Correlation between brain tryptophan and plasma neutral amino acid levels following food consumption in rats

Brain tryptophan increases significantly within two hr of the time that rats begin to consume a diet containing carbohydrate and fat, but fails to rise if the diet also contains 18–24% protein. The effects of particular diets on brain tryptophan are not well correlated with plasma tryptophan concentrations alone, but do correlate well with the ratio of plasma tryptophan to individual neutral amino acids (leucine, isoleucine, valine, tyrosine, phenylalanine) or to their sums. (These amino acids compete with tryptophan for uptake into the brain.) Carbohydrate ingestion raises brain tryptophan by elevating plasma tryptophan and depressing the plasma levels of the competing neutral amino acids; protein consumption prevents an increase in brain tryptophan by raising the plasma concentrations of the competing amino acids more than of tryptophan.     

Differential amino acid utilization by Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) and its regulatory effect on chlamydial growth

The effect of omission of individual amino acids from growth medium on the multiplication of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) in cycloheximide-treated McCoy cells has been examined. Marked differences were observed in the amounts of particular amino acids required for normal chlamydial multiplication: omission of either leucine, phenylalanine or valine completely inhibited multiplication, whereas absence of any one of another 10 amino acids had no effect on numbers of cells infected. Threshold concentrations of 80, 80 and approx. 8 nmol ml-1 for leucine, valine and phenylalanine, respectively, were needed for normal chlamydial multiplication. These requirements could not be related either to unusually high content in the whole organism, to degradation in the medium, or, from studies with leucine, to deficient association of leucine with host cells. Leucine deprivation at late stages of the developmental cycle also appeared to regulate multiplication. Possible mechanisms responsible for these effects are discussed.     

Characterization of system L and system y+ amino acid transport activity in cultured vascular smooth muscle cells

The uptake of L-leucine and L-lysine into vascular smooth muscle cells cultured from the aortas of rats has been investigated. Both amino acids are taken up by saturable systems that are independent of the presence of a ^·Na⁺ gradient and can be stimulated in trans by neutral bulky amino acids for leucine and cationic amino acids for lysine. Leucine uptake is inhibited competitively in cis by several neutral amino acids, whereas lysine uptake is inhibited strongly by other cationic amino acids but also significantly by neutral amino acids such as leucine. The leucine inhibition is noncompetitive. Cells preloaded with leucine and lysine could also export these amino acids and the rate of efflux was stimulated by the presence of appropriate amino acids in trans. These data are all consistent with leucine being transported largely if not entirely by System L and lysine by the System y⁺ transporter. © 1993 Wiley-Liss, Inc.     

EFFLUX OF PHENYLALANINE FROM RAT CEREBRAL CORTEX SLICES AS INFLUENCED BY EXTRA- AND INTRACELLULAR AMINO ACIDS

Abstract— Effects of other amino acids on the efflux of l -[³H]phenylalanine from rat cerebral cortex slices were studied in a superfusion system. Extracellular large neutral amino acids caused a strong trans-stimulation of [³H]phenylalanine efflux. Some small neutral amino acids were less effective, whereas acidic and basic amino acids and the amino acids without an amino group in the α-position were ineffective. Any trans -inhibition was not detected. The stimulatory trans -effects of phenylalanine and tryptophan were additive, reversible and concentration-dependent. They were apparently mediated by the same mechanisms. The efflux of [³H]phenylalanine was much slower at 273 K than at 310 K, but the effects of unlabelled phenylalanine and tryptophan on it were qualitatively similar at both temperatures. Amino acids accumulated intracellularly at moderately high concentrations did not inhibit [³H]phenylalanine efflux, but phenylalanine, leucine, isoleucine and norleucine caused an enhancement. Spontaneous efflux of [³H]phenylalanine showed some similarities to physical diffusion, but its selective and specific modification by other amino acids strongly suggests the involvement of mediated processes.     

