L-tryptophan Transport Research Articles

Transport of aromatic amino acids l-tryptophan, l-tyrosine, and l-phenylalanine by the organic anion transporting polypeptide (OATP) 3A1.

Amino acids are important for cellular metabolism. Their uptake across the plasma membrane is mediated by transport proteins. Despite the fact that the organic anion transporting polypeptide 4C1 (OATP4C1, Uniprot: Q6ZQN7) mediates transport of l-arginine and l-arginine derivatives, other members of the OATP family have not been characterized as amino acid transporters. The OATP family member OATP3A1 (gene symbol SLCO3A1, Uniprot: Q9UIG8) is ubiquitously expressed in human cells and highly expressed in many cancer tissues and cell lines. However, only a few substrates are known for OATP3A1. Accordingly, knowledge about its biological relevance is restricted. Our aim was to identify new substrates of OATP3A1 to gain insights into its (patho-)physiological function. In an LC-MS-based untargeted metabolomics assay using untreated OATP3A1-overexpressing HEK293 cells and control cells, we identified several amino acids as potential substrates of OATP3A1. Subsequent uptake experiments using exogenously added substrates revealed OATP3A1-mediated transport of l-tryptophan, l-tyrosine, and l-phenylalanine with 194.8 ± 28.7% (P < 0.05), 226.2 ± 18.7% (P < 0.001), and 235.2 ± 13.5% (P < 0.001), respectively, in OATP3A1-overexpressing cells compared to control cells. Furthermore, kinetic transport parameters (Km values) were determined (Trp = 61.5 ± 14.2 μm, Tyr = 220.8 ± 54.5 μm, Phe = 234.7 ± 20.6 μm). In summary, we identified the amino acids l-tryptophan, l-tyrosine, and l-phenylalanine as new substrates of OATP3A1. These findings could be used for a better understanding of (patho-)physiological processes involving increased demand of amino acids, where OATP3A1 should be considered as an important uptake transporter of l-tryptophan, l-tyrosine, and l-phenylalanine.

The high-affinity tryptophan uptake transport system in human cells.

The L-tryptophan (Trp) transport system is highly selective for Trp with affinity in the nanomolar range. This transport system is augmented in human interferon (IFN)-γ-treated and indoleamine 2,3-dioxygenase 1 (IDO1)-expressing cells. Up-regulated cellular uptake of Trp causes a reduction in extracellular Trp and initiates immune suppression. Recent studies demonstrate that both IDO1 and tryptophanyl-tRNA synthetase (TrpRS), whose expression levels are up-regulated by IFN-γ, play a pivotal role in high-affinity Trp uptake into human cells. Furthermore, overexpression of tryptophan 2,3-dioxygenase (TDO2) elicits a similar effect as IDO1 on TrpRS-mediated high-affinity Trp uptake. In this review, we summarize recent findings regarding this Trp uptake system and put forward a possible molecular mechanism based on Trp deficiency induced by IDO1 or TDO2 and tryptophanyl-AMP production by TrpRS.

A nanocomposite film with layer-by-layer self-assembled gold nanospheres driven by cucurbit[7]uril for the selective transport of l-tryptophan and lysozyme

Selective separation of amino acids and proteins is crucial in various areas of research, including proteomics, protein structure and function studies, protein purification and drug development, and biosensing and biodetection. A nanocomposite film is formed by combining layer-by-layer self-assembled gold nanospheres (AuNPs) driven by cucurbit[7]uril (CB[7]) and polymethyl methacrylate (PMMA) film. Due to the host-guest interactions, the selective transmission of l-tryptophan in the nanocomposite film is confirmed by the current-voltage measurements using a picoammeter. Furthermore, by adjusting the particle size of AuNPs to increase channel size, lysozyme containing multiple tryptophan residues can selectively pass through the nanocomposite film, indicating the high versatility and adaptability of the nanocomposite film. This study will provide a new direction for the selective separation of amino acids and proteins.

