Acute Alterations of Glutamate,Glutamine, GABA,and Other Amino Acids After Spinal Cord Contusion in Rats

Spinal cord injury (SCI) leads to an alteration of energetic metabolism. As a consequence, glutamate, glutamine, aspartate and other important amino acids are altered after damage, leading to important disregulation of the neurochemical pathways. In the present study, we characterized the acute-phase changes in tissue concentration of amino acids involved in neurotransmitter and non-neurotransmitter actions after SCI by contusion in rats. Animals were submitted to either laminectomy or SCI by contusion and sacrificed at 2, 4, 8, and 12 h after lesion, for the analysis of tissue amino acids by HPLC. Results showed that both aspartate and glutamate contents diminished after SCI, while glutamine concentrations raised, however, the sum of molar concentrations of glutamate plus glutamine remained unchanged at all time points. GABA concentrations increased versus control group, while glycine remained unchanged. Finally, citrulline levels increased by effect of SCI, while taurine-increased only 4 h after lesion. Results indicate complex acute-phase changes in amino acids concentrations after SCI, reflecting the different damaging processes unchained after lesion.     

Time-Related Loss of Glutamine from Hippocampal Slices and Concomitant Changes in Neurotransmitter Amino Acids

Abstract: A dramatic, time-dependent loss of l -glutamine was observed in mouse and rat hippocampal slices equilibrated in normal artificial CSF under static (no-flow) and super-fused (constant-flow) conditions. Concomitant with the decline in l -glutamine, there was a significant, but less pronounced, decrease in levels of the neurotransmitter amino acids, γ-aminobutyric acid, l -aspartate, and l -glutamate. The disappearance of l -glutamine was a result of diffusion from the tissue to the artificial CSF rather than chemical or biochemical transformation. The loss of amino acids from the hippocampal slices was prevented to different degrees by the addition of 0.5 m M exogenous l -glutamine to the artificial CSF. The levels of newly synthesized amino acids were also determined, because they may be more indicative of the neuronal activity than the total tissue levels of amino acids. The effects of perturbations in glutamine (length of the equilibration time and addition of exogenous. glutamine) on newly synthesized glutamate were more pronounced under 4-aminopyridine-stimulated than control (unstimulated) conditions. Therefore, a loss of l -glutamine from the hippocampal slices may have neurophysiological effects and warrants further investigation.     

Interaction Between the Glutamine Cycle and the Uptake of Large Neutral Amino Acids in Astrocytes

Abstract: When astrocyte cultures are incubated with glutamate and ammonium, the clearance of these substrates followed by the formation and export of glutamine simulates the action of the "glutamine cycle" that is believed to function in the CNS. In the present study this process was found to increase the uptake of large neutral amino acids (LNAAs), namely, histidine, kynurenine, leucine, phenylalanine, and tryptophan, by two-to threefold in mouse cerebral astrocytes. The enhancement of kynurenine uptake was shown to depend on the formation of glutamine and to saturate at low levels of glutamine. LNAAs transiently lowered the glutamine content of astrocytes that were incubated with glutamate and ammonium, but they did not affect net export of glutamine to the solution at normal physiological pH. However, on adjustment of the pH of the solution to 7.8, which causes a large increase in glutamine content without affecting export, kynurenine now significantly increased net glutamine export. These findings relate to proposed mechanisms of cerebral dysfunction in hyperammonemia.     