Kinetics of the absorption of amino acids by the rat intestine in vivo

The kinetics of l-phenylalanine and l-lysine absorption by the rat small intestine in vivo have been studied by perfusing intestinal segments and monitoring simultaneously the uptake of the substrate into the intestinal tissue and its disappearance from the perfusate.The rate of phenylalanine disappearance is a linear function of the substrate concentration. Its uptake into the tissue is rapid and obeys saturation kinetics, but is not concentrative. Both tissue uptake and disappearance rate can be inhibited by leucine or methionine, but are not influenced by hydrophilic neutral or dibasic amino acids.Lysine disappearance from the perfusate and its uptake into the tissue both display saturation kinetics. Lysine transport is quantitatively smaller than that of phenylalanine. Both uptake and disappearance are inhibited by arginine and leucine, but are unaffected by other neutral amino acids or sugars.To analyse the kinetic results, integrated equations were developed to express the final concentration in the perfusate in terms of the original concentration. The disappearance rate was considered as a mixed process (saturable and non-saturable in parallel) in a one-compartment system, and the uptake by the tissue was treated as a two-compartment system in which the amino acid entered the cells by a mixed process but left them by a pure non-saturable mechanism.The results concerning disappearance from the lumen are compatible with the one-compartment model. Phenylalanine absorption can be described by a major non-saturable component and a minor saturable one, while lysine absorption occurs almost entirely by a saturable process. The two-compartment model does not adequately describe the tissue uptake results.     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

Regional Blood-Brain Barrier Permeability to Amino Acids After Portacaval Anastomosis

Abstract: The influx of phenylalanine, tryptophan, leucine, and lysine across the blood-brain barrier of individual brain structures was studied in rats 7–8 weeks after a portacaval shunt or sham operation. The method involved a brief infusion of labeled amino acid in tracer quantity and quantitative autoradiography. The clearance rates of phenylalanine, tryptophan, and leucine were increased in proportion to each other in every region examined, but not by the same factor. Tryptophan clearance increased the most (about 200%) and leucine the least (about 30%), compared with phenylalanine (about 80%). This was unexpected, as all three amino acids are believed to be transported by the same mechanism. The changes were most marked in several limbic structures and the reticular formation, whereas the hypothalamus was least affected. Plasma clearance of lysine was decreased in all areas by about 70%. Since the circulating lysine concentration was decreased by 13%, the actual rate of lysine influx was even more reduced. The results demonstrate specific alterations in two different amino acid transport systems. The resulting excess brain neutral amino acids, some of which are neurotransmitter precursors, as well as reduced basic amino acid availability, may be of etiological significance in hepatic encephalopathy.     

Transport of amino acids in Lactobacillus casei by proton-motive-force-dependent and non-proton-motive-force-dependent mechanisms.

Lactobacillus casei 393 cells which were energized with glucose (pH 6.0) took up glutamine, asparagine, glutamate, aspartate, leucine, and phenylalanine. Little or no uptake of several essential amino acids (valine, isoleucine, arginine, cysteine, tyrosine, and tryptophan) was observed. Inhibition studies indicated that there were at least five amino acid carriers, for glutamine, asparagine, glutamate/aspartate, phenylalanine, or branched-chain amino acids. Transport activities had pH optima between 5.5 and 6.0, but all amino acid carriers showed significant activity even at pH 4.0. Leucine and phenylalanine transport decreased markedly when the pH was increased to 7.5. Inhibitors which decreased proton motive force (delta p) nearly eliminated leucine and phenylalanine uptake, and studies with de-energized cells and membrane vesicles showed that an artificial electrical potential (delta psi) of at least -100 mV was needed for rapid uptake. An artificial delta p was unable to drive glutamine, asparagine, or glutamate uptake, and transport of these amino acids was sensitive to a decline in intracellular pH. When intracellular pH was greater than 7.7, glutamine, asparagine, or glutamate was transported rapidly even though the proton motive force had been abolished by inhibitors.     

Na+-dependent transport of large neutral amino acids occurs at the abluminal membrane of the blood-brain barrier

