age because of neurological signs possibly related to arginine deficiency. Twenty-five SLC3A1 polymorphisms were observed in the sequenced cats when compared to the feline reference sequence. The cystinuric cat was homozygous for 5 exonic and 8 noncoding SLC3A1 polymorphisms, and 1 of them was a unique missense mutation (c.1342C>T). This mutation results in a deleterious amino acid substitution (p.Arg448Trp) of a highly conserved arginine residue in the rBAT protein encoded by the SLC3A1 gene. This mutation was found previously in cystinuric human patients, but was not seen in any other tested cats. CONCLUSIONS AND CLINICAL IMPORTANCE: This study is the first report of an SLC3A1 mutation causing cystinuria in a cat, and could be used to characterize other cystinuric cats at the molecular level.

Expression of cationic amino acid transporters, carcass traits, and performance of growing pigs fed low-protein amino acid-supplemented versus high protein diets.

Free amino acids (AA) appear to be absorbed faster than protein-bound AA (PB-AA). We conducted an experiment to assess the effect of feeding pigs with a partially free (F-AA) or totally PB-AA diet on expression of selected genes and performance of pigs. The expression of cationic AA transporters b(0,+) and CAT-1 in intestinal mucosa, liver, and longissimus (LM) and semitendinosus (SM) muscles, as well as that of myosin in LM and SM, was analyzed. Twelve pigs (31.7 ± 2.7 kg) were used. The F-AA diet was based on wheat, supplemented with 0.59% L-Lys, 0.33% L-Thr, and 0.10% DL-Met. The PB-AA diet was formulated with wheat-soybean meal. Average daily feed intake was 1.53 kg per pig. The expression of b(0,+) and CAT-1 was analyzed in jejunal and ileal mucosa, liver, LM, and SM; myosin expression was also analyzed in both muscles. Pigs fed the PB-AA diet tended to have higher weight gain and feed efficiency (P < 0.10), and had thinner back fat (P = 0.02). The expression of b(0,+) was higher (P < 0.01) in jejunum but lower (P < 0.01) in the liver of pigs fed the F-AA diet; CAT-1 tended to be lower in liver but higher in LM of PB-AA pigs. Myosin expression was not affected. Intestinal AA absorption was faster in pigs fed the F-AA diet, but AA uptake by the liver seemed to be faster in pigs fed the PB-AA. Performance and expression of AA transporters and myosin suggest that the dietary content of free or protein-bound AA does not affect their availability for protein synthesis in pigs.

SLC3A1 and SLC7A9 mutations in autosomal recessive or dominant canine cystinuria: a new classification system.

BACKGROUND: Cystinuria, one of the first recognized inborn errors of metabolism, has been reported in many dog breeds. HYPOTHESIS/OBJECTIVES: To determine urinary cystine concentrations, inheritance, and mutations in the SLC3A1 and SLC7A9 genes associated with cystinuria in 3 breeds. ANIMALS: Mixed and purebred Labrador Retrievers (n = 6), Australian Cattle Dogs (6), Miniature Pinschers (4), and 1 mixed breed dog with cystine urolithiasis, relatives and control dogs. METHODS: Urinary cystinuria and aminoaciduria was assessed and exons of the SLC3A1 and SLC7A9 genes were sequenced from genomic DNA. RESULTS: In each breed, male and female dogs, independent of neuter status, were found to form calculi. A frameshift mutation in SLC3A1 (c.350delG) resulting in a premature stop codon was identified in autosomal-recessive (AR) cystinuria in Labrador Retrievers and mixed breed dogs. A 6 bp deletion (c.1095_1100del) removing 2 threonines in SLC3A1 was found in autosomal-dominant (AD) cystinuria with a more severe phenotype in homozygous than in heterozygous Australian Cattle Dogs. A missense mutation in SLC7A9 (c.964G>A) was discovered in AD cystinuria in Miniature Pinschers with only heterozygous affected dogs observed to date. Breed-specific DNA tests were developed, but the prevalence of each mutation remains unknown. CONCLUSIONS AND CLINICAL IMPORTANCE: These studies describe the first AD inheritance and the first putative SLC7A9 mutation to cause cystinuria in dogs and expand our understanding of this phenotypically and genetically heterogeneous disease, leading to a new classification system for canine cystinuria and better therapeutic management and genetic control in these breeds.

