重复经颅磁刺激对创伤性颅脑损伤患者脑脊液中兴奋性氨基酸含量的影响

目的：探讨重复经颅磁刺激（rTMS）对急性颅脑损伤患者脑脊液中兴奋性氨基酸（EAA）含量的影响。方法：30例创伤性颅脑损伤（TBI）病人按格拉斯哥昏迷评分分为轻型组（rTMS3）、中型组（rTMS2）、重型组（rTMS1）,每组10例,各组病人分别随机分为rTMS对照亚组（A组）及治疗亚组（B组）,每亚组5例。于TBI后第15天行腰椎穿刺采集脑脊液,采用高效液相色谱法测定脑脊液中谷氨酸（ASP）及门冬氨酸（GLU）含量。结果：脑脊液ASP和GLU水平随着脑损伤程度的加重而升高,各rTMS治疗组与相应各对照组的EAA相比,rTMS治疗组EAA的水平均低于相应对照组。结论：rTMS可通过降低TBI后脑脊液EAA水平发挥脑保护作用。脑脊液EAA的含量变化可作为TBI严重程度的生化指标。     

Severity of experimental traumatic brain injury modulates changes in concentrations of cerebral free amino acids

In this study, concentrations of free amino acids (FAA) and amino group containing compounds (AGCC) following graded diffuse traumatic brain injury (mild TBI, mTBI; severe TBI, sTBI) were evaluated. After 6, 12, 24, 48 and 120 hr aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), histidine (His), glycine (Gly), threonine (Thr), citrulline (Cit), arginine (Arg), alanine (Ala), taurine (Tau), γ‐aminobutyrate (GABA), tyrosine (Tyr), S‐adenosylhomocysteine (SAH), l ‐cystathionine (l ‐Cystat), valine (Val), methionine (Met), tryptophane (Trp), phenylalanine (Phe), isoleucine (Ile), leucine (Leu), ornithine (Orn), lysine (Lys), plus N‐acetylaspartate (NAA) were determined in whole brain extracts (n = 6 rats at each time for both TBI levels). Sham‐operated animals (n = 6) were used as controls. Results demonstrated that mTBI caused modest, transient changes in NAA, Asp, GABA, Gly, Arg. Following sTBI, animals showed profound, long‐lasting modifications of Glu, Gln, NAA, Asp, GABA, Ser, Gly, Ala, Arg, Citr, Tau, Met, SAH, l ‐Cystat, Tyr and Phe. Increase in Glu and Gln, depletion of NAA and Asp increase, suggested a link between NAA hydrolysis and excitotoxicity after sTBI. Additionally, sTBI rats showed net imbalances of the Glu‐Gln/GABA cycle between neurons and astrocytes, and of the methyl‐cycle (demonstrated by decrease in Met, and increase in SAH and l ‐Cystat), throughout the post‐injury period. Besides evidencing new potential targets for novel pharmacological treatments, these results suggest that the force acting on the brain tissue at the time of the impact is the main determinant of the reactions ignited and involving amino acid metabolism.     

Identification of candidate biomarkers of brain damage in a mouse model of closed head injury: a metabolomic pilot study

We aim to identify candidate brain biomarkers for, and to elucidate the pathophysiology of closed traumatic brain injury (TBI). Nuclear magnetic resonance (NMR) based metabolomic analysis was performed on the whole brain of mice undergoing TBI using a validated technique. There were 10 TBI mice compared to 8 sham operated controls. A total of 45 metabolites were evaluated. There was a statistically significant alteration in concentrations of 29 metabolites in TBI brains as compared to controls (FDR <0.05). Profound disturbances of several metabolic pathways (FDR <1E-07), including pathways associated with purine, alanine, aspartate and glutamine and glutathione metabolism were observed. Also, a significant elevation in glutamate (the main excitatory neurotransmitter) and depression of GABA (the main inhibitory neurotransmitter) was observed. Four metabolites, ADP, AMP, NAD+, and IMP were the most important indicators of TBI, relative to normal controls. All were elevated in the TBI mice. A combination of these 4 biomarkers produced a perfect predictor of TBI status, AUC (95 % CI) = 1.0 (1.0, 1.0). We also detected significant disturbances in mitochondrial function, energy metabolism, neurotransmitter metabolism and other important biochemical pathways in TBI mouse brains. Further studies to assess the utility of metabolomics to detect and classify the severity of and assess the prognosis of TBI is warranted.     

Lactate Storm Marks Cerebral Metabolism following Brain Trauma

Brain metabolism is thought to be maintained by neuronal-glial metabolic coupling. Glia take up glutamate from the synaptic cleft for conversion into glutamine, triggering glial glycolysis and lactate production. This lactate is shuttled into neurons and further metabolized. The origin and role of lactate in severe traumatic brain injury (TBI) remains controversial. Using a modified weight drop model of severe TBI and magnetic resonance (MR) spectroscopy with infusion of ¹³C-labeled glucose, lactate, and acetate, the present study investigated the possibility that neuronal-glial metabolism is uncoupled following severe TBI. Histopathology of the model showed severe brain injury with subarachnoid and hemorrhage together with glial cell activation and positive staining for Tau at 90 min post-trauma. High resolution MR spectroscopy of brain metabolites revealed significant labeling of lactate at C-3 and C-2 irrespective of the infused substrates. Increased ¹³C-labeled lactate in all study groups in the absence of ischemia implied activated astrocytic glycolysis and production of lactate with failure of neuronal uptake (i.e. a loss of glial sensing for glutamate). The early increase in extracellular lactate in severe TBI with the injured neurons rendered unable to pick it up probably contributes to a rapid progression toward irreversible injury and pan-necrosis. Hence, a method to detect and scavenge the excess extracellular lactate on site or early following severe TBI may be a potential primary therapeutic measure.     

