Neutral Aromatic Amino Acids Research Articles

Structure of the Blood Brain Barrier and its Role in the Transporters for the Movement of Substrates across the Barriers.

For the brain to operate normally and to develop with structural integrity in addition to neuronal function, blood-brain barrier present in brain capillaries serves as a vital barrier mechanism. In addition to the transport barrier created by membranes, transporters, and vesicular processes, the structure and function of the BBB are summarised. The physical barrier is created by endothelial tight junctions. The permeability and transport of molecules between extracellular fluid and plasma are constrained by the presence of tight junctions between neighbouring endothelial cells. Each solute must pass through both membranes in the luminal and abluminal divisions. The functions of the neurovascular unit are described, with special emphasis on the pericytes, microglia, and astrocyte endfeet. The luminal membrane contains five separate facilitative transport mechanisms, each of which is exclusive to a few substrates. Nevertheless, the import of big-branched and aromatic neutral amino acids is facilitated by two key carriers (System L and y+) in the plasma membrane. It is asymmetrically present in both membranes. The sodium pump Na+/K+-ATPase is highly expressed in the abluminal membrane, where many Na+ dependent transport mechanisms move amino acids against its concentration gradient. The trojan horse strategy, which uses molecular tools to bind the medication and its formulations, is also preferred in drug delivery. The BBB's cellular structure, the transport systems unique to each substrate, and the necessity to identify transporters with changes that assist the transfer of various medications have all been changed in the current work. Nevertheless, to rule out the BBB passage for the new class of neuroactive medications, the mixing of traditional pharmacology and nanotechnology needs to be focused on outcomes that show promise.

Self-healing hydrogels triggered by amino acids

Nine amino acids with different chemical properties have been chosen to promote the formation of hydrogels based on the bolamphiphilic gelator A: three basic amino acids (arginine, histidine and lysine), one acidic amino acid (aspartic acid), two neutral aliphatic amino acids (alanine and serine) and three neutral aromatic amino acids (phenylalanine, tyrosine and tryptophan).

5-(2-18F-Fluoroethoxy)-l-Tryptophan as a Substrate of System L Transport for Tumor Imaging by PET

Large neutral l-amino acids are substrates of system L amino acid transporters. The level of one of these, LAT1, is increased in many tumors. Aromatic l-amino acids may also be substrates of aromatic l-amino acid decarboxylase (AADC), the level of which is enhanced in endocrine tumors. Increased amino acid uptake and subsequent decarboxylation result in the intracellular accumulation of the amino acid and its decarboxylation product. (18)F- and (11)C-labeled neutral aromatic amino acids, such as l-3,4-dihydroxy-6-(18)F-fluorophenylalanine ((18)F-FDOPA) and 5-hydroxy-l-[β-(11)C]tryptophan, are thus successfully used in PET to image endocrine tumors. However, 5-hydroxy-l-[β-(11)C]tryptophan has a relatively short physical half-life (20 min). In this work, we evaluated the in vitro and in vivo characteristics of the (18)F-labeled tryptophan analog 5-(2-(18)F-fluoroethoxy)-l-tryptophan ((18)F-l-FEHTP) as a PET probe for tumor imaging. (18)F-l-FEHTP was synthesized by no-carrier-added (18)F fluorination of 5-hydroxy-l-tryptophan. In vitro cell uptake and efflux of (18)F-l-FEHTP and (18)F-FDOPA were studied with NCI-H69 endocrine small cell lung cancer cells, PC-3 pseudoendocrine prostate cancer cells, and MDA-MB-231 exocrine breast cancer cells. Small-animal PET was performed with the respective xenograft-bearing mice. Tissues were analyzed for potential metabolites. (18)F-l-FEHTP specific activity and radiochemical purity were 50-150 GBq/μmol and greater than 95%, respectively. In vitro cell uptake of (18)F-l-FEHTP was between 48% and 113% of added radioactivity per milligram of protein within 60 min at 37°C and was blocked by greater than 95% in all tested cell lines by the LAT1/2 inhibitor 2-amino-2-norboranecarboxylic acid. (18)F-FDOPA uptake ranged from 26% to 53%/mg. PET studies revealed similar xenograft-to-reference tissue ratios for (18)F-l-FEHTP and (18)F-FDOPA at 30-45 min after injection. In contrast to the (18)F-FDOPA PET results, pretreatment with the AADC inhibitor S-carbidopa did not affect the (18)F-l-FEHTP PET results. No decarboxylation products of (18)F-l-FEHTP were detected in the xenograft homogenates. (18)F-l-FEHTP accumulates in endocrine and nonendocrine tumor models via LAT1 transport but is not decarboxylated by AADC. (18)F-l-FEHTP may thus serve as a PET probe for tumor imaging and quantification of tumor LAT1 activity. These findings are of interest in view of the ongoing evaluation of LAT1 substrates and inhibitors for cancer therapy.