Functional domains of a ribosome arresting peptide are affected by surrounding nonconserved residues

Expression of the Escherichia coli tnaCAB operon, responsible for L-tryptophan (L-Trp) transport and catabolism, is regulated by L-Trp-directed translation arrest and the ribosome arresting peptide TnaC. The function of TnaC relies on conserved residues distributed throughout the peptide, which are involved in forming an L-Trp binding site at the ribosome exit tunnel and inhibiting the ribosome function. We aimed to understand whether nonconserved amino acids surrounding these critical conserved residues play a functional role in TnaC-mediated ribosome arrest. We have isolated two intragenic suppressor mutations that restore arrest function of TnaC mutants; one of these mutations is located near the L-Trp binding site, while the other mutation is located near the ribosome active site. We used reporter gene fusions to show that both suppressor mutations have similar effects on TnaC mutants at the conserved residues involved in forming a free L-Trp binding site. However, they diverge in suppressing loss-of-function mutations in a conserved TnaC residue at the ribosome active site. With ribosome toeprinting assays, we determined that both suppressor mutations generate TnaC peptides, which are highly sensitive to L-Trp. Puromycin-challenge assays with isolated arrested ribosomes indicate that both TnaC suppressor mutants are resistant to peptidyl-tRNA cleavage by puromycin in the presence of L-Trp; however, they differ in their resistance to puromycin in the absence of L-Trp. We propose that the TnaC peptide two functionally distinct segments, a sensor domain and a stalling domain, and that the functional versatility of these domains is fine-tuned by the nature of their surrounding nonconserved residues.

Pharmacologic inhibition of L‐tyrosine degradation ameliorates cerebral dopamine deficiency in murine phenylketonuria (PKU)

Monoamine neurotransmitter deficiency has been implicated in the etiology of neuropsychiatric symptoms associated with chronic hyperphenylalaninemia in phenylketonuria (PKU). Two proposed explanations for neurotransmitter deficiency in PKU include first, that chronically elevated blood L-phenylalanine (Phe) inhibits the transport of L-tyrosine (Tyr) and L-tryptophan (Trp), the substrates for dopamine and serotonin synthesis respectively, into brain. In the second hypothesis, elevated Phe competitively inhibits brain tyrosine hydroxylase (TH) and tryptophan hydroxylase (TPH) activities, the rate limiting steps in dopamine and serotonin synthesis. Dietary supplementation with large neutral amino acids (LNAA) including Tyr and Trp has been recommended for individuals with chronically elevated blood Phe in an attempt to restore amino acid and monoamine homeostasis in brain. As a potential alternative treatment approach, we demonstrate that pharmacologic inhibition of Tyr degradation through oral administration of nitisinone (NTBC) yielded sustained increases in blood and brain Tyr, decreased blood and brain Phe, and consequently increased dopamine synthesis in a murine model of PKU. Our results suggest that Phe-mediated inhibition of TH activity is the likely mechanism of impaired dopamine synthesis in PKU. Pharmacologic inhibition of Tyr degradation may be a promising adjunct therapy for CNS monoamine neurotransmitter deficiency in hyperphenylalaninemic individuals with PKU.

Separation of Two Amino Acids by Microemulsion Bulk Liquid Membrane

In this research work the potentialities of microemulsion bulk liquid membrane for the selective transport of L-tryptophan (L-Trp) and L-tyrosine (L-Tyr) are investigated at 298.15 K. Reversed micelle formed by sodium bis(2-ethylhexyl)sulfosuccinate (AOT) in dichloroethane, was used as mobile carrier to transport amino acids between a source and a receiving aqueous phase. The effects of pH, surfactant concentration and initial amino acid concentration on the extraction efficiency and transfer rate of the amino acids were studied. It is verified that for a mixture of two amino acids, L-Trp can be extracted selectively by using this type of the bulk liquid membrane with optimized condition.

Functional characterization of L-tryptophan transport across mammalian cornea.

