Compartmentation and exchangeability of brain amino acids: evidence from studies of transport into tissue slices.

Compartmentation of the free amino acid pool of brain slices was investigated by measuring the approach to isotopic equilibrium between tissue and medium when slices were incubated with traces of radioactive amino acids. Trace quantities were used to minimize the effects of uptake, which could make the detection of slowly equilibrating pools difficult by greatly increasing tissue amino acid levels. Small, sequestered compartments were found. After 2 h in 20 vol of glucose-containing, oxygenated medium, the nonequilibrating compartments for lysine, leucine, tyrosine, histidine, valine, and threonine were 41, 20, 17, 16, 11, and 6% of their final tissue concentrations, respectively. The data for rapidly metabolized, nonessential, amino acids were more difficult to interpret. Considerable mixing of incoming glutamic and aspartic acids with their endogenous pools was observed and tissue glycine reached isotopic equilibrium within 1 h. With higher concentrations of amino acids, equilibration was complete in 30 min with 2 mm glycine in the medium; 83% in 30 min with 2 mm glutamic acid, and 95% in 60 min with 5 mm glutamic acid in the medium. The amino acid composition of protein free extracts of slices and medium was determined. During incubation, despite a large efflux of amino acids into the medium, most tissue amino acids remained close to their initial concentrations. Net increases in essential amino acids were accounted for by the breakdown of 0.7% of total tissue protein during the first hour and 0.3% during the second hour of incubation.     

Uptake of leucine,lysine, aspartic acid,and glycine into isolated neurons and astrocytes

The uptake of tritium-labeledl-leucine,l-lysine,l-aspartic acid, and glycine by neurons and astrocytes isolated from the cerebral cortex of 3-week-old rats was followed for varying periods up to 40 min at amino acid concentrations from 1 to 2000 mol/liter in medium. The effects of a low-sodium (15.5 mmol/liter) medium on the uptake were also studied. The influx of the amino acids was faster into astrocytes than into neurons. Leucine penetrated into the cells faster than the other amino acids. Amino acid transport was mainly saturable at the lowest amino acid concentrations studied, whereas nonsaturable penetration into the cells dominated in the millimolar concentration range. The saturable transport comprised only one transport system with relatively small transport constants, resembling in nature the so-called high-affinity transport. The maximal velocities of transport were about two times higher in astrocytes than in neurons. In neurons the partial substitution of sodium by choline in medium had the most effect in reducing the influx of glycine and aspartic acid. In astrocytes the effects were generally less pronounced. The results suggest that extracellular amino acids generally penetrate more readily into astrocytes than into neurons. Both cell types transport essential amino acids more effectively than other amino acids.     

Temperature dependence of amino acid transport in brain slices

A decrease in amino acid influx and exit in incubated slices when the temperature was lowered from 37 to 20°C was observed with all 16 amino acids examined at two concentrations (1 mM and 10 M). The temperature dependence of cellular amino acid influx observed in slices in vitro contrasts with the absence of temperature dependence of capillary amino acid influx in the brain in vivo that we recently reported. The temperature effects in slices varied some-what among the various amino acid transport classes. With some amino acids (Phe, Val, Tyr, Asp, Glu), especially at lower concentrations, the greater inhibition of exit than of uptake resulted in an actual increase in tissue amino acid uptake at lower temperatures in long-term experiments. The results indicate heterogeneity in the effect of temperature on the various transport classes and show a difference between capillary and cellular amino acid transport in the brain.     