LAT1 expression in non-small-cell lung carcinomas: Analyses by semiquantitative reverse transcription-PCR (237 cases) and immunohistochemistry (295 cases)

Objective System l-amino acid transport mediates the uptake of aromatic neutral amino acids and nutritionally essential amino acids from extracellular fluids. Little is known about the role of l-amino acid transporter 1 (LAT1), a member of the system l-amino acid transporter family, in non-small-cell lung carcinomas (NSCLCs). Patients and methods We examined (i) LAT1 mRNA levels in 40 normal lung tissues (NLTs) and 237 NSCLCs using semiquantitative RT-PCR, (ii) LAT1 protein expression in 295 NSCLCs using immunohistochemistry, and (iii) whether LAT1 mRNA and protein expressions were related to clinicopathologic findings and outcome. Results The LAT1 mRNA level was significantly higher in all NSCLCs (6.81 ± 1.13) than in NLT (1.00 ± 0.18). The LAT1 mRNA level showed no association with clinicopathologic findings or outcome. LAT1 protein was detected with a diffuse or granular appearance within the cytoplasm and/or on the plasma membrane of tumor cells. When tumors were graded as positive if staining indicating a plasma membrane expression of LAT1 protein made up more than 10% of the tumor, the frequency of this membrane expression was found to be associated with tumor histology, differentiation grade, pathologic stage, T classification, pleural invasion, lymph-vessel invasion, and overall survival rate. Conclusion Detection of a plasma membrane expression of LAT1 protein would appear to be of value in informing the prognosis in NSCLC cases.

Cation−π Interactions: Structures and Energetics of Complexation of Na+ and K+ with the Aromatic Amino Acids, Phenylalanine, Tyrosine, and Tryptophan

Threshold collision-induced dissociation of M(+)(AAA) with Xe is studied using guided ion beam tandem mass spectrometry. M(+) include the alkali metal ions Na(+) and K(+). The three aromatic amino acids are examined, AAA = phenylalanine, tyrosine, or tryptophan. In all cases, endothermic loss of the intact aromatic amino acid is the dominant reaction pathway. The threshold regions of the cross sections are interpreted to extract 0 and 298 K bond dissociation energies for the M(+)-AAA complexes after accounting for the effects of multiple ion-neutral collisions, internal energy of the reactant ions, and dissociation lifetimes. Density functional theory calculations at the B3LYP/6-31G level of theory are used to determine the structures of the neutral aromatic amino acids and their complexes to Na(+) and K(+) and to provide molecular constants required for the thermochemical analysis of the experimental data. Theoretical bond dissociation energies are determined from single-point energy calculations at the B3LYP/6-311++G(3df,3pd) level using the B3LYP/6-31G geometries. Good agreement between theory and experiment is found for all systems. The present results are compared to earlier studies of these systems performed via kinetic and equilibrium methods. The present results are also compared to the analogous Na(+) and K(+) complexes to glycine, benzene, phenol, and indole to elucidate the relative contributions that each of the functional components of these aromatic amino acids make to the overall binding in these complexes.

System L: heteromeric exchangers of large, neutral amino acids involved in directional transport.

The plasma membrane transport system L is in many cells the only (efficient) pathway for the import of large branched and aromatic neutral amino acids. The corresponding transporters are hetero(di)mers composed of a catalytic subunit (LAT1 or LAT2=light chain=glycoprotein-associated amino acid transporter) associated covalently with the glycoprotein 4F2hc/CD98 (heavy chain). The tissue distribution of LAT1 suggests that it is involved mainly in transporting amino acids into growing cells and across some endothelial/epithelial secretory barriers, whereas the localization of LAT2 indicates that it is mainly involved in the basolateral efflux step of transepithelial (re)absorptive amino acid transport. However, system L transporters are obligatory amino acid exchangers with 1:1 stoichiometry, with similar (but not identical) intra- and extracellular substrate selectivities and with highly asymmetrical apparent affinities (low affinity inside). Therefore, net directional transport of large, neutral amino acids by system L depends on the parallel expression of a unidirectional transporter with overlapping selectivity (for instance systems A or N) that provides/recycles amino acids that drive system L exchange function. By mediating the regulated flux of these exchange substrates, unidirectional transporters control the activity of system L.

Chemical modification of the neutral amino acid transport system L of Chinese hamster ovary cells with p-chloromercuribenzene sulfonate.