Repeated Administration of γ-VinylGABA Reduces Rat Brain Glutamine Synthetase Activity

Abstract: The glutamine cycle has been proposed as a pathway in which glutamine synthesized in glia provides substrate for synthesis of the neurotransmitters glutamate and GABA as they are lost from neurons. To test whether GABA may regulate this pathway, the effect of elevated GABA on the glial enzyme glutamine synthetase was examined in rat brain. Repeated subcutaneous injections of the antiepileptic GABA transaminase inhibitor γ-vinylGABA at a dose of 150 mg/kg per day for 21 days reduced glutamine synthetase activity by 36% in the cortex and 22% in the cerebellum. At 30 mg/kg per day, glutamine synthetase activity was reduced by 9.5% in the cortex but unchanged in the cerebellum. The reductions were brain specific because the skeletal muscle and liver enzymes were unaffected by γ-vinylGABA administration. Amino acid analysis of the cortex from γ-vinylGABA-treated rats demonstrated a 270% increase in GABA levels after 150 mg/kg but no change after 30 mg/kg. GABA levels and glutamine synthetase activity were inversely correlated. The 150 mg/kg dose significantly lowered cortical glutamine and glutamate levels. The decline in brain glutamine synthetase activity with chronic γ-vinylGABA administration developed gradually over time and may be due to the slow turnover of this enzyme in vivo.     

Excitatory Amino Acid Neurotransmitters in the Corticostriate Pathway: Studies Using Intracerebral Microdialysis In Vivo

The concentration of extracellular excitatory amino acids in the striatum of conscious, unrestrained rats was measured using intracerebral microdialysis, during chemical stimulation of the striatum in intact and hemidecorticate animals. Chemical stimulation of the striatum with tityustoxin (0.1 microM) evoked a rise in dialysate concentration of glutamate (to 383% of basal) and aspartate (to 156% of basal), accompanied by a drop in glutamine (to 55% of basal). These changes showed significant attenuation after treatment with L-proline (1 mM) or 2-chloroadenosine (15 microM). Unilateral degeneration of the corticostriate pathway, produced by frontal hemidecortication, caused a reduction in both basal and stimulated levels of glutamate in the lesioned side, whereas no effect was observed in the intact side. Similarly, basal and stimulated levels of glutamine were unchanged in the intact side, but were increased in the lesioned side. These results provide in vivo evidence for glutamate and possibly aspartate being neurotransmitters in the corticostriate pathway. In addition they lend support to previous studies in vitro, which implicated glutamine as the principal precursor for neurotransmitter glutamate.     

Effect of Amygdaloid Kindling on the Content and Release of Amino Acids from the Amygdaloid Complex: In Vivo and In Vitro Studies

Abstract: The tissue content and the interstitial fluid levels of glutamate, aspartate, GABA, glutamine, glycine, and serine were studied in amygdaloid-kindled rat brain. Interstitial levels were studied in vivo before and during stage 5 full limbic seizures using microdialysis. Slices of amygdala from kindled and sham-operated animals were used to study baseline and KCl-evoked release in vitro. The contents of these amino acids were measured in slices of amygdala, hippocampus, and cerebral cortex from kindled and sham-operated animals. Kindled brains showed two- to threefold higher levels of glutamate, aspartate, and GABA and 12-fold higher levels of glutamine than sham-operated controls. Correlating with this, interstitial fluid levels of glutamate were two- to threefold higher from kindled amygdala than from control both in vivo (microdialysis) and in vitro (superfusion). GABA levels in interstitial fluid from kindled amygdala were reduced by 67% compared with control amygdala.     

Brain Amino Acids During Hyponatremia In Vivo: Clinical Observations and Experimental Studies

Hyponatremia is a highly morbid condition, present in a wide range of human pathologies, that exposes patients to encephalopathic complication and the risk of permanent brain damage and death. Treating hyponatremia has proved to be difficult and still awaits safe management, avoiding the morbid sequelae of demyelinizing and necrotic lesions associated with the use of hypertonic solutions. During acute and chronic hyponatremia in vivo, the brain extrudes the excessive water by decreasing its content of electrolytes and organic osmolytes. At the cellular level, a similar response occurs upon cell swelling. Among the organic osmolytes involved in both responses, free amino acids play a prominent role because of the large intracellular pools often found in nerve cells. An overview of the changes in brain amino acid content during hyponatremia in vivo is presented and the contribution of these changes to the adaptive cell responses involved in volume regulation discussed. Additionally, new data are provided concerning changes in amino acid levels in different regions of the central nervous system after chronic hyponatremia. Results favor the role of taurine, glutamine, glutamate, and aspartate as the main amino acid osmolytes involved in the brain adaptive response to hyponatremia in vivo. Deeper knowledge of the adaptive overall and cellular brain mechanisms activated during hyponatremia would lead to the design of safer therapies for the hyponatremic patient.     

