Tyrosine Transport Research Articles

Aberrant amino acid transport in fibroblasts from children with autism

Autism is a developmental, cognitive disorder clinically characterized by impaired social interaction, communication and restricted behaviours. The present study was designed to explore whether an abnormality in transport of tyrosine and/or alanine is present in children with autism. Skin biopsies were obtained from 11 children with autism (9 boys and 2 girls) fulfilling the DSM-IV diagnostic criteria for autistic disorder and 11 healthy male control children. Transport of amino acids tyrosine and alanine across the cell membrane of cultured fibroblasts was studied by the cluster tray method. The maximal transport capacity, V max and the affinity constant of the amino acid binding sites, K m, were determined. Significantly increased V max for alanine ( p = 0.014) and increased K m for tyrosine ( p = 0.007) were found in children with autism. The increased transport capacity of alanine across the cell membrane and decreased affinity for transport sites of tyrosine indicates the involvement of two major amino acid transport systems (L- and A-system) in children with autism. This may influence the transport of several other amino acids across the blood–brain-barrier. The significance of the findings has to be further explored.

PH and Ionic Strength Effects on Amino Acid Transport through Au-Nanotubule Membranes Charged with Self-Assembled Monolayers

The transport of the amino acids tyrosine and phenylalanine through Au-nanotubule membranes with self-assembled monolayers of alkanethiols is theoretically and experimentally studied. The membranes are prepared by electroless deposition of gold on porous polycarbonate track-etched membranes with control of the inner pore size, followed by self-assembly of acid-functionalized thiols (final mean average pore radius: 7 nm). The capability of switchable ion-transport selectivity by external control (e.g., by changing the pH, ionic strength, and amino acid concentration) is discussed. The flux changes with the pH and the ionic strength of the solution clearly show that electrical charges play a key role in the amino acid transport through the nanopores. Remarkably, an uphill transport of the amino acid is observed when a pH difference is imposed in the external solutions. The theoretical approach based on the Nernst−Planck flux equations considers all of the charged species present in the system (the cationic, anionic, and zwitterionic forms of the amino acid, the hydrogen and hydroxide ions, and the two salt ions) and allows a qualitative understanding of the transport phenomena in the charged nanopores.

Tyrosine transport in fibroblasts from healthy volunteers and patients with schizophrenia

Aberrant tyrosine transport across the fibroblast membrane, as measured by lower V max and/or lower K m is a repeated finding in patients with schizophrenia. The aim of this study was to investigate the importance of two major transporters, the L- and A-systems and tyrosine transport in fibroblast cell lines from patients with schizophrenia and healthy volunteers. Fibroblast cell lines, n = 6 from healthy volunteers and n = 6 from patients with schizophrenia, were included in the study. Uptake of [14-C] l-tyrosine in fibroblasts was measured using the cluster tray method in absence and presence of inhibitors. The uptake of tyrosine by the L-system was evaluated with the inhibitor 2-aminobicyclo heptane-2-carboxylic acid (BCH) and the A-system with the inhibitor nonmetabolized methyl-aminoisobutyric acid (MeAIB). Using [14-C] MeAIB the functionality of system A isoform 2, ATA2, was tested. BCH inhibited the uptake of tyrosine with 90%, showing that tyrosine transport in fibroblasts is mainly transported by the L-system. Not more than 10% could be contributed by the A-system. Excess of MeAIB did not influence tyrosine kinetics. Moreover, MeAIB kinetics did not differ between the patients and the controls. In conclusion, aberrant tyrosine transport observed in patients with schizophrenia is probably linked to the one of the L-systems and does not seem to involve the ATA2 transporter.

Support for limited brain availability of tyrosine in patients with schizophrenia

Several mechanisms have been suggested to account for altered dopaminergic neurotransmission in schizophrenia. The brain is the only organ for which amino-acid transport is limited and competition for transport over the blood-brain barrier (BBB) occurs at physiological plasma concentrations. One line of research suggests that patients with schizophrenia have altered brain levels of the essential amino acid tyrosine, the precursor for the synthesis of dopamine. The most common hypothesis is that less tyrosine is available because of competition with elevated levels of other amino acids. By consequence, the synthesis of dopamine in the brain will decrease. In contrast, another line of evidence suggests a change in the affinity for one of the transport proteins. A limitation of this research has been that the systems for amino-acid transport across the BBB have not been fully characterized at a molecular or functional level. The L system is the major system for transport of tyrosine across cell membranes including the BBB. The A system is also involved in this transport. Earlier in-vitro studies using fibroblasts have demonstrated a normal L system in schizophrenia but nevertheless reduced tyrosine transport. The combination of molecular research, fibroblast techniques, and brain imaging provides a new basis for clinical research on the role of amino-acid membrane transport in schizophrenia.