DEVELOPMENTAL CHANGES IN Na+, K+ AND ATP AND IN THE LEVELS AND TRANSPORT OF AMINO ACIDS IN INCUBATED SLICES OF RAT BRAIN

Abstract—

• 1 Upon incubation, slices of brain tissue took up fluid; the degree of swelling increased with increasing age. No sweiling occurred in slices from foetal brain. Since this swelling was associated with increases in the inulin space, the percentage of inulin space in slices at the end of incubation increased during brain development.
• 2 Most of the capacity for ion transport seemed to be absent from foetal brain. In vivo and in slices, Na⁺ was very high and K⁺ was very low in comparison to levels at other ages. There was a rapid change around birth, but no significant change at later ages. Upon incubation, Na⁺ levels increased in other slices, but not in slices of foetal brain.
• 3 Upon incubation of the slices, ATP levels were restored to levels close to those in the living brain; there were no significant alterations in available energy during development to explain changes in amino acid transport.
• 4 The composition of the free pool of cerebral amino acids in vivo changed with development, with some compounds (glutamic acid and related compounds) increasing, others (mostly‘essential’amino acids) decreasing, with age. These changes were not linear with time, and the level of a compound might exhibit several peaks during development.
• 5 The uptake (influx) of taurine, glutamate and glycine into brain slices increased rapidly during the foetal and early neonatal periods, reached a maximum between 2 and 3 weeks of postnatal age and then declined to adult levels. The levels of steady-state uptake with glycine also exhibited a maximal peak at 2-3 weeks of postnatal age. Steady-state uptake of taurine and glutamate reached adult levels by about 3 weeks of age.
• 6 The pattern of inhibition of amino acid transport by two specific amino acid analogues changed during development for some amino acids (GABA, glycine and glutamate), indicating an alteration in substrate specificity.
• 7 The results demonstrate complex changes in cerebral amino acid transport during development, with several maxima or minima and with changes in specificity for at least some compounds.

     

Transport of Aminophosphonic Acids in Lactobacillus plantarum and Streptococcus faecalis

Aminophosphonic acids analogous to glutamic acid, aspartic acid, alanine, and valine were actively accumulated by Lactobacillus plantarum. Uptake was dependent on the availability of glucose and, in all cases, the estimated intracellular concentrations substantially exceeded extracellular levels. During uptake, there was little metabolism of tritiated 2-amino-3-phosphonopropionic acid (APP), the aspartic acid analogue, and a negligible incorporation of isotope from this substance into the nucleic acid, lipid, protein, or cell wall fractions of the cell. Competition studies with APP indicated that its transport in L. plantarum and in Streptococcus faecalis was antagonized only by structurally related compounds such as glutamic, aspartic, and cysteic acids. Kinetic studies showed that APP was taken up by a single catalytic system in S. faecalis. A mutant strain of this organism which lacks one of two kinetically distinguishable dicarboxylic amino acid transport systems failed to accumulate measurable amounts of APP. These experiments indicate that the aminophosphonic acids are accumulated by the amino acid transport systems in these bacteria with minimal metabolic changes.     

Characterization of amino-acid transport in Ricinus communis roots using isolated membrane vesicles

The mechanism and specificity of amino-acid transport at the plasma membrane of Ricinus communis L. roots was investigated using membrane vesicles isolated by phase partitioning. The transport of glutamine, isoleucine, glutamic acid and aspartic acid was driven by both a pH gradient and a membrane potential (internally alkaline and negative), created artificially across the plasma membrane. This is consistent with transport via a proton symport. In contrast, the transport of the basic amino acids, lysine and arginine, was driven by a negative internal membrane potential but not by a pH gradient, suggesting that these amino acids may be taken up via a voltage-driven uniport. The energized uptake of all of the amino acids tested showed a saturable phase, consistent with carrier-mediated transport. In addition, the membrane-potential-driven transport of all the amino acids was greater at pH 5.5 than at pH 7.5, which suggests that there could be a direct pH effect on the carrier. Several amino-acid carriers could be resolved, based on competition studies: a carrier with a high affinity for a range of neutral amino acids (apart from asparagine) but with a low affinity for basic and acidic amino acids; a carrier which has a high affinity for a range of neutral amino acids except isoleucine and valine, but with a low affinity for basic and acidic amino acids; and a carrier which has a higher affinity for basic and some neutral amino acids but has a lower affinity for acidic amino acids. The existence of a separate carrier for acidic amino acids is discussed.Abbreviations PM plasma membrane - TPP⁺ tetraphenylphosphonium ion - pH pH gradient - membrane potential This work was supported by the Agricultural and Food Research Council and The Royal Society. We would like to thank Mrs. Sue Nelson for help with some of the membrane preparations.     

Amino acid uptake systems in lizard and chick brain cells

The uptake of seven amino acids, -aminoisobutyric acid (AIB), cyclo-leucine (cyclo-Leu), -aminobutyric acid (GABA), glycine (Gly), glutamic acid (Glu), lysine (Lys), and taurine (Tau), representatives of different amino acid transport systems, was studied in slices of brain from Tokay lizards and White Leghorn chicks. In descending order, the rate of the initial uptake of the amino acids in both species was Glu>Gly>GABA>Cyclo-Leu>AIB>Lys>Tau. The substrate specificities and the differences in sodium and temperature dependence of the uptake of the amino acids indicate the presence of several distinct amino acid transport systems, some sodium-dependent and some sodium-independent. The structural specificity of amino acid transport classes in the brain of these species is similar to that in other vertebrate brain preparations.Special issue dedicated to Dr. Santiago Grisolia.     

