Differential effects of ammonia and β-methylene-dl-aspartate on metabolism of glutamate and related amino acids by astrocytes and neurons in primary culture

The effects of ammonium chloride (3 mM) and -methylene-dl-aspartate (BMA; 5 mM) (an inhibitor of aspartate aminotransferase, a key enzyme of the malate-aspartate shuttle (MAS)) on the metabolism of glutamate and related amino acids were studied in primary cultures of astrocytes and neurons. Both ammonia and BMA inhibited¹⁴CO₂ production from [U-¹⁴C]-and [1-¹⁴C]glutamate by astrocytes and neurons and their effects were partially additive. Acute treatment of astrocytes with ammonia (but not BMA) increased astrocytic glutamine. Acute treatment of astrocytes with ammonia or BMA decreased astrocytic glutamate and aspartate (both are key components of the MAS). Acute treatment of neurons with ammonia decreased neuronal aspartate and glutamine and did not apparently affect the efflux of aspartate from neurons. However, acute BMA treatment of neurons led to decreased neuronal glutamate and glutamine and apparently reduced the efflux of aspartate and glutamine from neurons. The data are consistent with the notion that both ammonia and BMA may inhibit the MAS although BMA may also directly inhibit cellular glutamate uptake. Additionally, these results also suggest that ammonia and BMA exert differential effects on astroglial and neuronal glutamate metabolism.This paper is dedicated to Professor E. Kvamme. Dr. Kvamme has conducted numerous pioneering studies on the regulation of the metabolism of glutamine, glutamate and ammonia in nervous and other tissues (see Refs. 1 and 3 for a complete discussion and citation of his many papers). Many important ideas in this exciting field of research have emerged from the work carried out in his laboratory.     

Effect of prostaglandins on α-aminoisobutylic acid uptake in cultured fibroblasts

Effect of various prostaglandins on the uptake of α-aminoisobutylic acid by cultured fibroblasts was studied. All the prostaglandins having an OH functional group in an intramolecular 5-membered ring showed an inhibitory effect on the amino acid uptake. The active compounds can be ranked in potency according to the values for the inhibition of the amino acid uptake per cent of control: prostaglandin F_2α(53 %) >F_1α(54 %) >D₂(56 %) >E₂(62 %) >thromboxane B₂ (66 %). Thus, prostaglandin F_2α was found to be the most potent inhibitor to membrane permeability and the inhibitory effect was dose dependent. The inhibition was maximal after 1 hour of exposure to prostaglandin F_2α, persisted at least up to 6 hours in the presence of prostaglandin F_2α.     

Effects ofγ-irradiation on embryonic development and nucleic acids in trigeminal neurons

Summary Pregnant rats were exposed to absorbed doses of 0.5, 1, 2, and 4 Gy of⁶⁰Co- rays either on day 8 or on day 12 p.c.. The embryos were collected on day 18 p.c.. Irradiations both on day 8 and 12 p.c. result in a dose dependent decrement of weight. The effect is larger for irradiation on day 8 p.c.. Caryometric studies of the neurons in the trigeminal ganglion show, on the other hand, that the reduction of nuclear sizes in this tissue is more substantial for an irradiation on day 12 p.c. when the ganglion is in a stage of formation. Cytophotometric determinations of the Feulgen-DNA content and determination of the RNA content by staining with chromic gallocyanin in combination with ribonuclease digestion lead to the conclusion that the irradiation induces no significant hyperploidy and that the irradiated neurons have the nuclear RNA content that is normal at this stage of gestation. This applies both to the irradiations on day 8 and on day 12 p.c.     

Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids

Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment is often not accurate if sequence identities between to-be-aligned se- quences are less than 30%. This is because that for these sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment using substitu- tion matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced alphabet with N around 9.     