Supplemental leucine and isoleucine affect expression of cationic amino acid transporters and myosin, serum concentration of amino acids, and growth performance of pigs.

Leucine (Leu) participates in the activity of cationic amino acid (aa) transporters. Also, branched-chain aa [Leu, isoleucine (Ile), and valine (Val)] share intestinal transporters for absorption. We conducted an experiment with 16 young pigs (body weight of about 16 kg) to determine whether Leu and Ile affect expression of aa transporters b(0,+) and CAT-1 in the jejunum and expression of myosin in muscle, as well as serum concentration of essential aa, and growth performance in pigs. Dietary treatments were: wheat-based diets fortified with Lys, Thr, and Met; basal diet plus 0.50% Leu; basal diet plus 0.50% Ile, and basal diet plus 0.50% Leu and 0.50% Ile. After 28 days, the pigs were sacrificed to collect blood, jejunum, and semitendinosus and longissimus muscle samples. The effects of single and combined addition of Leu and Ile were analyzed. Leu alone or combined with Ile significantly decreased daily weight gain and reduced feed conversion. Leu and Ile, alone or in combination, significantly decreased expression of b(0,+) and significantly increased CAT-1. Ile alone or combined with Leu significantly decreased myosin expression in semitendinosus and significantly decreased it in longissimus muscle. Leu alone significantly decreased Lys, Ile and Thr serum concentrations; Ile significantly decreased Thr serum concentration; combined Leu and Ile significantly decreased Thr and significantly increased Val serum concentration. We conclude that dietary levels of Leu and Ile affect growth performance, expression of aa transporters and myosin, and aa serum concentrations in pigs.

Mismatch recognition function of Arabidopsis thaliana MutSγ.

Genetic stability depends in part on an efficient DNA lesion recognition and correction by the DNA mismatch repair (MMR) system. In eukaryotes, MMR is initiated by the binding of heterodimeric MutS homologue (MSH) complexes, MSH2-MSH6 and MSH2-MSH3, which recognize and bind mismatches and unpaired nucleotides. Plants encode another mismatch recognition protein, named MSH7. MSH7 forms a heterodimer with MSH2 and the protein complex is designated MutSγ. We here report the effect the expression of Arabidopsis MSH2 and MSH7 alone or in combination exert on the genomic stability of Saccharomyces cerevisiae. AtMSH2 and AtMutSγ proteins failed to complement the hypermutator phenotype of an msh2 deficient strain. However, overexpressing AtMutSγ in MMR proficient strains generated a 4-fold increase in CAN1 forward mutation rate, when compared to wild-type strains. Can(r) mutation spectrum analysis of AtMutSγ overproducing strains revealed a substantial increase in the frequency of base substitution mutations, including an increased accumulation of base pair changes from G:C to A:T and T:A to C:G, G:C or A:T. Taken together, these results suggest that AtMutSγ affects yeast genomic stability by recognizing specific mismatches and preventing correction by yeast MutSα and MutSß, with subsequent inability to interact with yeast downstream proteins needed to complete MMR.

The response to postnatal stress: amino acids transporters and PKC activity.