Several Na+-dependent carriers of amino acids exist on the abluminal membrane of the blood-brain barrier (BBB). These Na+-dependent carriers are in a position to transfer amino acids from the extracellular fluid of brain to the endothelial cells and thence to the circulation. To date, carriers have been found that may remove nonessential, nitrogen-rich, or acidic (excitatory) amino acids, all of which may be detrimental to brain function. We describe here Na+-dependent transport of large neutral amino acids across the abluminal membrane of the BBB that cannot be ascribed to currently known systems. Fresh brains, from cows killed for food, were used. Microvessels were isolated, and contaminating fragments of basement membranes, astrocyte fragments, and pericytes were removed. Abluminal-enriched membrane fractions from these microvessels were prepared. Transport was Na+ dependent, voltage sensitive, and inhibited by 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid, a particular inhibitor of the facilitative large neutral amino acid transporter 1 (LAT1) system. The carrier has a high affinity for leucine (Km 21 +/- 7 microM) and is inhibited by other neutral amino acids, including glutamine, histidine, methionine, phenylalanine, serine, threonine, tryptophan, and tyrosine. Other established neutral amino acids may enter the brain by way of LAT1-type facilitative transport. The presence of a Na+-dependent carrier on the abluminal membrane capable of removing large neutral amino acids, most of which are essential, from brain indicates a more complex situation that has implications for the control of essential amino acid content of brain.     

Construction and characterization of Pichia pastoris strains for labeling aromatic amino acids in recombinant proteins

Strains of the methylotrophic yeast Pichia pastoris auxotrophic for the aromatic amino acids (tyrosine, phenylalanine, and tryptophan) have been constructed by targeted gene disruption for protein labeling applications. Three strains, with defects in ARO1 (coding for a homolog of the arom pentafunctional enzyme), ARO7 (coding for chorismate mutase), and TYR1 (coding for prephenate dehydrogenase), have been engineered in a P. pastoris ura3Delta1 parent strain using standard methods. The nutritional requirements of these auxotrophic strains have been characterized and their utility as expression hosts for labeling recombinant proteins has been demonstrated. All three strains show a surprising sensitivity to rich culture medium and must be grown in supplemented minimal medium. The tyr1::URA3 strain in particular is strongly inhibited by tryptophan, and to a lesser extent by phenylalanine, leucine, and isoleucine. Highly efficient incorporation of exogenously supplied amino acids by these three auxotroph strains has been demonstrated using recombinant galactose oxidase. Stereochemically pure l-amino acids and racemic d,l-mixtures serve nearly equally well to support protein expression and labeling. These strains allow efficient labeling of aromatic amino acids in recombinant proteins, supporting NMR structural biology and a wide range of other biophysical studies.     

Quantitative study of free amino acids in human eccrine sweat excreted from the forearms of healthy trained and untrained men during exercise

The free amino acids in eccrine sweat collected from the forearms of 20 healthy trained and 20 healthy untrained men during controlled exercise were determined quantitatively using ion exchange column chromatography. Sweat was deproteinized by adding an equal volume of 5% sulphosalicylic acid. The amino acid concentrations showed a constant qualitative pattern in sweat and large individual differences. Essential amino acids, such as isoleucine, leucine, lysine, methionine, phenylalanine, and valine were excreted in relatively small amounts. As compared to the trained men, untrained men showed statistically significantly higher concentrations in sweat for the following amino acids: Alanine, arginine, glycine, histidine, isoleucine, leucine, lysine, ornithine, phenylalanine, serine, taurine, threonine, tyrosine, and valine. No significant differences were found for citrulline, cystine, ethanolamine, and methionine. The comparison of the amino acid excretions in sweat obtained under controlled exercise and in urine showed that the amounts of amino acids excreted in sweat under controlled exercise were comparable to the losses of amino acids in urine.     

Regulation of RNA degradation in cultured rat hepatocytes: Effects of specific amino acids and insulin

The regulation of RNA degradation by specific amino acids and insulin was investigated in cultured rat hepatocytes from fed rats previously injected in vivo with [6-¹⁴C]orotic acid. The effects of three groups of amino acids were compared to those of a complete amino acid mixture. The first one consisted of the eight amino acids (leucine, proline, glutamine, histidine, phenylalanine, tyrosine, methionine, tryptophan) previously found to be particularly effective in the control of proteolysis. The two other groups were defined from our study with single additions of amino acids, one consisting of proline, asparagine, glutamine, alanine, phenylalanine, and leucine and the other including the latter group with serine, histidine, and tyrosine. The results showed that these three groups were able to strongly inhibit deprivation-induced RNA breakdown at one and ten times normal plasma concentrations but to a lower extent than the complete amino acid mixture. Six amino acids (proline, asparagine, glutamine, alanine, phenylalanine, leucine) inhibited individually RNA degradation by more than 20%. However, the deletions of proline, asparagine, glutamine, or alanine from the group of these six amino acids were not followed by a loss of inhibitory effect. On the contrary, an important loss of inhibition was observed when leucine and phenylalanine were deleted. Furthermore, only these two amino acids exhibited an additive inhibitory effect. Thus leucine and phenylalanine could be considered as important inhibitors of RNA breakdown in cultured rat hepatocytes. Finally, insulin which had no significant effect on RNA degradation in the absence of amino acids, was able to potentiate the inhibitory effect of different amino acid groups. © 1993 Wiley-Liss, Inc.     