γ-Aminobutyric acid and related amino acids in plant immune responses: Emerging mechanisms of action

The entanglement between primary metabolism regulation and stress responses is a puzzling and fascinating theme in plant sciences. Among the major metabolites found in plants, γ-aminobutyric acid (GABA) fulfils important roles in connecting C and N metabolic fluxes through the GABA shunt. Activation of GABA metabolism is known since long to occur in plant tissues following biotic stresses, where GABA appears to have substantially different modes of action towards different categories of pathogens and pests. While it can harm insects thanks to its inhibitory effect on the neuronal transmission, its capacity to modulate the hypersensitive response in attacked host cells was proven to be crucial for host defences in several pathosystems. In this review, we discuss how plants can employ GABA's versatility to effectively deal with all the major biotic stressors, and how GABA can shape plant immune responses against pathogens by modulating reactive oxygen species balance in invaded plant tissues. Finally, we discuss the connections between GABA and other stress-related amino acids such as BABA (β-aminobutyric acid), glutamate and proline.     

High-affinity uptake of γ-aminobutyric acid in cultured glial and neuronal cells

Both glial and neuronal cells maintained in primary culture were found to accumulate [³H]GABA by an efficient high-affinity uptake system (apparentK _m=9 M,V _max=0.018 and 0.584 nmol/mg/min, respectively) which required sodium ions and was inhibited by 1 mM ouabain. Strychnine and parachloromercuriphenylsulfonate (pCS) (both at 1 mM) also strongly inhibited uptake of [³H]GABA, but metabolic inhibitors (2,4-dinitrophenol, potassium cyanide, and malonate) were without effect. Only three structural analogs of GABA (nipecotate, -alanine, and 2,4-diaminobutyrate) inhibited uptake of [³H]GABA, while several other compounds with structural similarities to GABA (e.g. glycine,l-proline, and taurine) did not interact with the system. The kinetic studies indicated presence of a second uptake (K _m=92 M,V _max=0.124 nmol/mg/min) in the primary cultures containing predominantly glioblasts. On the other hand, only one of the neuronal cell lines transformed by simian virus SV40 appeared to accumulate [³H]GABA against a concentration gradient. ApparentK _m of this uptake was relatively high (819 M), and it was only weakly inhibited by 1 mM ouabain and 1 mM pCS. The structural specificity also differed from that of the uptake observed in the primary cultures. Significantly, none of the nontransformed continuous cell lines of either tumoral (glioma, C₆; neuroblastoma, Ml; MINN) or normal (NN; I₆) origin actively accumulated [³H]GABA. It is suggested that for the neurochemical studies related to GABA and requiring homogeneous cell populations, the primary cultures offer a better experimental model than the continuous cell lines.     

Stimulated uptake of amino acids by HeLa cells following ‘preloading’ in high concentrations of amino acids

HeLa cells exposed to 10mM leucine in medium for 30 mins at 37 °C showed greatly increased uptake of tritiated leucine when returned to medium containing 0.1mM levels of labelled amino acid. Further analysis of the phenomenon demonstrates that the steepness of the gradient is essential for rapid uptake to be observed, that no stereo-specificity is involved, and that the peak of uptake is followed by discharge with first order kinetics back to equilibrium. The effect of 14 different amino acids examined individually as 10mM supplements to the labelling medium ranged from slight reduction (lysine, proline) to complete suppression of the ‘stimulated uptake’ (phenylalanine, isoleucine).     

Metabolic engineering of microorganisms for the production of multifunctional non-protein amino acids: γ-aminobutyric acid and δ-aminolevulinic acid

Gamma-aminobutyric acid (GABA) and delta-aminolevulinic acid (ALA), playing important roles in agriculture, medicine and other fields, are multifunctional non-protein amino acids with similar and comparable properties and biosynthesis pathways. Recently, microbial synthesis has become an inevitable trend to produce GABA and ALA due to its green and sustainable characteristics. In addition, the development of metabolic engineering and synthetic biology has continuously accelerated and increased the GABA and ALA yield in microorganisms. Here, focusing on the current trends in metabolic engineering strategies for microbial synthesis of GABA and ALA, we analysed and compared the efficiency of various metabolic strategies in detail. Moreover, we provide the insights to meet challenges of realizing industrially competitive strains and highlight the future perspectives of GABA and ALA production.     