In last few years transporter targeted drug delivery has drawn attention of research to identify and explore various nutrient transport systems including amino acid transporters for better drug delivery. The aim of present research work is to investigate the transport characteristics of L-tryptophan (L-try) across goat cornea. Transport of L-try was investigated using a glass diffusion cell for effect of concentration, pH, presence of other amino acids or metabolic inhibitor or dipeptide and tripeptide. The amount of L-try transported increased as the pH of L-try aqueous solution increased from 5 to 9. Inhibition was observed in L-try transport in absence of sodium ions where L-try solution was made isotonic with dextrose. Amino acids like L-histidine, L-arginine, L-lysine (cationic), L-glutamic acid, L-aspartic acid (anionic), glycine and L-proline (neutral) inhibited the L-try transport as compared to control (L-try alone). In presence of sodium azide and Ouabain the inhibition in L-try transport across goat cornea was observed while no marked inhibition was observed on L-try transport across goat cornea in presence of aspartame and glutathione. The L-try transport was favored up to concentration 1% w/v and at higher pH in presence of sodium ions through excised goat cornea. Functional presence of a sodium dependent L-try transport system as inhibited by ouabain having affinity to cationic and neutral amino acid is evident on goat cornea. Keywords: Cornea; Amino acid; Tryptophan; Transport

L-Tryptophan transport through a hydrophobic liquid membrane using AOT micelles: Dynamics of the process as revealed by small angle X-ray scattering

Hydrophobic liquid membranes have a high technological potential in many fields of separation science. The dynamics of these systems is very complex and still not fully understood. In this work we studied the effect of the incorporation of cationic and anionic l-tryptophan at pH 1.8 and 10.0, respectively, in Aerosol-OT reverse micelles performing small angle X-ray scattering experiments. The use of a synchrotron radiation source allowed efficient in situ data acquisition. Several insights on l-tryptophan transport dynamics through hydrophobic membranes containing AOT could be obtained from these SAXS experiments, such as amino acid site localization and changes in the reverse micelle sizes.

Indoleamine 2,3-dioxygenase activity and l-tryptophan transport in human breast cancer cells

The activity and expression of indoleamine 2,3-dioxygenase together with l-tryptophan transport has been examined in cultured human breast cancer cells. MDA-MB-231 but not MCF-7 cells expressed mRNA for indoleamine 2,3-dioxygenase. Kynurenine production by MDA-MB-231 cells, which was taken as a measure of enzyme activity, was markedly stimulated by interferon-γ (1000 units/ml). Accordingly, l-tryptophan utilization by MDA-MB-231 cells was enhanced by interferon-γ. 1-Methyl-dl-tryptophan (1 mM) inhibited interferon-γ induced kynurenine production by MBA-MB-231 cells. Kynurenine production by MCF-7 cells remained at basal levels when cultured in the presence of interferon-γ. l-Tryptophan transport into MDA-MB-231 cells was via a Na+-independent, BCH-sensitive pathway. It appears that system L (LAT1/CD98) may be the only pathway for l-tryptophan transport into these cells. 1-Methyl-d,l-tryptophan trans-stimulated l-tryptophan efflux from MDA-MB-231 cells and thus appears to be a transported substrate of system L. The results suggest that system L plays an important role in providing indoleamine-2,3-dioxygenase with its main substrate, l-tryptophan, and suggest a mechanism by which estrogen receptor-negative breast cancer cells may evade the attention of the immune system.

The role of L-tryptophan transport in L-tryptophan degradation by indoleamine 2,3-dioxygenase in human placental explants.