Anilinonaphthalene sulfonate fluorescence and amino acid transport in yeast

Summary Fluorescence of 1-anilinonaphthalene-8-sulfonate in yeast membranes appears to be caused predominantly by binding to lipids (ANS_proteinANS_lipid120) as indicated by the fluorescence lifetime, degree of polarization, and excitation spectra. It was insensitive to short-circuiting the membrane potential. Fluorescence intensity increased as cells (especially after pretreatment with energy donors such as glucose) were exposed to some amino acids, in particular, aspartic and glutamic acids. The character of fluorescence shifted to that of protein-bound ANS, suggesting an exposure of new protein sites accessible to the probe. This shift could be prevented by inhibitors of energy transduction as well as of transport. TheK _1/2 of the shift was at 2.5mm aspartic acid.     

Effect of acute cyanide intoxication on the active transport of amino acids in brain slices

(1) Acute hypoxia was produced in adult rats by cyanide inhalation and the effect on the active transport of amino acids was studied in brain slices. (2) Initial and steady-state accumulation of amino acids and rates of amino acid exit were identical in brain slices from control and treated animals when a glucose-containing incubation medium was used. (3) When the incubation was carried out in a glucose-free incubation medium, the inhibition of initial and steady-state accumulation and the stimulation of amino acid exit observed in control slices were significantly reduced or abolished in slices from treated animals. (4) Tissue swelling, size of ‘inulin space’ and glucose consumption did not differ in the two groups of animals. (5) Also the respiration rate was identical in slices from control and treated animals incubated in the presence of glucose. In the absence of added substrate, brain slices from treated animals consumed 15-20 per cent more oxygen than control slices. (6) A possible correlation between the effects observed on amino acid transport and on respiration is suggested. The reasons why cyanide given in vivo or added in vitro have different effects on amino acid transport in brain slices are discussed.     

Effect of ammonium and other ions on the glucose-dependent active transport of l-histidine in slices of rat-brain cortex

1. The influence of cations on the active transport into cells of rat-brain-cortex slices of l-histidine, an amino acid that is not metabolized by this tissue, has been studied. 2. Like other amino acids, l-histidine accumulated in the cells in the presence of glucose in concentrations up to over double that in the incubation medium. 3. The active transport of l-histidine was highest in a medium containing Ca²⁺ (3mm). The addition of K⁺ (27mm) led to a marked decrease in the intracellular concentration of l-histidine, though the oxygen uptake of the slices was higher. 4. The active l-histidine transport was inhibited by NH₄⁺. The inhibitory effect increased with the NH₄⁺ concentration, being about 25% at 8mm, 65% at 20mm, and 90% at 27 and 50mm. The oxygen uptake of the brain slices was depressed by only 25% by the highest NH₄⁺ concentration used, and less by lower concentrations.     

Optimization of a synthetic nutrient medium for culturing Bacillus thuringiensis

The requirement of Bacillus thuringiensis subsp. galleriae 69/6 for amino acids and vitamins was studied. The composition of a synthetic nutrient medium was optimized. Alanine and nicotinic acid were found to be necessary for growth while other amino acids (aspartic and glutamic acids, histidine, threonine) were not indispensable although they increased the population density (yield). A deficiency as well as an excess of individual amino acids (threonine and glutamic acid) inhibited growth and decreased the yield of biomass. Elevated concentrations of aspartic and glutamic acids inhibited the formation of spores and crystals. As was demonstrated using the method of mathematical planning of an experiment, the synthetic medium contained optimal concentrations of nicotinic and amino acids and was suitable for the growth of B. thuringiensis strains as well as for the formation of spores and crystals by them.     

