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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.20, #4)

Asparagine uptake in rat hepatocytes: Resolution of a paradox and insights into substrate-dependent transporter regulation by T. M. Pawlik; W. W. Souba; B. P. Bode (pp. 335-352).

Extracellular asparagine has previously been shown to markedly stimulate both ornithine decarboxylase and System N-mediated glutamine transport activities in hepatocytes by a transport-dependent mechanism. However, as a weak substrate of its inferred transporter System N, the specific route of asparagine uptake has remained enigmatic. In this study, asparagine transport was studied in detail and shown to be Na⁺-dependent, Li⁺-tolerant, stereospecific, and inhibited profoundly by glutamine and histidine. Coupled with competitive inhibition by glutamine (K_i = 2.63 ± 1.11 mM), the data indicated that asparagine was indeed slowly transported by System N in rat hepatocytes, albeit at rates an order of magnitude less than for glutamine. The differential substrate transport velocities were shown to be attributable to a low transporter asparagine affinity (K_m = 9.3 − 17.5 mM) compared to glutamine (K_m∼ 1 mM). Consistent with its slow uptake, asparagine accumulated to a fivefold lesser degree than glutamine after 60 min, yet stimulated System N activity to the same extent as glutamine. The transaminase inhibitor aminooxyacetate and starvation of the donor animal each enhanced asparagine uptake twofold and augmented subsequent transporter activation. Conversely, asparagine-dependent System N stimulation was abrogated by hyperosmotic media and blunted 30%–40% by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Collectively, the data suggest that System N-mediated asparagine uptake serves an autostimulatory role, mediated by cellular swelling and in part by a PI3K-dependent signal transduction pathway.

Keywords: Keywords: Amino acids; Transport; Asparagine; Glutamine; Hepatocytes; Cell volume; Phosphatidylinositol 3-kinase

Pharmacological elevation of endogenous kynurenic acid levels activates nigral dopamine neurons by S. Erhardt; H. Öberg; J. M. Mathé; G. Engberg (pp. 353-362).

Inhibitors of kynurenine 3-hydroxylase have previously been used to increase endogenous levels of kynurenic acid, an excitatory amino acid receptor antagonist. In the present electrophysiological study PNU 156561A was utilized to elevate endogenous concentrations of kynurenic acid and subsequent effects on the firing pattern of dopamine (DA) neurons of rat substantia nigra (SN) were analyzed. Pretreatment with PNU 156561A (40 mg/kg, i.v., 5–7 h) caused a five-fold increase in endogenous kynurenic acid levels in whole brain five to seven hours after administration and also evoked a significant increase in firing rate and bursting activity of nigral DA neurons. The results of the present study show that a moderate increase in endogenous kynurenic acid levels produces significant actions on the tonic glutamatergic control of the firing pattern of nigral DA neurons, and implicate kynurenine 3-hydroxylase inhibitors as novel antiparkinsonian agents.

Keywords: Keywords: Amino acids; Kynurenic acid; Firing rate; Burst activity; PNU 156561A; Substantia nigra; Dopamine

The effect of heat shock on amino acid transport and cell volume in 3T3 cells by P. G. Petronini; A. E. Caccamo; R. R. Alfieri; M. A. Bonelli; A. F. Borghetti (pp. 363-380).

In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response.

Keywords: Keywords: Amino acids; Amino acid transport; Cell volume; Heat shock; HSP70

New quinone-amino acid conjugates linked via a vinylic spacer by M. Alnabari; S. Bittner (pp. 381-387).

Chloranil and 2,3-dichloro-1,4-naphthoquinone have been linked to different natural and unnatural amino acids via a vinylic spacer. Two routes were developed for the facile preparation of these novel modified amino acids: the direct method, which can only be applied to secondary amines, and the indirect method (transamination reaction), which can be applied to any amino acid or ester.

Keywords: Keywords: Amino acids; Quinones; Chloranil; 2; 3-Dichloro-1; 4-naphthoquinone; Transamination

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents by H. A. Johnson; R. L. Baldwin; K. C. Klasing; C. C. Calvert (pp. 389-400).

Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR.

Keywords: Keywords: Amino acids; Protein turnover model; Protein synthesis rate; Amino acid recycling

Assignment of d-amino-acid oxidase gene to a human and a mouse chromosome by R. Konno (pp. 401-408).