The physiological importance of L-tryptophan transport for placental indoleamine 2,3-dioxygenase-mediated degradation of L-tryptophan has been studied using human placental chorionic villous explants. L-Tryptophan influx into villous explants is supported exclusively by transport system L and is substantially inhibited by the L-system-specific substrate 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH) and also by 1-methyl-tryptophan which is also an inhibitor of indoleamine 2,3-dioxygenase. L-Tryptophan influx is enhanced 2.3-fold following in vitro culture of the villous explant. Interferon-gamma, which increases villous explant indoleamine 2,3-dioxygenase expression, has no effect on L-tryptophan influx. In explants both BCH and 1-methyl-tryptophan inhibit indoleamine 2,3-dioxygenase-mediated L-tryptophan degradation. This also applies when L-tryptophan degradation has been stimulated by interferon-gamma. These findings show transport of L-tryptophan into the trophoblast to be a rate-limiting step for indoleamine 2,3-dioxygenase-mediated L-tryptophan degradation and therefore for the normal physiology of mammalian pregnancy.

Role of serum free fatty acids in L-tryptophan transport into the liver.

A large part of L-tryptophan (Trp) present in the plasma or serum is bound to albumin (McMenamy et al., 1957). It is known (Curzon et al., 1974; Kragh-Hansen, 1983) that this binding of Trp to albumin is affected by various substances such as free fatty acids (FFA). It has been shown (Madras et al., 1974; Curzon et al., 1972) that in the plasma of rats under feeding of a high fat diet, starvation, and immobilization, free Trp levels are enhnaced by an increase in FFA levels. Madras et al. (1974) have demonstrated that in rats fed a protein-car-bonhydrate diet with various amounts of fat, liver Trp content dose not correlate with an increase in free serum Trp levels consequent to increased serum FFA levels. Recently, we have reported (Sasaki et al, 1993) that in rats fed a 40% lard-containing diet, there are little changes in the disappearance of blood Trp and the content of liver Trp under a marked increase in free serum Trp levels, and have suggested that under physiological levels of serum Trp and albumin, increased serum FFA levels do not have a large influence on Trp transport into the liver. However, it is still unclear whether or not serum FFA plays a facilitating role in Trp transport into the liver of rats, although increased serum FFA levels seem to have little effect on Trp transport into the liver under physiological levels of serum Trp and albumin. We, therefore, attempted to further clarify the role of serum FFA in Trp transport into the liver using analbuminemic rats, which are the mutant strain established from Sprague-Dawley rats and are genetically lacking in plasma albumin (Nagase et al., 1979).

Transport of various forms of L-tryptophan in the small intestine of chicks. Mechanism and regulation.

Tryptophan belongs under the irreplacable amino acids, the presence of which is needed in the ration for keeping up the life of organism. The ways of their absorption in mammal intestines were studied by Munck and Rasmussen (1975). They had stated that the entrance of L-tryptophan across the apical membrane of enterocytes was performed by the carriers of neutral amino acids. At its exit across the basolateral membrane a mechanism is used common with L-lysine. The study of mechanism for L-tryptophan absorption has been carried out on chicks in our laboratory as well. At this investigation the active character of L-tryptophan transport process was found out, its stereospeciflcity was revealed as well as dependence of the presence of sodium and hydrogen ions in the incubation medium and the presence of oxygen in it. A stimulating impact of a number of L-amino acids and carbohydrates was observed on the absorption of this irreplacable amino acid (Kushak et al., 1984; Kushak and Basova, 1987; Basova and Kushak, 1987).KeywordsSmall IntestineNeutral Amino AcidRabbit JejunumMammal IntestineSensitive Spectrophotometric MethodThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Kinetic characterization of a carrier-mediated transport system for L-tryptophan in human blood platelets.