A Family of Basic Amino Acid Transporters of the Vacuolar Membrane from Saccharomyces cerevisiae

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.

Kinetics of tyrosine transport and cognitive functioning in schizophrenia

Background Tyrosine supplementation in humans has been shown to improve cognitive functioning. Several studies have demonstrated a decreased maximal transport capacity of tyrosine ( V max) across the cell membrane and an increased affinity ( K m) of tyrosine to membrane binding sites in schizophrenic patients. A lack of tyrosine for dopamine synthesis with impairment of dopaminergic transmission could impair cognitive functioning. Aberrant tyrosine kinetics in patients with schizophrenia might therefore be associated with cognitive dysfunction—a core feature of schizophrenia. Methods Tyrosine kinetics was determined in cultured fibroblasts from 36 schizophrenic patients. The kinetic parameters V max and K m were calculated and then the patients were divided into two groups according to the median of the kinetic parameters. A comprehensive neuropsychological test battery was used to evaluate cognitive functioning. Results Patients with low K m (below the median) had poorer cognitive performance than patients with high K m (above the median). V max did not discriminate schizophrenic patients with cognitive dysfunction to the same extent. Conclusions Changes in tyrosine transport probably influence cognitive functioning via the dopamine system. However, our findings of a relation between low K m and cognitive dysfunction may have a more complex background. It is suggested that the connection is related to genetically determined membrane factors that disturb communication/transmission among neurons.

Elevated free tyrosine in rhinoceros erythrocytes

Red blood cells of African black rhinoceroses ( Diceros bicornis) are highly sensitive to oxidant-induced hemolysis and they possess a number of enzymatic and biochemical features that differ radically from other mammals. Here we show concentrations of free tyrosine in rhinoceros red blood cells which can approach levels as high as 1 mM, 50-fold higher than in human red blood cells. Elevated levels of tyrosine are also observed in red blood cells of other members of the order Perissodactyla such as the horse and zebra. Captive black rhinoceroses have significantly lower levels of red blood cell tyrosine than black rhinoceroses in the wild. Tyrosine transport studies indicate that black rhinoceros red blood cells have lost the ability to transport tyrosine as efficiently as human red blood cells.

3-[ 123I]Iodo-L-α-methyl tyrosine transport into human fibroblasts and comparison with Ewing’s sarcoma cells

The cellular transport systems and the transport kinetics of [ 123I]IMT uptake into non-malignant extracranial cells were characterized for the first time. Human fibroblasts were chosen as non-malignant extracranial cells as they are found ubiquitous in the body. [ 123I]IMT is exclusively transported into fibroblasts via the sodium independent system L. An apparent Michaelis constant K m = 116.2 ± 18.9 μM and a maximum transport velocity V max = 191.6 ± 13.9 pmol ∗ (10 6 cells) −1 ∗ min −1 were calculated for the sodium-independent transport. These results were compared with those determined in two malignantly transformed extracranial cell lines, the human Ewing’s sarcoma cell lines VH-64 and CADO-ES-1.

Aberrant tyrosine transport across the cell membrane in schizophrenia

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptors family. PPARs are a family of 3 ligand-activated transcription factors: PPARα (NR1C1), PPARβ/δ (NUC1; NR1C2), and PPARγ (NR1C3). PPARα, -β/δ, and -γ are encoded by different genes but show substantial amino acid similarity, especially within the DNA and ligand binding domains. All PPARs act as heterodimers with the 9-cis-retinoic acid receptors (retinoid X receptor; RXRs) and play important roles in the regulation of metabolic pathways, including those of lipid of biosynthesis and glucose metabolism, as well as in a variety of cell differentiation, proliferation, and apoptosis pathways. Recently, there has been a great deal of interest in the involvement of PPARs in inflammatory processes. PPAR ligands, in particular those of PPARα and PPARγ, inhibit the activation of inflammatory gene expression and can negatively interfere with pro-inflammatory transcription factor signaling pathways in vascular and inflammatory cells. Furthermore, PPAR levels are differentially regulated in a variety of inflammatory disorders in man, where ligands appear to be promising new therapies.