It is well known that animals exposed to stressful stimuli during their early life develop different neurological disorders when they become adults. In this study, we evaluated the effect of acute cold stress on gamma-aminobutyric acid (GABA) and L-Serine (L-Ser) transporters in vitro, using the uptake of [(3)H]-GABA and [(3)H]L-Ser by synaptosomes-enriched fractions isolated from rat cerebral cortex during postnatal development. GABA and L-Ser uptake studies in vitro will be used in this investigation as a colateral evidence of changes in the expression of transporters of GABA and L-Ser. We observed that the maximum velocity (V (max)) in L-Ser and GABA uptake after stress session increased in all stages studied. In contrast, K (m) values of L-Ser uptake enhancent in almost age calculated, excluding at PD21 after cold stress during development, at the same time as K (m) (uptake affinity) values of GABA increased in just about age considered but not at PD5 compared with the control group. Finally we investigated the mechanism by which cells regulate the substrate affinity of L-Ser and GABA transporters. We demonstrated a significantly increase in total PKC activity to PD5 from PD21. Pretreatment with PKC inhibitor: staurosporine (SP) led to a restoration of control uptake in several postnatal-days suggesting a relationship between amino acids system and PKC activation. These findings suggest that a single exposure to postnatal cold stress at different periods after birth modifies both GABA and L-Ser transporters and the related increase in total PKC activity could be intracellular events that participate in neuronal plasticity by early life stress, which could be relevant to function of transporters in the adult rat brain.

Trypanosoma cruzi amino acid transporter TcAAAP411 mediates arginine uptake in yeasts.

Trypanosoma cruzi, the aetiological agent of Chagas' disease, is exposed to extremely different environment conditions during its life cycle, and transporters are key molecules for its adaptive regulation. Amino acids, and particularly arginine, are essential components in T. cruzi metabolism. In this work, a novel T. cruzi arginine permease was identified by screening different members of the AAAP family (amino acid/auxin permeases) in yeast complementation assays using a toxic arginine analogue. One gene candidate, TcAAAP411, was characterized as a very specific, high-affinity, l-arginine permease. This work is the first identification of the molecular components involved specifically in amino acid transport in T. cruzi and provides new insights for further validation of the TcAAAP family as functional permeases.

DNA repair mutant pso2 of Saccharomyces cerevisiae is sensitive to intracellular acetaldehyde accumulated by disulfiram-mediated inhibition of acetaldehyde dehydrogenase.

Blocking aldehyde dehydrogenase with the drug disulfiram leads to an accumulation of intracellular acetaldehyde, which negatively affects the viability of the yeast Saccharomyces cerevisiae. Mutants of the yeast gene PSO2, which encodes a protein specific for repair of DNA interstrand cross-links, showed higher sensitivity to disulfiram compared to the wild type. This leads us to suggest that accumulated acetaldehyde induces DNA lesions, including highly deleterious interstrand cross-links. Acetaldehyde induced the expression of a PSO2-lacZ reporter construct that is specifically inducible by bi- or poly-functional mutagens, e.g., nitrogen mustard and photo-activated psoralens. Chronic exposure of yeast cells to disulfiram and acute exposure to acetaldehyde induced forward mutagenesis in the yeast CAN1 gene. Disulfiram-induced mutability of a pso2Delta mutant was significantly increased over that of the isogenic wild type; however, this was not found for acetaldehyde-induced mutagenesis. Spontaneous mutability at the CAN1 locus was elevated in pso2Delta, suggesting that growth of glucose-repressed yeast produces DNA lesions that, in the absence of Pso2p-mediated crosslink repair, are partially removed by an error-prone DNA repair mechanism. The use of disulfiram in the control of human alcohol abuse increases cellular acetaldehyde pools, which, based on our observations, enhances the risk of mutagenesis and of other genetic damage.

L-arginine signalling potential in the brain: the peripheral gets central.

L-arginine is a basic amino acid that has versatile metabolic roles, being involved in the generation of a wide range of biologically active intermediates such as nitric oxide (NO), polyamines, creatine and L-amino acids [1]. Because the levels of L-arginine reflect a metabolic crossroads, the mechanisms of its synthesis and degradation in peripheral tissues are very well described. However, there is an increasing amount of data also implicating this amino acid as a mediator of central nervous system activities and those are not yet fully understood. Here we shall summarize the tissue-specific pathways controlling L-arginine intracellular and blood levels and also the emerging evidence pointing to a role of this metabolite in the central regulation of diverse physiological processes, as blood pressure control and inflammatory response. As a conclusion we shall discuss the advantages of targeting L-arginine metabolism over other NO donors, as a general strategy to correct NO deficiency related diseases and discuss a few new patents recently deposited which take into account the rational perspective outlined in the present review.