Desmethylimipramine Enhances the Release of Endogenous GABA and Other Neuro trans mitter Amino Acids from the Rat Thalamus

The influence of desmethylimipramine (DMI) on the release of endogenous gamma-aminobutyric acid (GABA) and some other amino acids from the rat thalamus was studied with a push-pull perfusion technique. Following HPLC the amino acids were fluorimetrically estimated. Added to the perfusion medium at a concentration of 10 mumol L-1, DMI caused a 5- to 10-fold increase in the release of GABA. Similar effects were found with imipramine, trimeprimine, haloperidol, and propranolol. The elevation of GABA release induced by DMI was Ca dependent. The release of aspartate and glutamate was also increased by DMI, but in contrast to K ions, DMI did not reduce the thalamic output of glutamine.     

Extracellular Levels of Amino Acids and Choline in Human High Grade Gliomas: An Intraoperative Microdialysis Study

The concentrations of endogenous amino acids and choline in the extracellular fluid of human cerebral gliomas have been measured, for the first time, by in vivo microdialysis. Glioblastoma growth was associated with increased concentrations of choline, GABA, isoleucine, leucine, lysine, phenylalanine, taurine, tyrosine, and valine. There was no difference between grade III and grade IV tumors in the concentrations of phenylalanine, isoleucine, tyrosine, valine, and lysine, whereas the concentrations of choline, aspartate, taurine, GABA, leucine, and glutamate were significantly different in the two tumor-grade subgroups. In contrast to the other compounds, the concentration of glutamate was decreased in glioma. The parenchyma adjacent to the tumor showed significant changes only in the extracellular concentration of glutamate, isoleucine, and valine. The concentrations of choline and the amino acids, glutamate, leucine, taurine, and tyrosine showed significant positive correlations with the degree of cell proliferation. Epilepsy, which is relatively common in subjects with gliomas, was shown to be a significant confounding variable when the extracellular concentrations of aspartate, glutamate and GABA were considered.     

Glutamine Stimulates Biosynthesis and Secretion of Insulin-like Growth Factor 2 (IGF2), an Autocrine Regulator of Beta Cell Mass and Function

IGF2 is an autocrine ligand for the beta cell IGF1R receptor and GLP-1 increases the activity of this autocrine loop by enhancing IGF1R expression, a mechanism that mediates the trophic effects of GLP-1 on beta cell mass and function. Here, we investigated the regulation of IGF2 biosynthesis and secretion. We showed that glutamine rapidly and strongly induced IGF2 mRNA translation using reporter constructs transduced in MIN6 cells and primary islet cells. This was followed by rapid secretion of IGF2 via the regulated pathway, as revealed by the presence of mature IGF2 in insulin granule fractions and by inhibition of secretion by nimodipine and diazoxide. When maximally stimulated by glutamine, the amount of secreted IGF2 rapidly exceeded its initial intracellular pool and tolbutamide, and high K⁺ increased IGF2 secretion only marginally. This indicates that the intracellular pool of IGF2 is small and that sustained secretion requires de novo synthesis. The stimulatory effect of glutamine necessitates its metabolism but not mTOR activation. Finally, exposure of insulinomas or beta cells to glutamine induced Akt phosphorylation, an effect that was dependent on IGF2 secretion, and reduced cytokine-induced apoptosis. Thus, glutamine controls the activity of the beta cell IGF2/IGF1R autocrine loop by increasing the biosynthesis and secretion of IGF2. This autocrine loop can thus integrate changes in feeding and metabolic state to adapt beta cell mass and function.     