Biosynthesis of amino acids from sucrose and Krebs cycle metabolites by Rhizobium lupini bacteroids

Summary The possibility of amino acids biosynthesis from sucrose, metabolites of Krebs cycle or glyoxylate and ammonium by intact bacteroids has been studied. The suspension of intact Rhizobium lupini bacteroids in phosphate buffer solution pH 7.8 was shown to catalyse the biosynthesis from sucrose and ammonium of some amino acids, such as alanine, aspartic and glutamic acids, glycine and serine. The yield of alanine and aspartic acid was 2.5–3 times higher than that of other amino acids, which were formed in almost equal quantities. Intact bacteroids were also found to catalyse the biosynthesis of aspartic and glutamic acids, alanine and glycine from ammonium and Krebs cycle metabolites such as fumaric acid (FA), oxaloacetic acid (OAA), pyruvic acid (PA), a-ketoglutaric acid (a-KGA), malic acid (MA), as well as from glyoxylic acid (GOA). The biosynthesis of aspartic acid from fumaric acid was dominant. Besides that, the suspension of intact bacteroids catalysed transamination of aspartic and glutamic acids, the transamination of aspartic acid being especially intense with -KGA and GOA. Aspartic acid was synthesized most efficiently through the amination of fumaric acid, while glutamic acid was better synthesized through the transamination of aspartic acid with -KGA than through reductive amination of -KGA.The experimental data proved that intact bacteroids posess Krebs cycle enzymes and primary ammonia assimilation enzymes. This enzyme complex permits bacteroids to detoxify ammonia, which they produce using sucrose and metabolites of Krebs cycle as the sources of carbon.The data obtained are of great interest as they prove the importance of bacteroids in the synthesis of amino acids from ammonium which is formed in the course of N₂-fixation, and sucrose available from leaves.     

Ischemia and reoxygenation induced amino acid release release and tissue damage in the slices of rat corpus striatum

Summary. Ischemic incubation significantly increased amino acid release from rat striatal slices. Reoxygenation (REO) of the ischemic slices, however, enhanced only taurine and citrulline levels in the medium. Ischemia-induced increases in glutamate, taurine and GABA outputs were accompanied with a similar amount of decline in their tissue levels. Tissue final aspartic acid level, however, was doubled by ischemia. Lactate dehydrogenase (LDH) leakage was not altered by ischemia, but enhanced during REO. Presence of tetrodotoxine (TTX) during ischemic period caused significant decline in ischemia-induced glutamate output, but not altered REO-induced LDH leakage. Although omission of extracellular calcium ions from the medium during ischemic period protected the slices against REO-induced LDH leakage, this treatment failed to alter ischemia-induced glutamate and GABA outputs. The release of other amino acids, however, declined 50% in calcium-free medium. Blockade of the glutamate uptake transporter by L-trans-PDC, on the other hand, doubled ischemia induced glutamate and aspartic acid outputs. These results indicate that more than one mechanisms probably support the ischemia-evoked accumulation of glutamate and other amino acids in the extracellular space. Although LDH leakage enhanced during REO, processes involved in this increment were found to be dependent on extracellular calcium ions during ischemia but not REO period.     

Formation of amino acids on heating glycine with alumina

The conversion of glycine into amino acids on heating at 240°C with basic manganous carbonate and alumina is investigated. Alanine, -aminobutyric acid, norvaline, norleucine, sarcosine, N-ethylglycine, N-methylalanine, N-ethylalanine, aspartic acid and glutamic acid are identified among the products of the reaction. Paper chromatography, ion-exchange chromatography and nuclear magnetic resonance are used for the analysis. A scheme for the observed transformations is presented and it is suggested that it may have been a pathway for the synthesis of amino acids from glycine under primitive Earth conditions.     

Possible involvement of glutamic and/or aspartic acid residue(s) and requirement of mitochondrial integrity for the protective effect of creatine against inhibition of cardiac mitochondrial respiration by methylglyoxal

We had previously shown that creatine exerted a protective effect against inhibition of cardiac mitochondrial respiration by methylglyoxal (SinhaRoy S, Biswas S, Ray M, Ray S. Biochem J 372: 661–669, 2003). In the present study, we have investigated the mechanism of this protective effect by specific amino acid modifying reagent and by several compounds, which are structurally related to creatine. The results show that the compounds, which contain guanidine group such as arginine and guanidinopropionic acid, exert a protective effect, which is quantitatively similar to creatine. This result suggests the presence of carboxylic acid(s) such as glutamic and/or aspartic acid(s) in the creatine-binding site, which has been further supported by experiments with N-ethyl-5-phenyl isoxazolium-3-sulfonate a reagent known to modify these amino acids. Both polarographic and spectrophotometric assays were performed with NADH as respiratory substrate by using a) submitochondrial particles by sonication, b) freeze-thawed mitochondria and c) mitochondria permeabilized by alamethicin treatment. The results of these studies as compared to that of intact mitochondria indicate that structural integrity of mitochondria is essential for the protective effect of creatine. (Mol Cell Biochem 271: 167–176, 2005)     

Identification and application of amino acids as chelators in phytoremediation of rare earth elements lanthanum and yttrium

Aims

This study aimed to identify amino acids that could act as chelators in enhancing absorption and translocation capabilities of a rare earth element (REE) lanthanum and yttrium in a non-hyperaccumulator plant.