The Human T-Type Amino Acid Transporter-1: Characterization, Gene Organization, and Chromosomal Location

System T is a Na+-independent transport system that selectively transports aromatic amino acids. Here, we determined the structure of the human T-type amino-acid transporter-1 (TAT1) cDNA and gene (SLC16A10). The human TAT1 cDNA encoded a 515-amino-acid protein with 12 putative membrane-spanning domains. Human SLC16A10 was localized on human chromosome 6, mapped to 6q21–q22. SLC16A10 contains six exons spanning 136 kb. In contrast to rat TAT1, which is mainly present in the intestine, human TAT1 was strongly expressed in human kidney as well as in human intestine. Expression of human TAT1 in Xenopus laevis oocytes demonstrated the Na+-independent transport of tryptophan, tyrosine, phenylalanine, and L-dopa, indicating that human TAT1 is a transporter subserving system T. Because human TAT1 is proposed to be crucial to the efficient absorption of aromatic amino acids from intestine and kidney, its defect could be involved in the disruption of aromatic amino-acid transport, such as in blue diaper syndrome.

Transport of aspartic acid, arginine, and tyrosine by the opportunistic protist Pneumocystis carinii

In order to improve culture media and to discover potential drug targets, uptake of an acidic, a basic, and an aromatic amino acid were investigated. Current culture systems, axenic or co-cultivation with mammalian cells, do not provide either the quantity or quality of cells needed for biochemical studies of this organism. Insight into nutrient acquisition can be expected to lead to improved culture media and improved culture growth. Aspartic acid uptake was directly related to substrate concentration, Q 10 was 1.10 at pH 7.4. Hence the organism acquired this acidic amino acid by simple diffusion. Uptake of the basic amino acid arginine and the aromatic amino acid tyrosine exhibited saturation kinetics consistent with carrier-mediated mechanisms. Kinetic parameters indicated two carriers ( K m=22.8±2.5 μM and K m=3.6±0.3 mM) for arginine and a single carrier for tyrosine ( K m=284±23 μM). The effects of other L-amino acids showed that the tyrosine carrier was distinct from the arginine carriers. Tyrosine and arginine transport were independent of sodium and potassium ions, and did not appear to require energy from ATP or a proton motive force. Thus facilitated diffusion was identified as the mechanism of uptake. After 30 min of incubation, these amino acids were incorporated into total lipids and the sedimentable material following lipid extraction; more than 90% was in the cellular soluble fraction.

Characterization of 3-[ 123I]iodo-L-α-methyl tyrosine ([ 123I]IMT) transport into human Ewing’s sarcoma cells in vitro

3-[ 123I]Iodo-L-α-methyl tyrosine ([ 123I]IMT) scintigraphy of extracranial malignant tumors has been described, but little is known about the transport systems involved in [ 123I]IMT uptake into extracranial tumor cells. Here, the precise kinetics of [ 123I]IMT transport into human Ewing’s sarcoma cells (VH-64) was determined. The apparent Michaelis constant was of high affinity value ( K m =41.7±3.9 μM) and maximum transport velocitiy amounted to V max=20.7±0.6 nmol·mg protein −1·10 min −1. Inhibition experiments revealed the predominance of [ 123I]IMT uptake via sodium-independent system L.

Characterization of 3‐[123I]iodo‐L‐α‐methyl tyrosine transport in astrocytes of neonatal rats

3-[(123)I]Iodo-L-alpha-methyl tyrosine ((123)I-IMT) is used for diagnosis and monitoring of brain tumours by means of single-photon emission tomography. As recently shown, (123)I-IMT is predominantly mediated into rat C6 glioma cells by sodium-independent system L for large neutral amino acids. Until now, (123)I-IMT transport in non-neoplastic glial cells has not been examined. Therefore, the aim of this study was to examine the cellular pathways and precise transport kinetics of (123)I-IMT uptake into astrocytes of neonatal rats. In particular sodium-independent (123)I-IMT transport into neonatal astrocytes was compared with sodium-independent (123)I-IMT uptake into neoplastic rat C6 glioma cells. Competitive inhibition experiments showed that (123)I-IMT is exclusively transported via sodium-independent system L into the neonatal astrocytes (92%). Kinetic analysis of sodium-independent (123)I-IMT uptake into neonatal astrocytes and into C6 glioma cells revealed apparent Michaelis constants K(M) = 13.9 +/- 0.5 microM and K(M) = 33.9 +/- 4.1 microM, respectively, which are in the same range of K(M) values as those recently determined for amino acid transport into neoplastic and non-neoplastic glial cells. Indeed, the K(M) values in the micromolar range correspond to the expression of the LAT-1 subunit of system L both in the neonatal astrocytes and in C6 glioma cells. However, sodium-independent maximum transport velocities (V(max)) differed significantly between neonatal astrocytes and C6 glioma cells (11.1 +/- 0.3 and 39.9 +/- 3.3 nmol/mg protein/10 min, respectively).