We have characterized a membrane transport system on washed human blood platelets for tritiated L-tryptophan (L-TRP). This transport was extremely rapid, temperature dependent and markedly reduced by disruption of the platelet membranes. Kinetic studies within a large range of L-TRP concentrations have revealed the presence of a high affinity saturable transport system which follows simple Michaelis-Menten kinetics, with an apparent Km value of 10 microM and a Vmax of 200 pmol/min/10(8) cells. Platelet L-TRP accumulation was insensitive to changes in sodium concentrations and to the inclusion of ouabain in the incubation medium. Furthermore, uptake was unmodified by the presence of the metabolic inhibitor dinitrophenol, suggesting that it is mediated by facilitated diffusion. D-Tryptophan was a very poor inhibitor of L-TRP uptake. Transport was insensitive to serotonin and imipramine but was inhibited in a dose-dependent manner by L-tyrosine, L-phenylalanine and L-leucine, implying that it may be mediated by a system that is specific for aromatic and long chain amino acids. The results were compared to reports examining the L-TRP transport in other cell types.

Facilitating Effect of Albumin on L-Tryptophan Transport into Isolated Rat Hepatocytes.

The direct role of albumin in L-tryptophan transport into hepatocytes was studied by examining the effect of bovine serum albumin (BSA) on L-tryptophan uptake by isolated rat hepatocytes in comparison with that on the uptake of L-phenylalanine and L-leucine, which are known to be taken up into hepatocytes via the transport systems common to L-tryptophan. When the uptake rates of these amino acids were determined in an incubation medium containing BSA and at the same free-form substrate concentration, the rate of L-tryptophan uptake increased, while the rates of L-phenylalanine and L-leucine uptakes remained unchanged. L-Tryptophan was well bound to BSA, while L-phenylalanine and L-leucine were scarcely bound to the protein. BSA was clearly bound to the incubated cells. From the kinetic analysis of L-tryptophan uptake by hepatocytes with and without BSA, BSA was found to enhance the affinity between L-tryptophan and its transport site on the surface of liver cells. In hepatocytes pretreated with BSA, only L-tryptophan uptake was stimulated, in which the affinity between L-tryptophan and its transport site was enhanced. A significant amount of BSA was bound to the cells pretreated with the protein. When the effects of BSA co-addition and its pretreatment on the binding of L-tryptophan to plasma membranes prepared from rat livers were examined, the protein was found to promote the binding of L-tryptophan to the membranes. The present results suggest that albumin can facilitate L-tryptophan transport into hepatocytes by enhancing the affinity between the amino acid and its transport site present on the cell surface through its direct interaction with both the amino acid and the plasma membrane.

Transport of L-Tryptophan across the Rat Nasal Mucosa

Transport of L-Tryptophan across the Rat Nasal Mucosa

Effect of Albumin on the Disappearance of L-Tryptophan from the Perfusate into Isolated Perfused Rat Livers.

In order to clarify the role of albumin in the transport of L-tryptophan (Trp) into the liver, we examined the effect of bovine serum albumin (BSA) on the disappearance of Trp from the perfusate into isolated perfused rat livers. In liver perfusion with the perfusion medium containing 100μM Trp and 0.5, 2, or 4% BSA, the disappearance of Trp from the perfusate was depressed with a decrease in the concentration of free Trp (albumin-unbound form). The perfusion of livers with the medium containing 1mM Trp and 0.5, 2, or 4% BSA had little effect on the disappearance of Trp from the perfusate, though free-Trp concentrations decreased with perfusion time. In the perfusate of liver perfusion with the medium containing 100μM Trp and 0.5% BSA, total Trp concentrations correlated well with free Trp concentrations, whereas when the perfusion medium contained 100μM Trp and 2 or 4% BSA, total Trp concentrations correlated well with bound Trp concentrations. When liver perfusion was conducted with the medium containing 1mM Trp with 0.5, 2, or 4% BSA, there was a good correlation between total Trp and free Trp concentrations in the perfusate. These results suggest that under physiological conditions (ca., 100μM total Trp and 4% albumin), albumin contributes to the maintenance of total serum Trp concentrations and to the constant supply of serum Trp to the liver by lowering changes in free serum Trp concentrations through its binding to the amino acid.