Characteristics of a lysosomal membrane transport system for tyrosine and other neutral amino acids in rat thyroid cells

Tyrosine countertransport was used to demonstrate the existence of a carrier system for neutral amino acids in the lysosomal membrane of FRTL-5 thyroid cells. In addition to tyrosine, the carrier system recognized the neutral amino acids leucine, histidine, phenylalanine, and tryptophan. Cystine and lysine, amino acids for which a lysosomal carrier system has been demonstrated, showed no competition with tyrosine for countertransport. The tyrosine system showed stereospecificity and cation independence. It did not require an acidic lysosome or the availability of free thiols. The apparent Km for tyrosine was approximately 100 microM; the energy of activation of the system was approximately 9.7 kcal/mol. This new lysosomal membrane carrier system for neutral amino acids resembles the plasma membrane L system in 3T3 Chinese hamster ovary cells and melanoma B-16 cells.     

Leucine transport in membrane vesicles from Chironomus riparius larvae displays a mélange of crown-group features

Leucine uptake into membrane vesicles from larvae of the midge Chironomus riparius was studied. The membrane preparation was highly enriched in typical brush border membrane enzymes and depleted of other membrane contaminants. In the absence of cations, there was a stereospecific uptake of l-leucine, which exhibited saturation kinetics. Parameters were determined both at neutral (Km 33 +/- 5 microM and Vmax 22.6 +/- 6.8 pmol/7s/mg protein) and alkaline (Km 46 +/- 5 microM and Vmax 15.5 +/- 2.5 pmol/7s/mg protein) pH values. At alkaline pH, external sodium increased the affinity for leucine (Km 17 +/- 1 microM) and the maximal uptake rate (Vmax 74.0 +/- 12.5 pmol/7s/mg protein). Stimulation of leucine uptake by external alkaline pH agreed with lumen pH measurements in vivo. Competition experiments indicated that at alkaline pH, the transport system readily accepts most L-amino acids, including branched, unbranched, and alpha-methylated amino acids, histidine and lysine, but has a low affinity for phenylalanine, beta-amino acids, and N-methylated amino acids. At neutral pH, the transport has a decreased affinity for lysine, glycine, and alpha-methylleucine. Taken together, these data are consistent with the presence in midges of two distinct leucine transport systems, which combine characters of the lepidopteran amino acid transport system and of the sodium-dependent system from lower neopterans.     

Absorption of neutral amino acids by the gut ofArenicola marina

Summary The absorption of neutral amino acids byArenicola marina was studied using anin vitro preparation of the alimentary canal. Regional variation in absorption was observed, with the intestine being the region of greatest uptake. The L enantiomorphs of the neutral amino acids alanine and leucine were shown to be actively absorbed by the intestine as was the D enantiomorph of alanine. A saturable component was demonstrated in the absorption of L-alanine and this was shared by L-methionine, which was found to competitively inhibit alanine uptake. Inhibition of L-alanine uptake also occurred in the presence of other neutral, basic and acidic amino acids. The greatest inhibition was found with the L stereoisomers of methionine, leucine, valine, histidine and phenylalanine, whilst proline, lysine and aspartic acid decreased uptake to a smaller extent.     

Competition between leucine and phenylalanine and its relation to p-fluorophenylalanine resistant mutations in Aspergillus nidulans

Summary Auxanography and growth kinetics of a leucine and phenylalaninerequiring strain of Aspergillus nidulans reveals that (a) a phenylalanine-requiring strain is competitively inhibited by leucine but a leucine-requiring strain is not inhibited by phenylalanine, (b) the molar ratio of the two amino acids is critical for inhibition, and (c) leucine is specific for the possible replacement of phenylalanine.Failure to isolate a leucine-resistant phenylalanine-auxotroph suggests that the competition between these two amino acids does not take place at the coding level. By mitotic and meiotic analysis the mutant fpaB37 has been located on the left arm of linkage group I and has been found to be distinct and different from the locus trypB.Interactions between p-fluorophenylllanine-resistance and amino acid requirements and uptake experiments indicate that there are at least two sites for which leucine competes with phenylalanine-one of them being the site of entry of these essential amino acids into the mycelium. Both of these interaction sites are common for leucine, phenylalanine and p-fluorophenylalanine.     