Branched-chain and aromatic neutral amino acids enter mammalian cells predominantly through a Na(+)-independent transport agency called System L. The sulfhydryl specific reagent p-chloromercuribenzene sulfonate (pCMBS) has been shown to be a potent inactivator of System L transport activity in Chinese hamster ovary cells, however, inactivation by pCMBS can be prevented by the presence of System L-specific substrate amino acids during the inactivation reaction. In addition, the presence of amino acids that are not substrates for System L have no effect on pCMBS inactivation of System L. Inactivation of System L activity by pCMBS was sensitive to pH and reversible by incubation with dithiothreitol. These findings suggest that there is a sulfhydryl group in, or very near, the amino acid-binding site of the System L transporter of CHO cells. Substrate protection, however, could be explained by conformational changes in the transporter associated with substrate binding. The presence of a substrate protectable sulfhydryl group on the System L transporter would aid in the attempt to identify this transporter using the technique of differential labeling.

Uptake of l-leucine and l-phenylalanine across the basolateral cell surface in isolated oxyntic glands

The time course kinetic specificity and sodium-dependence of l-leucine and l-phenylalanine uptake by rabbit isolated oxyntic glands were studied in order to identify the systems involved in the transport of branched-chain and aromatic neutral amino acids through the basolateral cell membrane. The uptake was measured directly in the disrupted cells after incubation of the glands with the 3H-labelled amino acid both in a sodium-containing and a sodium-free medium. The uptake of l-leucine was largely carrier-mediated whilst l-phenylalanine was taken up by either carrier-mediated and nonsaturable processes. Both amino acids were taken up by a Na +-independent process. The kinetic parameters of l-leucine and l-phenylalanine carrier-mediated influx were, respectively: K t = 2.71 mM and J max = 1390 nmol mg −1 s −1, K t = 1.03 mM and J max = 176 nmol mg −1 s −1. From cross-inhibition studies it can be inferred that l-leucine is primarily transported by a Na +-independent system which shows specificity for bulky side chains dipolar amino acids. The system displays similar affinities for l-phenylalanine ( K i = 2.81 mM) and l-isoleucine ( K i = 2.62 mM). Similar results were obtained from self-inhibition experiments: the K i of the carrier-mediated uptake of l-leucine and phenylalanine were 2.12 and 2.40 mM (from a Hanes plot) or 3.2 and 0.8 mM (from a Dixon plot), respectively. It is concluded that a sodium-independent transport system, like Christensen's L′ type, is shared by branched-chain and aromatic dipolar amino acids, which only shows slight differences in their affinities for the carrier.

Expression cloning of a Na(+)-independent neutral amino acid transporter from rat kidney.

Uptake of long-chain and aromatic neutral amino acids into cells is known to be catalyzed by the Na(+)-independent system L transporter, which is ubiquitous in animal cells and tissues. We have used a Xenopus oocyte expression system to clone the cDNA of a system L transporter from a rat kidney cDNA library. The 2.3-kilobase cDNA codes for a protein of 683 amino acids. The transporter has four putative membrane-spanning domains and bears no sequence or structural homology to any known animal or bacterial transporter. When transcribed and expressed in Xenopus oocytes, the transporter exhibits many, but not all, of the characteristics of L-system transporters, suggesting that this represents one of several related L-system transporters.

High-affinity uptake of L-kynurenine by a Na+-independent transporter of neutral amino acids in astrocytes

L-Kynurenine (KYN), an intermediary product in the kynurenine pathway of tryptophan metabolism, is the common precursor from which are formed both quinolinic acid, a potent endogenous "excitotoxin," and kynurenic acid, a nonselective antagonist of excitotoxins. The present work examines 3H-KYN transport in primary astrocyte cultures derived from the cerebra of newborn mice. Influx and efflux of 3H-KYN were attributable almost entirely to carrier-mediated transport. The tritium recovered in uptake experiments was identifiable as 3H-KYN, indicating a low rate of KYN metabolism during incubations up to 30 min. KYN uptake decreased in the presence of extracellular Na+, at least in part because KYN efflux was accelerated. Marked trans stimulation of KYN efflux by extracellular KYN provided evidence of the exchanging nature of the carrier. Saturation curves for the initial velocity of KYN uptake conformed to a 1-component saturable system with Km of 32 microM and Vmax of 2.1 nmol mg-1 protein min-1. KYN was notably concentrated by the astrocytes, with an estimated steady-state distribution ratio of 180-fold for 1 microM KYN. Analog inhibition studies showed that the KYN transporter exhibited a clear preference for large neutral amino acids; leucine, tryptophan, and phenylalanine were recognized with relatively higher affinity than KYN. In summary, KYN is concentratively transported into astrocytes by a Na+-independent exchanger with high affinity for branched-chain and aromatic neutral amino acids. The substrate specificity and high affinity of this transport system resemble the properties of neutral amino acid transport across the blood-brain barrier in the rat and human.