A part of d-amino-acid oxidase gene was amplified in the human and mouse by polymerase chain reaction. The amplified fragments were ligated to plasmids and then cloned. The plasmids containing the parts of d-amino-acid oxidase gene were biotinylated and hybridized to human and mouse metaphase chromosomes. The chromosomal slides were treated with fluorescein isothiocyanate (FITC)-conjugated avidin. The hybridized signals were amplified with biotinylated anti-avidin antibody and FITC-avidin. The chromosomes were counter-stained with diamidino-phenylindole for assignment of the signal to a specific band. Using this fluorescence in situ hybridization (FISH), d-amino-acid oxidase gene was assigned to human chromosome 12q23–24.1 and mouse chromosome 5E3-F. Since these regions are syntenic between human and mouse, the present results indicate that the locus for this enzyme has been conserved through evolution.

Keywords: Keywords:d-Amino-acid oxidase; Chromosomal mapping; FISH; Human; Mouse

Homocysteine concentrations in a German cohort of 500 individuals: Reference ranges and determinants of plasma levels in healthy children and their parents by M. Rauh; S. Verwied; I. Knerr; H. G. Dörr; A. Sönnichsen; B. Koletzko (pp. 409-418).

Elevated plasma homocysteine is a risk factor for cardiovascular disease and a sensitive marker of inadequate vitamin B12 and folate status. We studied 257 pupils (120 boys, 137 girls, aged 6–17 years) and their parents (88 males, 172 females, aged 26–50 years). Our measurements were part of a national Bavarian health and nutrition examination survey evaluating cardiovascular risk factors. A mild hyperhomocysteinemia (Hcys >15 μmol/l) occurred in 7% of the adults, but in none of the children. Men had significantly higher Hcys levels than women (p < 0.0001), boys and girls had comparable concentrations. For adults and children, Hcys correlated inversely with vitamin B12 and folate and positively with the lean body mass and creatinine in serum, but not with cystatin C. Genetic and nutritional factors are determinants of Hcys metabolism. The correlation of Hcys and serum creatinine is dependent on the metabolic link between Hcys production and creatine synthesis.

Keywords: Keywords: Amino acids; Cobalamin; Cystatin C; Folate; Healthy population; Homocysteine; Reference values

Taurine is a weak scavenger of peroxynitrite and does not attenuate sodium nitroprusside toxicity to cells in culture by T. R. Mehta; R. Dawson, Jr. (pp. 419-433).

Many studies have suggested an antioxidant role for taurine, but few studies have directly measured its free radical scavenging activity. The aim of the present study was to directly determine the action of taurine and taurine analogs to inhibit peroxynitrite-mediated oxidation of dihydrorhodamine 123 (DHR) to rhodamine. Taurine was also tested to determine if it could attenuate the toxicity of sodium nitroprusside (SNP) to neuronal cultures. Taurine at concentrations above 30 mM had a modest ability to inhibit peroxynitrite formation derived from SIN-1. Hypotaurine could inhibit peroxynitrite formation from both SIN-1 (↓75%) and SNP (↓50%) at 10 mM. Other taurine analogs (homotaurine, β-alanine & isethionic acid) slightly potentiated DHR oxidation by SIN-1. Short-term (1-hour) treatment of PC12 cultures with either SNP (1–2 mM) or taurine (20–40 mM) appeared to induce cellular proliferation. In contrast, 24-hour treatment with SNP (1 mM) induced cell death. Combination treatments with taurine and SNP appeared to interact in an additive fashion for both cell proliferation and neurotoxic actions. It appears unlikely that taurine is a major endogenous scavenger of peroxynitrite.

Keywords: Keywords: Amino acids; Taurine; Peroxynitrite; Hypotaurine; Sodium nitroprusside; Neurotoxicity; Nitric oxide

Detection of moderate hyperhomocysteinemia: Comparison of the Abbott fluorescence polarization immunoassay with the Bio-Rad and SBD-F high-performance liquid chromatographic assays by F. Barbé; I. Abdelmouttaleb; A. Chango; P. Gérard; D. Quilliot; M. Klein; D. Lambert; J.-P. Nicolas; J.-L. Guéant (pp. 435-440).

The importance of accurate methods for homocysteine measurement has been emphasized. We compared the results obtained with the most commonly used high-performance liquid chromatography (HPLC) assay, and two recently commercially available methods: another HPLC and a fluorescence polarization immunoassay, in plasmas from normo- or hyper-homocysteinemic patients. A significant agreement between the different methods in classifying the results as hyper or normal-homocysteinemia was observed. However, a significant difference between the results was found. Standardization is urgently necessary to improve the concordance of homocysteine determination.

Keywords: Keywords: Amino acids; Homocysteine; High-performance liquid chromatography; Fluorescence polarization immunoassay

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Amino Acids: The Forum for Amino Acid, Peptide and Protein Research (v.20, #4)