Albumin synthesis in cultured hempatoma cells. Regulation by essential amino acids.

The effects of essential amino acids on albumin synthesis by a mouse hepatoma cell line have been investigated. The amino acids tested were tryptophan, phenylalanine, histidine, isoleucine and leucine. Cellular rates of synthesis (molecules albumin/cell per min) were determined from rates of [3H]leucine incorporation into immunoprecipitable albumin in the culture medium. The effects of amino acids on albumin synthesis fall into three distinct groups. The concentration of tryptophan producing half-maximal synthesis is 4 micronM. The corresponding concentration for leucine is 100 micronM. Histidine, phenylalanine and isoleucine were very similar, the half-maximal concentrations being approximately 15 micronM. The concentrations of amino acids producing half-maximal synthesis correlate directly with the amino acid composition of albumin. The levels of these essential amino acids necessary to saturate albumin synthesis have been compared with amino acid levels in normal plasma.     

The effects of chronic hyperphenylalaninaemia on mouse brain protein synthesis can be prevented by other amino acids.

A prolonged elevation in the concentrations of circulating phenylalanine was maintained in newborn mice by daily injections of phenylalanine and a phenylalanine hydroxylase inhibitor, alpha-methylphenylalanine. The result of this chronic hyperphenylalaninaemia was an accumulation of vacant or inactive monoribosomes that persisted for 18 h of each day. An elongation assay in vitro with brain postmitochondrial supernatants demonstrated that, in addition, there was an equally prolonged decrease in the rates of polypeptide-chain elongation by the remaining brain polyribosomes. Analyses of the free amino acid composition in the brains of hyperphenylalaninaemic mice showed a loss of several amino acids from the brain, particularly the large, neutral amino acids, which are co- or counter-transported across plasma membranes with phenylalanine. When a mixture of these amino acids (leucine, isoleucine, valine, threonine, tryptophan, tyrosine, methionine) was injected into hyperphenylalaninaemic mice, there was an immediate cessation of monoribosome accumulation in the brain and there was no inhibition of the rates of polypeptide-chain elongation. Although the concentrations of the large, neutral amino acids in the brain were partially preserved by treatment of hyperphenylalaninaemic mice with the amino acid mixture, the elevated concentrations of phenylalanine remained unaltered. The amino acid mixture had no detectable effect on brain protein synthesis in the absence of the hyperphenylalaninaemic condition.     

EVIDENCE FOR SEPARATE SYSTEMS FOR THE TRANSPORT OF NEUTRAL AND BASIC AMINO ACIDS ACROSS THE BLOOD-BRAIN BARRIER

Abstract— The effects of high circulating concentrations of several amino acids on the free amino acids of rat brain were measured, to see whether or not the results followed any consistent pattern. High circulating concentrations of large, neutral amino acids (phenylalanine, valine or isoleucine) caused significantly decreased values only of other large, neutral amino acids in the brains. High circulating concentrations of the basic amino acids lysine or arginine caused significantly decreased values only of each other. The data suggest that there are separate systems for the transport of neutral and basic amino acids across the blood-brain barrier. The effects of valine and lysine on the uptake by brain and the con-vulsant action of allylglycine (a neutral amino acid) were consistent with the concept of separate systems for the transport of amino acids across the blood-brain barrier. Valine inhibited the uptake by brain and the convulsant action of allylglycine in mice, but lysine did not. The data suggest that allylglycine and valine are transported into the brain by a common mechanism that does not transport lysine.     

Classification of essential amino acids for the weanling pig

Feeding experiments with mixtures of purified amino acids show that arginine, leucine, phenylalanine, and valine are required for good growth of weanling pigs. The pig resembles the rat in its ability to synthesize part, but not all of the arginine required. It is now possible to tentatively classify the known amino acids with respect to major growth effects in weanling pigs. The amino acids which must be present in the diet for good growth are arginine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. The remaining amino acids may be tentatively classed as dispensable.