Evidence for a defective platelet L-tryptophan transport in depressed patients

The kinetic characteristics of platelet L-tryptophan uptake were investigated in 23 untreated depressed patients and 18 healthy volunteers. A significant increase of 50% in the apparent Michaelis constant (Km) was observed in depressed patients compared with controls, without significant change in the maximal velocity (Vmax). After 1 month of successful antidepressant treatment the mean Km value decreased significantly and reached the control value. This result raises the possibility that the decrease in platelet tryptophan uptake affinity is a state-dependent marker for depression. It is likely that the transport alteration results in a decrease in platelet tryptophan accumulation. The effect of this peripheral membrane defect on brain serotonin function is discussed.

L-tryptophan uptake by segment-specific membrane vesicles from the proximal tubule of rabbit kidney.

1. The mechanism of the renal transport of L-tryptophan by basolateral and luminal membrane vesicles prepared from either the pars convoluta or the pars recta of the rabbit proximal tubule was studied. The uptake of L-tryptophan by basolateral membrane vesicles from the pars convoluta was found to be an Na(+)-dependent transport event. The Na(+)-conditional influx of the amino acid was stimulated in the presence of an inwardly directed H+ gradient. Lowering the pH without an H+ gradient had no effect, indicating that L-tryptophan is co-transported with H+. 3. On the other hand, no transient accumulation of L-tryptophan was observed in the presence or absence of Na+ in basolateral membrane vesicles from the pars recta. 4. In luminal membrane vesicles from the pars recta, the transient Na(+)-dependent accumulation of L-tryptophan occurred via a dual transport system. In addition, an inwardly directed H+ gradient could drive the uphill transport of L-tryptophan into these vesicles in both the presence and the absence of an Na+ gradient. 5. By contrast, the uptake of L-tryptophan by luminal membrane vesicles from the pars convoluta was a strictly Na(+)-dependent and electrogenic transport process, mediated by a single transport component. 6. Investigation of the coupling ratio in luminal membrane vesicles suggested that 1 Na+:1 L-tryptophan are co-transported in the pars convoluta. In the pars recta, examination of the stoichiometry indicated that approx. 1 H+ and 2 Na+ (high affinity) or 1 Na+ (low affinity) are involved in the uptake of L-tryptophan.

L-Tryptophan supplementation does not affect postoperative pain intensity or consumption of analgesics

Aiming at optimizing serotonin levels in endogenous pain-modulating systems, l-tryptophan supplementation pre- and postoperatively (2 g/day for 7 days) did not affect pain development or analgesic consumption after third molar surgery. Biological effects related to an increase in mood were observed, however, indicating transport of l-tryptophan to the central nervous system. The findings are discussed in relation to earlier reports on favorable effects of l-tryptophan on experimental pain in healthy subjects and in chronic pain patients.

Characterization of the L-tryptophan transport system in the liver of growing rats

In order to characterize the system of L-tryptophan (TRP) transport into liver during the growing period of 10 to 42 days, the changes of tryptophan 2,3-dioxygenase (TDO) activity, levels of serum, liver, brain, and muscle TRP, and the rate and mode of TRP uptake into isolated hepatocytes were examined in male Wistar rats. Liver TDO activity increased rapidly at 16 days of age. A marked and rapid decrease in free serum TRP level occurred before weanling, while a small decrease in total serum TRP level was found after weanling. The change of liver TRP level was similar to that of free serum TRP level and correlated well. There was a significant inverse correlation between liver TDO activity and either free serum TRP level or liver TRP level. A rapid change in TRP level did not occur in brain and muscle during the growing period. The concentrations of brain and muscle TRP correlated better with those of total serum TRP than with those of free serum TRP. The rate of TRP uptake into hepatocytes isolated from rats aged 10 days was lower than that from rats aged 21 and 42 days. The former hepatocytes were lacking in a high-affinity saturable transport component for TRP uptake which was present in the latter ones. The present results indicate that a great change in the system of TRP transport into liver occurs in growing rats, and that in suckling rats a high level of free serum TRP contributes to the efficient transport of the amino acid into the liver.