Glutamine synthetases of green and etiolated leaves ofSinapis alba

Studies on the glutamine synthetases (GS, EC 6.3.1.2) of green (GS₂) and etiolated leaves (GS_et) ofSinapis alba L. (cv. Steinacher) revealed striking similarities between the respective enzyme proteins. The enzymes showed corresponding chromatographic properties, both on dimethylaminoethyl-Sephacel and on hydroxylapatite columns. The purified GS proteins were also identical with regard to the molecular weight of their subunits. Isoelectrofocusing of pure GS_et yielded two distinct polypeptide bands in the pH 5.6 region of the gels. This pattern corresponded to the two strong bands of GS₂. Two charge variants of GS polypeptides could be detected by Western-blot analysis of the soluble protein of green leaves using antibodies against mustard GS₂. In immunoprecipitation experiments, the holoenzymes of GS₂ and GS_et were recognized with identical affinities by this antiserum. We conclude that strong similarities exist between the proteins of the GS enzymes in green and etiolated leaves of mustard. Most probably only one GS form, namely the plastidic enzyme, can be found in the epigeal organs ofSinapis. The polypeptides of the GS₂ subunits showed no differences in the hydrophobicity of the polypeptide chains. Neither glucosyl nor mannosyl residues could be detected. Dedicated to Professor Dr. H. Mohr on the occasion of his 60th birthday     

Excitatory Amino Acids: Studies on the Biochemical and Chemical Stability of Ibotenic Acid and Related Compounds

The complex pharmacological profile (excitation/inhibition) of ibotenic acid on single neurons in the mammalian CNS prompted studies on the stability of ibotenic acid and a number of structurally related excitatory amino acids under different in vitro conditions in the presence or absence of enzymes. Ibotenic acid, (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-7-carboxylic acid (7-HPCA), (RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and (RS)-alpha-amino-3-hydroxy-4-bromo-5-isoxazolepropionic acid (4-Br-homoibotenic acid) were all inhibitors of (S)-glutamic acid decarboxylase (GAD) in mouse brain homogenates, but only ibotenic acid was shown to undergo decarboxylation during incubation with brain homogenates. The formation of the decarboxylated product, muscimol, which primarily occurred in a synaptosomal fraction, was dependent on the presence of pyridoxal-5-phosphate (PALP) and was inhibited by (S)-glutamic acid, 3-mercaptopropionic acid (3MPA), aminooxyacetic acid (AOAA), and allyglycine, suggesting that ibotenic acid is a substrate for GAD. The overall decomposition rate for ibotenic acid (8.7 nmol min-1 mg-1 of protein), which apparently embraces other reactions in addition to decarboxylation to muscimol, was higher than the rate of decarboxylation of (S)-glutamic acid (3.2 nmol min-1 mg-1 of protein). At pH 7.4 and 37 degrees C, but in the absence of enzymes, none of the excitatory amino acids under study underwent any detectable decomposition, whereas ibotenic acid and 7-HPCA, but not AMPA and 4-Br-homoibotenic acid, decomposed, partially by decarboxylation, at 100 degrees C in a pH-dependent manner. In the presence of liver homogenates, ibotenic acid was also shown to decompose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Severity of Hyperammonemic Encephalopathy Correlates with Brain Ammonia Level and Saturation of Glutamine Synthetase In Vivo