“Hypotyrosinemia” in Phenylketonuria

It has been postulated that the significant incidence of learning disabilities in well-treated patients with phenylketonuria (PKU) may be due, in part, to reduced production of neurotransmitters as a result of deficient tyrosine transport across the neuronal cell membrane. Hypotyrosinemia has been reported in treated and untreated PKU but virtually no data are available. We decided to examine this in our patient population and to compare it with the published norms, patient data from our hospital clinical biochemical laboratory database, and a group of normal children and adolescents in a private pediatric practice. We found that the mean nonfasting plasma tyrosine in 99 classical PKU patients was 41.1 μmol/L, in 26 mild (atypical) PKU patients 53.3 μmol/L, and in 35 non-PKU mild hyperphenylalaninemia patients 66.6 μmol/L. This compared to nonfasting plasma tyrosine levels in 102 non-PKU subjects of 64.0 μmol/L in our hospital biochemistry database, 69.1 μmol/L in 58 volunteers in the private office practice, and 64–78.8 μmol/L in infants, children, and adolescents in the literature review. Our data support the previously undocumented statements in the literature that plasma tyrosine levels are low in PKU.

Tyrosine transport is regulated differently in patients with schizophrenia

Previous PET studies of tyrosine transport have suggested that the transport of tyrosine from blood to brain compartment is not dependent on its plasma concentration in patients with schizophrenia. In order to examine this relationship, the transport constant ( K 1) of tyrosine was determined in five patients with schizophrenia and five normals. l-[1- 11C]Tyrosine was injected i.v. and arterial blood samples were taken during PET scanning. The tyrosine transport was assessed during baseline conditions and after oral administration of l-tyrosine at a dose (175 mg/kg) that significantly elevated the plasma levels. K 1 was determined from tracer kinetic modelling . The transport rate dropped in the normals after tyrosine loading, which is consistent with the prevailing notion that the brain transport system for neutral amino acids works close to saturation, whereas it was virtually unchanged in the schizophrenics. The results demonstrated that tyrosine transport was not saturated in the patients with schizophrenia and thus could lead to elevated brain concentrations of tyrosine.

Effect of UV irradiation on cell surface protease activity and amino acid uptake

The surface of most cells includes a coterie of resident proteins which act as receptors for a wide variety of ligands and other proteins which are potentially bioactive on cell–cell contact (juxtacrine effects), or else are released by enzyme activity to influence cell behaviour by autocrine or paracrine mechanisms. We previously found that UVC irradiation stimulates the release of TGFα from its membrane-bound preprocursor form whereby it acts as a stimulus to rapid, reparative cell multiplication; clearly this runs the risk hastening mitosis before UV-induced DNA damage is fully corrected, which in turn may increase the likelihood of residual lesions persisting and hence of new mutations being generated. We found that sublethal UVC irradiation (10 J m −2) of HeLa cell cultures also resulted in activation of ecto-aminopeptidase and ecto-endopeptidases which were maximal 16 and 20–24 h after irradiation, respectively. Both of these classes of protease were shown to be metalloproteases using a nonapeptide substrate (called P9) which is cognate to the N-terminal cleavage site of preproTGFα except for a reporter 125 I -tyrosine [Piva et al., J. Cell. Biochem. 64 (1997) 353–368]. We now show that the N-terminal tyrosine cleaved from P9 by cell surface aminopeptidase activity, was found to be taken up by the cell resulting in its 10–25-fold concentration intracellularly, some two- to threefold higher than from a reservoir source, and may represent a novel salvage pathway for recovery of essential amino acids. Aminopeptidase activity was found to be both temperature- and FBS-dependent but was not reliant on ATP for its activity. Tyrosine transport across the cell membrane was also temperature and FBS-dependent but required ATP for maximal activity. UVC irradiation enhanced aminopeptidase activity but not tyrosine uptake by the cultures. The fraction of HeLa cells undergoing apoptosis increased in those cultures which were exposed to higher doses of UVC. The levels of ecto-aminopeptidase and ecto-endopeptidase activity in apoptotic cells were elevated compared to viable cells receiving the same dose of UVC. These results suggest that increased levels of cell surface protease activity in apoptotic cells would increase the amounts of free amino acids and growth factors in the extracellular medium and hence stimulate the proliferation of surrounding cells to replace those killed by UV irradiation.