Synthetic amino acids and the nature of L-DOPA transport at the blood-brain barrier.

Abstract—The blood‐brain barrier transport of amino acids has been measured using the carotid injection technique in the rat. The synthetic amino acids, 2‐aminobicyclo(2,2,1)heptane‐2‐carboxylic acid (BCH) and α‐(methylamino)isobutyric acid (MeAIB), were model substrates in the Ehrlich cell for the leucine (L) and alanine (A) neutral amino acid transport mechanisms, respectively. The uptake (±)b‐[carboxyl‐14C]BCH at the same rate for the five brain regions tested suggested a similarity between regions for the L transport mechanism. At injectant concentrations of 0·1 mm (similar to naturally occurring aromatic neutral amino acids), BCH was mainly taken up by a saturable mediated transport mechanism (K1, 0·16 mm and Vmax, 0·03/μmol/g per min). At higher concentrations, uptake by a nonsaturable or diffusional mechanism could be demonstrated. When BCH was added as a second amino acid to l‐[3‐14C]DOPA, the saturable component of l‐DOPA transport was significantly inhibited. MeAIB had no measurable effect on the rate of l‐DOPA transport. These results suggested that the mediated transport mechanism for l‐DOPA at the cerebral capillaries is similar to the l‐neutral amino acid transport system.

Molecular transport: I. In vivo studies of transport mutants of Neurospora crassa with altered amino acid competition patterns

The active transport of metabolites by Neurospora crassa conidia has been studied by both kinetic and genetic methods. Transport, reported to be dependent on temperature, pH, and an energy source, is shown to accumulate amino acids by (1) a system specific for neutral-aromatic amino acids, the neutral transport system; (2) a system specific for basic amino acids, the basic transport system; and (3) a non-specific general amino acid transport system. Transport by any of these systems, separately or in combination, results in the accumulation of the respective amino acid against a concentration gradient. The p-fluorophenylalanine-resistant mutant, Pm − N 22, is deficient in transport of l-phenylalanine, but not l-arginine. Analogous to this is the canavanine-resistant mutant, Pm − B 37, which is deficient in transport of l-arginine, but not l-phenylalanine. The double mutant, Pm − NB (derived from a cross of Pm − N 22 and Pm − B 37) has reduced transport of both l-arginine and l-phenylalanine. This double mutant retains some residual transport activity for both the basic and neutral amino acids. The kinetic properties of this residual activity indicate a capability for transport of either neutral or basic amino acids and further suggests that it is under separate genetic control.

Taenia crassiceps: Uptake of basic and aromatic amino acids and imino acids by larvae

A study was made of the uptake of lysine, tyrosine, phenylalanine, and proline by metacestode larvae of Taenia crassiceps (cestoda, cyclophyllidea) by observing in each case the rate of uptake, the kinetics of uptake, and the effects upon uptake of the presence of a variety of other compounds. Lysine was absorbed by a mechanism of active transport specific for molecules with a charged basic group. Tyrosine and phenylalanine were absorbed by an active mechanism specific for neutral aromatic amino acids. Proline was absorbed by a mechanism showing affinity for both imino and amino acids, but the involvement of active processes was not conclusively demonstrated for this mechanism.

Studies on the absorption of amino acids by larval tapeworms (Cyclophyllidea: Taenia crassiceps).

Larvae of Taenia crassiceps have been shown to absorb L-valine and L-methionine by a process of active transport. Uptake is maximal at about 43 °C and a pH of about neutrality, and concentration against a gradient has been demonstrated. In advanced larvae, the absorption mechanism has been shown to be specific for neutral aliphatic amino acids, with an uncharged amino group in the α-position. Competitive inhibitions of uptake have been demonstrated in the presence of other amino acids.Comparison has been made with a mechanism present in Hymenolepis diminuta for the uptake of neutral amino acids, demonstrated by Read et al. (1963), and the absorption of L-methionine by the two species has been compared in detail.The uptake of valine and methionine by the two types of larvae has been compared in detail: the presence of two types of sites for the uptake of these aminoacids has been suggested.The absorption of the L-isomers of glutamic acid, proline, serine, glycine and leucine has been demonstrated in advanced larvae. Separate mechanisms for the uptake of acidic amino acids, basic amino acids and neutral aromatic amino acids have been suggested.Thanks are due to Professor G. Chapman, in whose department this work was carried out, and to Miss E. Clarry for her willing assistance. We would also like to thank Dr G. Ayrey, of the Queen Elizabeth College Isotope Unit, for his valuable help, especially in the development of a technique for the simultaneous counting of 14C and 3H. The work was supported by a research grant from the Wellcome Trustees, to whom we are most grateful.