Methods

We analysed correlations between amino acid, La and Y concentrations in xylem saps of the REE hyperaccumulator Phytolacca americana L., to identify functional amino acids in long-distance transportation processes. These were used as chelators to observe the efficacy of La uptake and translocation in REE non-hyperaccumulator tomato seedlings. Pot culture experiments were conducted using modified Hoagland solution artificially contaminated with REEs.

Results

Eighteen xylem sap amino acids were identified and measured in the xylem sap, using reversed-phase high-performance liquid chromatography. Aspartic acid, asparagine, histidine and glutamic acid may be related to xylem La and Y long-distance transportion in P. americana L. Extraneous aspartic acid, asparagine, histidine and glutamic acid enhanced La absorption in the whole tomato, compared with La alone. Moreover, the whole tomato La content increased by 449 μg and 139 μg in the presence of aspartic acid and asparagine as compared to P. americana L. Tryptophan insignificantly affected La uptake and translocation in tomato seedlings.

Conclusions

Aspartic acid and asparagine could potentially promote remediation of La contamination in soil when used as a chelator.     

Effects of dorsal root section and occlusion of dorsal spinal artery on the neurotransmitter candidates in rat spinal cord

In order to obtain further evidence of putative neurotransmitters in primary sensory neurons and interneurons in the dorsal spinal cord, we have studied the effects of unilateral section of dorsal roots and unilateral occlusion of the dorsal spinal artery on cholinergic enzyme activity and on selected amino acid levels in the spinal cord. One week after sectioning dorsal roots from caudal cervical (C₇) to cranial thoracic (T₂) levels, the specific activity of choline acetyltransferase (ChAT) was significantly decreased and acetylcholinesterase (AChE) showed a tendency to decrease in the dorsal quadrant on the operated side of the spinal cord. Dorsal root sectioning had little effect on the levels of free glutamic acid or other amino acids in the dorsal spinal cord. These results suggest that primary sensory neurons may include some cholinergic axons, and that levels of putative amino acid transmitters are not regulated by materials supplied by axonal transport from the dorsal root ganglia. By contrast, one week following unilateral occlusion of the dorsal spinal artery, the activities of ChAT and AChE were unchanged in the operated quadrant of the spinal cord, while decreases of Asp, Glu, and GABA, and an increase in Tau were detected. These findings are consistent with the proposals that such amino acids, but not ACh, may function as neurotransmitter candidates in interneurons of the dorsal spinal cord.Abbreviation used ACh acetylcholine - AChE acetylcholinesterase - Asp aspartic acid - ChAT choline acetyltransferase - GABA -aminobutyric acid - Glu glutamic acid - Gly glycine - SP substance P - Tau taurine     

The effects of sediment slurrying on microbial processes,and the role of amino acids as substrates for sulfate reduction in anoxic marine sediments

In sediment slurry experiments with anoxic marine sediments collected in Cape Lookout Bight, NC, and a site in mid-Chesapeake Bay, the rates of sulfate reduction and ammonium production decrease with increasing dilution of sediment with oxygen-free sea-water. The effect of sediment dilution on the rates of these processes can be described by a simple mathematical relationship, and when these rates are corrected for sediment dilution they yield values which agree well with direct measurements of these processes.In sediment slurry studies of amino acid utilization in Cape Lookout Bight sediments, the fermentative decarboxylation of glutamic acid (to -aminobutyric acid) or aspartic acid (to alanine or -alanine) did not occur when either of these amino acids were added to Cape Lookout Bight slurries. The addition of glutamic acid did however lead to a small (1) transient build-up of -aminoglutaric acid. Measured rates of glutamic acid uptake in these slurries also decreased with increasing sediment dilution.Molybdate inhibition experiments demonstrated that dissolved free amino acids represent 1–3% of the carbon sources/electron donors used for sulfate reduction in Cape Lookout Bight sediments. The direct oxidation of amino acids by sulfate reducing bacteria also accounts for 13–20% of the total ammonium produced. Glutamic acid, alanine, -aminoglutaric acid, aspartic acid and asparagine are the major amino acids oxidized by sulfate reducing bacteria in Cape Lookout Bight sediments.