Abstract: Correlation among in vivo glutamine synthetase (GS) activity, brain ammonia and glutamine concentrations, and severity of encephalopathy was examined in hyperammonemic rats to obtain quantitative information on the capacity of GS to control these metabolites implicated in the etiology of hepatic encephalopathy. Awake rats were observed for neurobehavioral impairments after ammonium acetate infusion to attain a steady-state blood ammonia concentration of 0.9 (group A) or 1.3 µmol/g (group B). As encephalopathy progressed from grade III to IV, brain ammonia concentration increased from 1.9 to 3.3 µmol/g and then decreased to 1.3 µmol/g on recovery to grade III. In contrast, brain glutamine concentration was 26, 23, and 21 µmol/g, respectively. NH₄⁺-infused rats pretreated with l -methionine dl -sulfoximine reached grade IV when brain ammonia and glutamine concentrations were 3.0 and 5.5 µmol/g, respectively; severity of encephalopathy correlates with brain ammonia, but not glutamine. In vivo GS activity, measured by NMR, was 6.8 ± 0.7 µmol/h/g for group A and 6.2 ± 0.6 µmol/h/g for group B. Hence, the in vivo activity, shown previously to increase with blood ammonia over a range of 0.4–0.64 µmol/g, approaches saturation at blood ammonia >0.9 µmol/g. This is likely to be the major cause of the observed accumulation of brain ammonia and the onset of grade IV encephalopathy.     

Effect of Intracellular Glutamine on the Uptake of Large Neutral Amino Acids in Astrocytes: Concentrative Na+-Independent Transport Exhibits Metastability

To examine whether the concentration gradient of glutamine (Gln) drives concentrative Na(+)-independent uptake of neutral amino acids (NAA) in mouse cerebral astrocytes, uptake was compared in "Gln-depleted" and "Gln-replete" cultures. Uptake (30 min in Na(+)-free buffer) of histidine, kynurenine, leucine, tyrosine, and a model substrate for System L transport was 70-150% greater in Gln-replete cultures. Phenylalanine uptake was not affected. All of these NAA trans-stimulated the export of Gln from astrocytes. However, the increase in NAA uptake was sustained even though the Gln content of Gln-replete cultures declined. Also, uptake of Gln itself was enhanced in Gln-replete cultures. Thus, countertransport of Gln was insufficient to explain the enhancement of NAA uptake. Enhanced uptake was restored, and could be magnified, by reloading Gln-depleted cultures either with Gln or with histidine. It is suggested that substrate-induced asymmetry and molecular hysteresis in the Na(+)-independent carrier could account for the sustained enhancement of NAA uptake. Only histidine and kynurenine were concentrated comparably to Gln (15- to 29-fold at 1 mM in Na(+)-free buffer). The other NAA were four to six times less concentrated. At least two Na(+)-dependent transport systems also supported the concentration gradient of Gln in regular buffer.     

Studies on the Mechanism of Strengthening Fish Gel by Basic Amino Acids

An electro-energizing fermentation (E-E F) method has been developed. In this method, a direct electrical current is applied to a microbial culture to accelerate the reductive metabolism of microorganisms or to impart profitable effects to microbial cells. This E-E F method was applied to l-glutamic acid fermentation by Brevibacterium flavum No. 2247. When glucose was used as a substrate, the addition of 0.01 mm neutral red (NR), redox dye (electron carrier), to the fermentation broth at the beginning of cultivation was effective for l-glutamate (l-Glu) production. A direct current of 200~300 μA/cm² at 1.5 V was applied through out the cultivation of this bacterium. This resulted in about a 10% increase in yield of l-Glu.     

Discovery of a potent,selective, and orally bioavailable histamine H3 receptor antagonist SAR110068 for the treatment of sleep–wake disorders

Previous studies have shown that compound 1 displayed high affinity towards histamine H₃ receptor (H₃R), (human (h-H₃R), K_i = 8.6 nM, rhesus monkey (rh-H₃R), K_i = 1.2 nM, and rat (r-H₃R), K_i = 16.5 nM), but exhibited high affinity for hERG channel. Herein, we report the discovery of a novel, potent, and highly selective H₃R antagonist/inverse agonist 5a(SS) (SAR110068) with acceptable hERG channel selectivity and desirable pharmacological and pharmacokinetic properties through lead optimization sequence. The significant awakening effects of 5a(SS) on sleep–wake cycles studied by using EEG recording in rats during their light phase support its potential therapeutic utility in human sleep–wake disorders.     