Effects of γ-aminobutyric acid on feed intake,growth performance and expression of related genes in growing lambs

This study was conducted to investigate the effects of rumen-protected γ-aminobutyric acid (GABA) on feed intake, growth performance and expression of related genes in growing lambs. A total of 24 lambs weaned at age of 50 days were divided into four block of six based on their BW, six lambs within a block were allocated to three pairs, which were then assigned randomly to three treatments with addition of rumen-protected GABA at levels of 0, 70 or 140 mg/day for 6 weeks. Dry matter intake was recorded weekly in three consecutive days, and BW was recorded every two weeks. At the end of the trial, four lambs from each group were slaughtered, and duodenum and ileum mucosa were obtained for measurement of mRNA abundance of GABA receptor and cholecystokinin receptor. Dry matter intake was higher (P<0.01) in the lambs fed 140 mg/day GABA than that in the control or 70 mg GABA-fed lambs. Average daily gain and nutrients digestibility were not different (P>0.05) among treatments. Lambs fed 140 mg/day GABA had higher mRNA abundance of GABA-B receptor (P<0.01) and lower mRNA abundance of cholecystokinin-2 receptor (P<0.01) in duodenum mucosa. Serum CCK content was lower (P<0.01) in lambs fed 140 mg/day GABA than that in control. It is indicated that GABA may enhance feed intake by regulating GABA- and cholecystokinin-related genes.     

Gender related differences in the effect of aging on blood amino acid compartmentation☆

This work has been focused on the study of the variations in blood amino acid compartmentation (plasma and blood cells) with aging, both in men and women. Aging is a situation which, under the influence of gender, involves a decline in body weight functions and variations in energy metabolism with a deterioration of muscular metabolism leading to changes in amino acid handling. We determined the blood levels of individual amino acids in whole blood, plasma compartment and blood cell compartment of 51 men and 51 women. Subjects were classified in three age groups—AG1 (18 to 35 y), AG2 (35–50 y) and AG3 (more than 50 y). Aging was accompanied by significant changes in blood levels of amino acids showing gender-linked differences which were distinct for both blood compartments (plasma and blood cells). In men, aging was accompanied by a drop in blood levels of several amino acids, due mainly to the plasma compartment, whereas in women aging brought about a rise in blood levels of various amino acids mainly in blood cell compartment. This paper contributes to enhancing the physiological importance of the blood cell pool in the handling of amino acids.     

Effects of ornithine on neutrophil (PMN) free amino acid and α-keto acid profiles and immune functions in vitro

Summary. The objective of this study was to determine the effects of ornithine on polymorphonuclear leucocyte (PMN) free amino- and -keto acid profiles, superoxide anion (O₂^–) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase acitivity (MPO). Exogenous ornithine significantly increased PMN asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, -ketoglutarate and pyruvate as intracellular ornithine increased. Concerning PMN immune function markers ornithine increased H₂O₂-generation and MPO acitivity while O₂^–-formation was decreased. We believe therefore that ornithine is important for affecting PMN susceptible free amino- and -keto acid pool although the mechanisms are not yet clear. This may be one of the determinants in PMN nutrition considerably influencing and modulating PMN host defense capability.     

The effects of α- and β-adrenergic agents,Ca2+ and insulin on 2-deoxyglucose uptake and phosphorylation in perfused rat heart