Effect of ocular hypertension on retinal nitridergic pathway activity.

PURPOSE: Understanding the mechanisms of neuronal cell death in glaucoma is important for devising new treatments. Excitatory amino acids, excessive Ca(2+) influx, and formation of nitric oxide (NO) via NO synthase (NOS)-1 could be involved in glaucomatous neuropathy. The purpose of the present study was to examine the retinal nitridergic pathway activity in rats exposed to experimentally elevated intraocular pressure. METHODS: Weekly injections of HA were performed unilaterally in the rat anterior chamber, whereas the contralateral eye was injected with saline solution. At 3 or 6 weeks of treatment, retinal NOS activity was assessed through the conversion of (3)H-L-arginine to (3)H-L-citrulline, whereas NOS-1, -2, and -3 levels were assessed by Western blotting. L-Arginine uptake was measured using (3)H-l-arginine, whereas mRNA levels of L-arginine transporters were determined by semiquantitative RT-PCR. In addition, cyclic guanosine monophosphate (cGMP) levels were quantified by radioimmunoassay. RESULTS: At both 3 and 6 weeks of treatment, NOS activity significantly increased in HA-injected eyes although no changes in retinal NOS-1, -2, or -3 levels were observed in eyes injected with HA. L-Arginine influx and mRNA levels of cationic amino acid transporter type (CAT)-1 and -2 significantly increased in retinas from hypertensive eyes. Retinal cGMP levels significantly increased in eyes injected with HA for 3 but not 6 weeks. CONCLUSIONS: These results suggest a significant activation of the retinal nitridergic pathway in hypertensive eyes.

Equilibrative nucleoside (ENTs) and cationic amino acid (CATs) transporters: implications in foetal endothelial dysfunction in human pregnancy diseases.

Gestational diabetes (GD, characterized by abnormal D-glucose metabolism), intrauterine growth restriction (IUGR, a disease associated with reduced oxygen delivery (hypoxia) to the foetus), and preeclampsia (PE, a pregnancy complication characterized by high blood pressure, proteinuria and increased vascular resistance), induce foetal endothelial dysfunction with implications in adult life and increase the risk of vascular diseases. Synthesis of nitric oxide (NO) and uptake of L-arginine (the NO synthase (NOS) substrate) and adenosine (a vasoactive endogenous nucleoside) by the umbilical vein endothelium is altered in pregnancies with GD, IUGR or PE. Mechanisms underlying these alterations include differential expression of equilibrative nucleoside transporters (ENTs), cationic amino acid transporters (CATs), and NOS. Modulation of ENTs, CATs, and NOS expression and activity in endothelium involves protein kinase C (PKC), mitogen-activated protein kinases p42 and p44 (p42/44(mapk)), calcium, and phosphatidyl inositol 3 kinase (PI3k), among others. Elevated extracellular D-glucose and hypoxia alter human endothelial function. However, information regarding the transcriptional modulation of ENTs, CATs, and NOS is limited. This review focuses on the effect of transcriptional and post-transcriptional regulatory mechanisms involved in the modulation of ENTs and CATs, and NOS expression and activity, and the consequences for foetal endothelial function in GD, IUGR and PE. The available information will contribute to a better understanding of the cell and molecular basis of the altered vascular endothelial function in these pregnancy diseases and will emphasize the key role of this type of epithelium in placental function and the normal foetal development and growth.

Characterization of L-arginine transport in adrenal cells: effect of ACTH.