Glutamine and Aspartate Loading of Synaptosomes: A Reevaluation of Effects on Calcium-Dependent Excitatory Amino Acid Release

Guinea-pig cerebral cortical synaptosomes were preincubated for 60 min with 100 microM D-aspartate, L-aspartate, or L-glutamate. The total D- plus L-aspartate content of the synaptosomal fraction increased to 235%, 195%, or 164%, respectively, of the control. Despite this no increase was seen in the very low KCl evoked, Ca2+-dependent release of aspartate. Preincubation with the three amino acids changed the synaptosomal glutamate content to 78% (D-aspartate), 149% (L-aspartate), or 168% (L-glutamate) of control. However there was no statistically significant effect of these preincubations on the extent of Ca2+-dependent glutamate release. Thus the Ca2+-dependent release of aspartate and glutamate is not determined by the total synaptosomal content of these amino acids. The addition of 0.1-0.5 mM glutamine to the incubation caused a massive appearance of glutamate in the extrasynaptosomal medium. Analysis of specific activities showed that glutamine was hydrolysed directly by an extrasynaptosomal glutaminase, and that intrasynaptosomal glutamate was predominantly labelled by uptake of this glutaminase-derived glutamate. No increase was seen in the extent of Ca2+-dependent release of glutamate (by fluorimetry) either after preincubation with glutamine or in the continued presence of glutamine. Thus we are unable to confirm reports that glutamine expands the transmitter pool of glutamate. The extrasynaptosomal glutaminase activity in the synaptosomal preparation was inhibited by Ca2+ and activated by phosphate. Identical kinetics were obtained with "free" brain mitochondria, confirming the origin of the glutamine-derived glutamate.     

Acetoacetate and Glucose as Lipid Precursors and Energy Substrates in Primary Cultures of Astrocytes and Neurons from Mouse Cerebral Cortex

Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3-14C]acetoacetate or [2-14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, D-3-hydroxybutyrate, and 14CO2, and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only approximately 20% in the case of cultured astrocytes. This indicates that the rate at which 14C-labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.     

Selective Alterations of Extracellular Brain Amino Acids in Relation to Function in Experimental Portal-Systemic Encephalopathy: Results of an In Vivo Microdialysis Study

Abstract: Portal-systemic encephalopathy (PSE) is characterized by neuropsychiatric symptoms progressing through stupor and coma. Previous studies in human autopsy tissue and in experimental animal models of PSE suggest that alterations in levels of brain amino acids may play a role in the pathogenesis of PSE. To assess this possibility, levels of amino acids were measured using in vivo cerebral microdialysis in frontal cortex of portacaval-shunted rats administered ammonium acetate (3.85 mmol/kg, i.p.) to precipitate severe PSE. Sham-operated rats served as controls. Portacaval shunting resulted in significant increases of levels of extracellular glutamine (threefold, p < 0.001), alanine (38%, p < 0.01), aspartate (44%, p < 0.05), phenylalanine (170%, p < 0.001), tyrosine (140%, p < 0.001), tryptophan (63%, p < 0.001), leucine (75%, p < 0.001), and serine (60%, p < 0.001). Administration of ammonium acetate to sham-operated animals led to a significant increase in extracellular glutamine and taurine content, but this response was absent in shunted rats. The lack of taurine release into extracellular fluid following ammonium acetate administration in portacaval-shunted rats could relate to the phenomenon of brain edema in these animals. Ammonium acetate administration resulted in significant increases in the extracellular concentrations of phenylalanine and tyrosine in both sham-operated and portacaval-shunted rats. Severe PSE was not accompanied by significant increases in extracellular fluid concentrations of glutamate, aspartate, GABA, tryptophan, leucine, or serine, suggesting that increased spontaneous release of these amino acids in cerebral cortex is not implicated in the pathogenesis of hepatic coma.