Insulin (0.1 μM) and 1 μM epinephrine each increased the uptake and phosphorylation of 2-deoxyglucose by the perfused rat heart by increasing the apparent V_max without altering the K_m. Isoproterenol (10 μM), 50 μM methoxamine and 10 mM CaCl₂ also increased uptake. Lowering of the perfusate Ca²⁺ concentration from 1.27 to 0.1 mM Ca²⁺, addition of the Ca²⁺ channel blocker nifedipine (1 μM) or addition of 1.7 mM EGTA decreased the basal rate of uptake of 2-deoxyglucose and prevented the stimulation due to 1 μM epinephrine. Stimulation of 2-deoxyglucose uptake by 0.1 μM insulin was only partly inhibited by Ca²⁺ omission, nifedipine or 1 mM EGTA. Half-maximal stimulation of 2-deoxyglucose uptake by insulin occurred at 2 nM and 0.4 nM for medium containing 1.27 and 0.1 mM Ca²⁺, respectively. Maximal concentrations of insulin (0.1 μM) and epinephrine (1 μM) were additive for glucose uptake and lactate output but were not additive for uptake of 2-deoxyglucose. Half-maximal stimulation of 2-deoxyglucose uptake by epinephrine occurred at 0.2 μM but maximal concentrations of epinephrine (e.g., 1 μM) gave lower rates of 2-deoxyglucose uptake than that attained by maximal concentrations of insulin. The addition of insulin increased uptake of 2-deoxyglucose at all concentrations of epinephrine but epinephrine only increased uptake at sub-maximal concentrations of insulin. The role of Ca²⁺ in signal reversal was also studied. Removal of 1 μM epinephrine after a 10 min exposure period resulted in a rapid return of contractility to basal values but the rate of 2-deoxyglucose uptake increased further and remained elevated at 20 min unless the Ca²⁺ concentration was lowered to 0.1 mM or nifedipine (1 μM) was added. Similarly, removal of 0.1 μM insulin after a 10 min exposure period did not affect the rate of 2-deoxyglucose uptake, which did not return to basal values within 20 min unless the concentration of Ca²⁺ was decreased to 0.1 mM. Insulin-mediated increase in 2-deoxyglucose uptake at 0.1 mM Ca²⁺ reversed upon hormone removal. It is concluded that catecholamines mediate a Ca²⁺-dependent increase in 2-deoxyglucose transport from either α or β receptors. Insulin has both a Ca²⁺-dependent and a Ca²⁺-independent component. Reversal studies suggest an additional role for Ca²⁺ in maintaining the activated transport state when activated by either epinephrine or insulin.     

Effects of beta-amyloid on behavioral and amino acids spectrum in rats’ brain and their modulation by embryonic proteins

One of the crucial events in the pathogenesis of neurodegenerative disorders linked with dementia-like Alzheimer’s Disease (AD) is the disturbance in neurotransmission based on progressive deficit of neuromediators that is manifested by marked decrease in cognitive behavior, loss of memory and inability to learn as a result of impairment in synaptic plasticity of neurons.In this study we have used a new complex of proteoglycans of embryonic genesis (PEG) created by Prof. L. Mkrtchyan, as a possible therapeutic approach that can rescue neurons from further degeneration caused by beta-amyloid (Aβ). We attempt to reveal the biochemical (determination of neuroactive amino acids such as glutamate, GABA, taurine, glycine and aspartate) changes and behavior on Y-maze and avoidance/exploratory activity on elevated plus-maze task in rats’ brain after modeling Alzheimer’s disease by i.c.v. injection of Aβ25–35. Furthermore, in this study we analyzed the neuroprotective properties of PEG.Under the influence of PEG the concentration of all investigated amino acids both in cerebral cortex and hippocampus (except striatum changes) increased. In the present study we demonstrated that bilateral i.c.v. injection of aggregated Aβ25–35 in dosage 30 nmol/rat resulted in impairment in spatial alternation behavior. Both preliminary (single) and double injection of PEG showed constant improvement of spatial memory after the first trial up to 90 days after i.c.v. injection of aggregated Aβ25–35.Our findings suggest that proteoglycans of embryonic genesis in neurodegenerative state show an expressed regulatory–protective effect.     

The effect of amino acids on the C₁₄-sphingosine-triggered germination of Nomuraea rileyi, and surface amino acids on Spodoptera litura fabricius

D-erythro-C??-Sphingosine (C??-Sph) accelerated the germination of Nomuraea rileyi in a solution containing peptone, but activity declined to a large degree in water. This suggests the presence of a co-factor in C??-Sph-triggered germination. Since the main role of peptone is to supply nitrogen constituents, we examined the effects of various nitrogen constituents. It was found that Ala and His were highly effective for C??-Sph-triggered germination.     

Effects of the amino acid derivatives, β-hydroxy-β-methylbutyrate,taurine, and N-methyltyramine,on triacylglycerol breakdown in fat cells

Journal of Physiology and Biochemistry - Various amino acid (AA) metabolites are used as supplements to facilitate metabolic control and enhance responsiveness of insulin-sensitive tissues....     