Nitric oxide synthesis depends on the availability of its precursor L-arginine, which could be regulated by the presence of a specific uptake system. In the present report, the characterization of the L-arginine transport system in mouse adrenal Y1 cells was performed. L-arginine transport was mediated by the cationic/neutral amino acid transport system y+L and the cationic amino acid transporter (CAT) y+ in Y1 cells. These Na+-independent transporters were identified by their selectivity for neutral amino acids in both the presence and absence of Na+ and by the effect of N-ethylmaleimide. Transport data correlated to expression of genes encoding for CAT-1, CAT-2, CD-98, and y+LAT-2. A similar expression profile was detected in rat adrenal zona fasciculata. In addition, cationic amino acid uptake in Y1 cells was upregulated by ACTH and/or cAMP with a concomitant increase in nitric oxide (NO) production.

L-Arginine transport in disease.

The importance of membrane transport in normal physiological cell function is unquestionable. However, to what extent alterations in the transport of amino acids are the cause and/or consequence of pathological changes observed in disease states is a question not yet completely clarified. Kinetic experiments with blood cells provide a simple and useful model for researching alterations in amino acid transport. The cationic amino acid L-arginine is the precursor of nitric oxide (NO), a key second messenger involved in functions such as endothelium-dependent vascular relaxation, immune defence and platelet activation. The transport of L-arginine, being rate-limiting for nitric oxide production, is extremely relevant to pathological conditions where NO synthesis and/or actions are affected. The current review provides an overview of L-arginine transport in disease, specifically in uraemia, heart failure, hypertension, diabetes mellitus, septic shock and sickle cell disease.

Biochemical characterization of a low-affinity arginine permease from the parasite Trypanosoma cruzi.

Trypanosoma cruzi, the etiological agent of Chagas disease, uses arginine for several metabolic processes, including energy reserves management. In the present work, a novel low-affinity arginine transport system has been studied. Maximum velocity (97 pmol min(-1) per 10(7) cells), and an estimate for the apparent Km value (350 microM) of this arginine transporter, were 6-fold and 80-fold higher respectively, when compared with the previously described high-affinity arginine transport system. This transport activity seems to be H+ -mediated, presents a broad specificity by other amino acids such as methionine, and is regulated along the parasite growth curve and life cycle.

Cell signalling-mediating insulin increase of mRNA expression for cationic amino acid transporters-1 and -2 and membrane hyperpolarization in human umbilical vein endothelial cells.

Insulin induces vasodilatation in human subjects and increases L-arginine transport and NO synthesis in human umbilical vein endothelial cells (HUVEC). Cell signalling events associated with insulin effects on activity and mRNA expression of the human cationic amino acid transporters 1 (hCAT-1) and 2B (hCAT-2B) are unknown. L-arginine transport and eNOS activity were determined in HUVEC exposed to insulin. mRNA levels for hCAT-1, hCAT-2B and eNOS were quantitated by real time RT-PCR and endothelial NO synthase (eNOS) protein was identified by Western blot analysis. Intracellular Ca2+, L-arginine and L-citrulline levels, L-[3H]citrulline formation from L-[(3)H]arginine, cGMP formation, nitrite level, ATP release and membrane potential were determined. Insulin increased L-arginine transport and the mRNA levels for hCAT-1 and hCAT-2B and eNOS expression and activity. Insulin also induced membrane hyperpolarization and increased intracellular Ca2+, L-[3H]citrulline, cGMP and nitrite formation. Insulin-mediated stimulation of the L-arginine/NO pathway is thus associated with increased hCAT-1 and hCAT-2B mRNA, and eNOS expression, via mechanisms involving membrane hyperpolarization, mitogen-activated protein kinases p42 and p44, phosphatidylinositol 3-kinase, NO and protein kinase C. We have characterized a cell signalling pathway by which hyperinsulinaemia could lead to vasodilatation in human subjects, and which could have implications in patients in whom plasma insulin levels are altered, such as in diabetes mellitus.