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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.31, #2)

Transport of peptides, amino acids, and derivatives by E. I. Closs; F. Leibach (pp. 91-91).

Chlorpromazine, clozapine and olanzapine inhibit anionic amino acid transport in cultured human fibroblasts by C. Marchesi; V. Dall’Asta; B. M. Rotoli; M. G. Bianchi; C. Maggini; G. C. Gazzola; O. Bussolati (pp. 93-99).

We report here that chlorpromazine, a first generation antipsychotic drug, inhibits anionic amino acid transport mediated by system X⁻ _AG (EAAT transporters) in cultured human fibroblasts. With 30 µM chlorpromazine, transport inhibition is detectable after 3 h of treatment, maximal after 48 h (>60%), and referable to a decrease in V_max. Chlorpromazine effect is not dependent upon changes of membrane potential and is selective for system X⁻ _AG since transport systems A and y⁺ are not affected. Among antipsychotic drugs, the inhibitory effect of chlorpromazine is shared by two dibenzodiazepines, clozapine and olanzapine, while other compounds, such as risperidon, zuclopentixol, sertindol and haloperidol, are not effective. Transport inhibition by clozapine and olanzapine, but not by chlorpromazine, is reversible, suggesting that the mechanisms involved are distinct. These results indicate that a subset of antipsychotic drugs inhibits EAAT transporters in non-nervous tissues and prompt further investigation on possible alterations of glutamate transport in peripheral tissues of schizophrenic patients.

Keywords: Keywords: Transport – Glutamate – Chlorpromazine – Glutamine – Olanzapine – Clozapine

y⁺ LAT-1 mediates transport of the potent and selective iNOS inhibitor, GW274150, in control J774 macrophages by A. R. Baydoun; J. Bertran; S. Thakur; J. Dawson; M. Palacín; R. G. Knowles (pp. 101-109).

This study has characterised the transport mechanism(s) for the novel and selective inhibitor of inducible nitric oxide synthase (iNOS), GW274150, in murine macrophage J774 cells. Transport of GW274150 was saturable (K_m = 0.24 ± 0.01 mM and V_max of 8.5 ± 0.12 pmol·µg protein⁻¹ min⁻¹), pH-insensitive and largely Na⁺-independent. Transport was also susceptible to trans-stimulation and was significantly inhibited by a 10-fold excess of L-arginine, L-lysine, L-leucine, L-methionine, L-glutamine and 6-diazo-5-oxo-L-norleucine but not by other amino acids or by N-ethylmaleimide. More importantly, the inhibitions caused by the neutral amino acids were critically dependent on Na⁺. These results strongly implicate system y⁺L in the transport of GW274150. Northern blot analysis confirmed this by revealing the presence of transcripts for y⁺LAT-1 but not y⁺LAT-2. Thus, taken together, our data show for the first time that J774 macrophages express y⁺LAT-1 transporters and that these carriers mediate transport of GW2741500 at least in these cells.

Keywords: Keywords: GW274150 – J774 macrophages – System y⁺ – y⁺LAT-1 – Nitric oxide – Inducible nitric oxide synthase – Amino acid transport

Substrate specificity of the amino acid transporter PAT1 by L. Metzner; K. Neubert; M. Brandsch (pp. 111-117).

The proton coupled amino acid transporter PAT1 expressed in intestine, brain, and other organs accepts L- and D-proline, glycine, and L-alanine but also pharmaceutically active amino acid derivatives such as 3-amino-1-propanesulfonic acid, L-azetidine-2-carboxylic acid, and cis-4-hydroxy-D-proline as substrates. We systematically analyzed the structural requirements for PAT1 substrates by testing 87 amino acids, proline homologs, indoles, and derivatives. Affinity data and effects on membrane potential were determined using Caco-2 cells. For aliphatic amino acids, a blocked carboxyl group, the distance between amino and carboxyl group, and the position of the hydroxyl group are affinity limiting factors. Methylation of the amino group enhances substrate affinity. Hetero atoms in the proline template are well tolerated. Aromatic α-amino acids display low affinity. PAT1 interacts strongly with heterocyclic aromatic acids containing an indole scaffold. The structural requirements of PAT1 substrates elucidated in this study will be useful for the development of prodrugs.

Keywords: Keywords: H⁺/Amino acid cotransporter – hPAT1 – Caco-2 cells – Substrate specificity – Proline – Membrane transport

Transport of L-proline, L-proline-containing peptides and related drugs at mammalian epithelial cell membranes by M. Brandsch (pp. 119-136).

Membrane transport of L-proline has received considerable attention in basic and pharmaceutical research recently. Of the most recently cloned members of the solute carrier family, two are “proline transporters”. The amino acid transporter PAT1, expressed in intestine, kidney, brain and other organs, mediates the uptake of proline and derivatives in a pH gradient-dependent manner. The Na⁺-dependent proline transporter SIT1, cloned in 2005, exhibits the properties of the long-sought classical IMINO system. Proline-containing peptides are of interest for several reasons. Many biologically important peptide sequences contain highly conserved proline residues. Xaa-Pro peptides are very often resistant to enzymatic hydrolysis and display, in contrast to Pro-Xaa peptides, a high affinity to the H⁺/peptide cotransporter PEPT1 which is expressed in intestinal, renal, lung and biliary duct epithelial cells. Furthermore, several orally available drugs are recognized by PEPT1 as Xaa-Pro analogues due to their sterical resemblance to small peptides.