Effects of γ-rays on Poly-L-glutamic acid and Poly-D,L-glutamic acid in the solid state

Summary Poly-L-glutamic acid and poly-D,L-glutamic acid, as models of proteins, were irradiated with⁶⁰Co--radiation in air and under vacuo to examine whether or not the changes caused by the exposure to ionizing radiation depend on the conformations of polypeptides.It was found that theG- values (yield of main-chain scissions per 100 eV of energy absorbed) of both polypeptides are approximately equal for the irradiation in air, while under vacuo theG- value of poly-D,L-glutamic acid is larger than that of poly-L-glutamic acid. This observation for irradiation under vacuo was ascribed to the stabilizing effect of intramolecular hydrogen bond bridges in poly-L-glutamic acid. It was also found that the-helical structure of poly-L-glutamic acid is destroyed by the exposure to ionizing radiation.     

Effects of α-ketoglutarate on neutrophil intracellular amino and α-keto acid profiles and ROS production

The aim of this study was to determine the effects of α-ketoglutarate on neutrophil (PMN), free α-keto and amino-acid profiles as well as important reactive oxygen species (ROS) produced [superoxide anion (O₂ ^?), hydrogen peroxide (H₂O₂)] and released myeloperoxidase (MPO) acitivity. Exogenous α-ketoglutarate significantly increased PMN α-ketoglutarate, pyruvate, asparagine, glutamine, asparatate, glutamate, arginine, citrulline, alanine, glycine and serine in a dose as well as duration of exposure dependent manner. Additionally, in parallel with intracellular α-ketoglutarate changes, increases in O₂ ^– formation, H₂O₂-generation and MPO acitivity have also been observed. We therefore believe that α-ketoglutarate is important for affecting PMN “susceptible free amino- and α-keto acid pools” although important mechanisms and backgrounds are not yet completely explored. Moreover, our results also show very clearly that changes in intragranulocytic α-ketoglutarate levels are relevant metabolic determinants in PMN nutrition considerably influencing and modulating the magnitude and quality of the granulocytic host defense capability as well as production of ROS.     

Effects of linoleic acid on intracellular calcium concentration in primarily cultured rat pancreatic β-cells and underlying mechanism

In this study, we investigated the mechanism of linoleic acid-stimulated increase in intracellular calcium concentration ([Ca(2+)](i)) in pancreatic islet β-cells. Pancreatic islet cells were primarily isolated from rats and cultured for the experiments. The cells were loaded with Fluo-3/AM, the indicator of [Ca(2+)](i), and the intensity of Fluo-3 was measured using confocal microscope. The islet β-cells were identified by immunocytochemical staining with insulin antibody after recording. The drugs were given by perfusion system. The results showed that linoleic acid (20 μmol/L) stimulated [Ca(2+)](i) increase with the first peak increase and the following plateau increase. Linoleic acid-stimulated [Ca(2+)](i) increase was partly inhibited by removal of extracellular calcium and by transient receptor potential (TRP) channel blocker, La(3+), and it was totally blocked by exhaustion of intracellular calcium stores and inhibition of phospholipase C. It is concluded that linoleic acid stimulates [Ca(2+)](i) increase in islet β-cells through both extracellular calcium influx via TRP channels and calcium release from intracellular calcium stores.     

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.     

Changes in γ-Methyleneglutamic acid, γ-Methyleneglutamine and other amino acids and amides during fruit growth of Tribulus terrestris

Abstract

Distribution and metabolism of γ-methyleneglutamic acid, γ-methyleneglutamine and other amino acids and amides has been studied during fruit growth of Tribulus terrestris. The largest concentration of free amino acids and amides has been observed in fruit stage 1. The marked decline in the amount of γ-methyleneglutamic acid and γ-methyleneglutamine after fruit stage 1 may indicate their rapid utilization along with asparagine and glutamine during fruit growth. In leaf and in different fruit growth stages, γ-methyleneglutamic acid dominated over γ-methyleneglutamine.