Normal Tyrosine Transport and Abnormal Tyrosinase Routing inPink-Eyed DilutionMelanocytes

The pink-eyed dilution phenotype in mice arises from mutations in thepgene; in humans, analogous mutations in thePgene result in oculocutaneous albinism type 2. Although the molecular mechanisms which underlie this phenotype remain obscure, it has been postulated that mutations inpresult in defective tyrosine transport into murine melanosomes, resulting in hypopigmentation and diminished coat color. However, we previously reported no difference in melanosomal tyrosine transport in unpigmented, melanoblast-like pink-eyed dilution (pcp/pcp), and in pigmented (melan-a) murine melanocytes. In this study, we utilized melan-p1 cells, more differentiated pink-eyed dilution (pcp/p25H) melanocytes which can be induced to produce melanin, to characterize the melanogenic lesion(s) more definitively. Uptake of [3H]tyrosine into melan-a melanosomes did not differ significantly from uptake into melanosomes derived from melan-p1 melanocytes, further arguing against its critical role as a tyrosine transporter. Pink-eyed dilution melanocytes incubated in high tyrosine concentrations became extremely pigmented as they became confluent and secreted large amounts of black material into the medium. Total cellular tyrosinase activity in melan-p1 melanocytes was significantly higher than that in melan-a melanocytes (which are wild-type at theplocus), but the localization of tyrosinase to melanosomes was impaired in melan-p1 melanocytes compared to melan-a melanocytes. These results indicate that mechanisms other than deficient tyrosine transport are involved in the pink-eyed dilution phenotype and that this protein may serve a chaperone-like or stabilizing function in melanocytes.

Tyrosine Transport into Melanosomes Is Increased Following Stimulation of Melanocyte Differentiation

A variety of physiological factors can stimulate differentiation of melanocytes to increase pigmentation, and critical to this process is the transport of the melanogenic substrate (tyrosine) into melanosomes. In this study, we examined whether stimulation of melanogenesis affects melanosomal tyrosine transport. Tyrosine uptake increased almost 2-fold in melanosomes derived from melanocytes treated with melanocyte-stimulating hormone (MSH), which acts to increase intracellular cAMP levels, resulting in the up-regulation of many genes involved in melanogenesis. Stimulation of melanoma cells with dibutyryl cAMP increased melanosomal tyrosine transport 2- to 3-fold after 24 to 48 hrs, with peak levels occurring after 3 to 5 days of treatment, suggesting thatde novogene expression may be required. The cAMP-induced increase in melanosomal tyrosine transport could be effectively competed with phenylalanine or tryptophan, but not with dopamine or proline, suggesting either that a pool of transporters with greater tyrosine transporting ability pre-exists, or that a greater number of tyrosine transporters reside within the melanosomal membrane. These results illustrate a rare example of hormonal plasma membrane stimulation which transduces a signal for increased vesicular transport of an amino acid.

65-128 - The transport of tyrosine is differently regulated in patients with schizophrenia than in normals

65-128 - The transport of tyrosine is differently regulated in patients with schizophrenia than in normals

A topological model for the general aromatic amino acid permease, AroP, of Escherichia coli.

The general aromatic amino acid permease, AroP, of Escherichia coli is responsible for the active transport of phenylalanine, tyrosine, and tryptophan. A proposed topological model for the AroP permease, consisting of 12 hydrophobic transmembrane spans connected by hydrophilic loops, is very similar to that of the closely related phenylalanine-specific permease. The validity of this model and its similarity to that of the PheP permease were investigated by studying fusion proteins of AroP permease and alkaline phosphatase. Based on the results obtained from the AroP-alkaline phosphatase sandwich fusions, we have significantly revised the proposed topological model for AroP in two regions. In this modified AroP topological model, the three charged residues E151, E153, and K160 are repositioned within the membrane in span 5. These three residues are conserved in a large family of amino acid transport proteins, and site-directed mutagenesis identifies them as being essential for transport activity. It is postulated that these residues together with E110 in transmembrane span 3 may be involved in a proton relay system.