Keywords: Keywords: Membrane transport – Imino acids – Proline derivatives – Peptides – PEPT1 – PEPT2 – PAT1 – SIT1 – Proton gradient

The renal type H⁺/peptide symporter PEPT2: structure-affinity relationships by A. Biegel; I. Knütter; B. Hartrodt; S. Gebauer; S. Theis; P. Luckner; G. Kottra; M. Rastetter; K. Zebisch; I. Thondorf; H. Daniel; K. Neubert; M. Brandsch (pp. 137-156).

The H⁺/peptide cotransporter PEPT2 is expressed in a variety of organs including kidney, lung, brain, mammary gland, and eye. PEPT2 substrates are di- and tripeptides as well as peptidomimetics, such as β-lactam antibiotics. Due to the presence of PEPT2 at the bronchial epithelium, the aerosolic administration of peptide-like drugs might play a major role in future treatment of various pulmonary and systemic diseases. Moreover, PEPT2 has a significant influence on the in vivo disposition and half-life time of peptide-like drugs within the body, particularly in kidney and brain. PEPT2 is known to have similar but not identical structural requirements for substrate recognition and transport compared to PEPT1, its intestinal counterpart. In this review we compiled available affinity constants of 352 compounds, measured at different mammalian tissues and expression systems and compare the data whenever possible with those of PEPT1.

Keywords: Keywords: PEPT1 – PEPT2 – Peptide transport – Dipeptides – Tripeptides – β-Lactam antibiotics – Peptidomimetics – Drugs – Drug-delivery

Taurine transporter is expressed in osteoblasts by L.-Q. Yuan; H. Xie; X.-H. Luo; X.-P. Wu; H.-D. Zhou; Y. Lu; E.-Y. Liao (pp. 157-163).

Taurine influences bone metabolism and is taken up by cells via a specific transport system, the taurine transporter (TAUT). We report a link between taurine and bone homeostasis by demonstrating transcription and translation of TAUT in bone-forming cells. TAUT was expressed in human primary osteoblasts, the human osteosarcoma osteoblast-like cell line MG63, and the mouse osteoblastic cell line MC3T3-E1. Immunostaining with polyclonal antibodies also demonstrated the presence of TAUT in both human and murine osteoblasts. TAUT mRNA expression and [³H]taurine uptake increased during differentiation of MG63 cells in culture. Supplementation of culture medium with taurine enhanced alkaline phosphatase activity and osteocalcin secretion. The regulation and detailed function of taurine and TAUT in bone remain unclear, but our findings suggest a functional role for them in bone homeostasis.

Keywords: Keywords: Taurine – Taurine transport – Osteoblast – Alkaline phosphatase – Osteocalcin

Indispensable amino acid concentrations decrease in tissues of stomachless fish, common carp in response to free amino acid- or peptide-based diets by Y. Zhang; K. Dabrowski; P. Hliwa; P. Gomulka (pp. 165-172).

The premise that free amino acid or dipeptide based diets will resolve the nutritional inadequacy of formulated feeds for larval and juvenile fish and improve utilization of nitrogen in comparison to protein-based diets was tested in stomachless fish, common carp (Cyprinus carpio L.) larvae. We examined the postprandial whole body free amino acid (FAA) pool in fish that were offered a FAA mixture based diet for the duration of 2 or 4 weeks. We found that the total amount and all indispensable amino acids concentrations in the whole body decreased after a meal. We then fed juvenile carp with dietary amino acids provided in the FAA, dipeptide (PP), or protein (live feed organisms; brine shrimp Artemia salina nauplii, AS) forms. Histidine concentrations in the whole fish body increased in all dietary groups after feeding whereas all other indispensable amino acids decreased in FAA and PP groups in comparison to the AS group. Taurine appears to be the major osmotic pressure balancing free amino acid in larval freshwater fish which may indicate a conditional requirement. We present the first evidence in larval fish that in response to synthetic FAA and PP diets, the whole body indispensable free AA concentrations decreased after feeding. This study shows that amino acids given entirely as FAA or PP cannot sustain stomachless larval fish growth, and may result in depletion of body indispensable AA and most of dispensable AA. The understanding of these responses will determine necessary changes in diet formulations that prevent accelerated excretion of amino acids without protein synthesis.

Keywords: Keywords: Protein – Dipeptide – Amino acids – Stomachless fish – Teleost

Acetohydroxyacid synthase and its role in the biosynthetic pathway for branched-chain amino acids by J. A. McCourt; R. G. Duggleby (pp. 173-210).

The branched-chain amino acids are synthesized by plants, fungi and microorganisms, but not by animals. Therefore, the enzymes of this pathway are potential target sites for the development of antifungal agents, antimicrobials and herbicides. Most research has focused upon the first enzyme in this biosynthetic pathway, acetohydroxyacid synthase (AHAS) largely because it is the target site for many commercial herbicides. In this review we provide a brief overview of the important properties of each enzyme within the pathway and a detailed summary of the most recent AHAS research, against the perspective of work that has been carried out over the past 50 years.

Keywords: Keywords: Acetohydroxyacid synthase – Acetolactate synthase – Branched-chain amino acids – Thiamin diphosphate – Flavin adenine dinucleotide – Herbicide – Inhibitor – Mechanism – Protein structure

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.31, #2)

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.31, #2)