A Comparative Study of Excitatory and Inhibitory Amino Acids in Three Different Brainstem Nuclei

This study was designed to shed more light onto the three different brainstem regions which are implicated in the pain pathway for the level of various excitatory and inhibitory neurotransmitters before and following neuronal stimulation. The in vivo microdialysis technique was used in awake, freely moving adult Sprague-Dawley rats. The neurotransmitters studied included aspartate, glutamate, GABA, glycine, and taurine. The three brainstem regions examined included the mid-brain periaqueductal gray (PAG), the medullary nucleus raphe magnus (NRM), and the spinal trigeminal nucleus (STN). Neuronal stimulation was achieved following the administration of the sodium channel activator veratridine. The highest baseline levels of glutamate (P < 0.0001), aspartate (P < 0.0001), GABA (P < 0.01), taurine (P < 0.0001), and glycine (P < 0.001) were seen in the NRM. On the other hand, the lowest baseline levels of glutamate, GABA, glycine, and taurine were found in the PAG, while that of aspartate was found in the STN. Following the administration of veratridine, the highest release of the above neurotransmitters except for the aspartate and glycine was found in the PAG where the level of glutamate increased by 1,310 ± 293% (P < 0.001), taurine by 1,008 ± 143% (P < 0.01), and GABA by 10,358 ± 1,920% (P < 0.0001) when comparison was performed among the three brainstem regions and in relation to the baseline levels. The highest release of aspartate was seen in the STN (2,357 ± 1,060%, P < 0.001), while no significant difference was associated with glycine. On the other hand, the lowest release of GABA and taurine was found in the STN (696 ± 91 and 305 ± 25%, respectively), and glutamate and aspartate in the NRM (558 ± 200 and 874 ± 315%, respectively). Our results indicate, and for the first time, that although some differences are seen in the baseline levels of the above neurotransmitters in the three regions studied, there are quite striking variations in the level of release of these neurotransmitters following neuronal stimulation in these regions. In our opinion this is the first study to describe the pain activation/modulation related changes of the excitatory and inhibitory amino acids profile of the three different brainstem areas.     

The role of excitatory amino acid receptors and intracellular messengers in persistent nociception after tissue injury in rats

Increased pain sensitivity (hyperalgesia) and persistent nociception following peripheral tissue injury depends both on an increase in the sensitivity of primary afferent nociceptors at the site of injury (peripheral sensitization), and on an increase in the excitability of neurons in the central nervous system (central sensitization). We will review evidence that central sensitization, and the persistent nociception it leads to, are dependent on an action of glutamate and aspartate at excitatory amino acid (EAA) receptors. Additional evidence will be presented implicating a role of various intracellular second messengers that are coupled to EAA receptors (nitric oxide, arachidonic acid, and protein kinase C) to central sensitization and persistent nociception following tissue injury. Finally, we will examine the evidence for a contribution of molecular events, including noxious stimulus-induced expression of immediate-early genes such as c-fos to persistent nociception.     

Contrasting Effects of D- and L-(E)-4-(3-Phospiono-2- Propenyl) Piperazine-2-Carboxylic Acid as Anticonvulsants and as Inhibitors of Potassium-Evoked Increases in Hippocampal Extracellular Glutamate and Aspartate Levels in Freely Moving Rats I

Abstract: Microdialysis experiments performed in the dorsal hippocampus of freely moving rats showed that L-( E )- 4-(3-phosphono-2-propenyl) piperazine-2-carboxylic acid (L-CPPene) is 10 times as potent as D-CPPene in inhibiting potassium-induced increases in extracellular levels of aspartate and glutamate. In control experiments, two 100 m M KCI stimuli (S1 and S2) applied for 10 min each (separated by a 40-min recovery period) produced substantial (300–500%) increases in the extracellular levels of aspartate, glutamate, taurine, and GABA and a 50% decrease in the glutamine level. S2/S1 ratios in the control groups were 0.67 (aspartate), 0.78 (glutamate), 0.83 (GABA), and 0.85 (taurine). In the experimental groups, D- or L-CPPene was applied via the probe during the second potassium stimulus (S2). L-CPPene (25 or 250 μ M ) produced selective suppression of potassium-induced increases of extracellular glutamate (S2/S1 ratio: 0.25) and aspartate (S2/S1 ratio: 0.20) levels, whereas 250 μ M D-CPPene was required to inhibit the extracellular aspartate and glutamate increases. Neither enantiomer of CPPene affected the potassium-induced increases of GABA and taurine or the decrease in extracellular glutamine concentration. An addtional study comparing the anticonvulsant potencies of D- and L-CPPene was performed using audiogenic DBA/2 mice. The anticonvulsant potency of D-CPPene, as assessed against sound-induced seizures in DBA/2 mice, was an order of magnitude higher than that of L-CPPene [ED₅₀ clonic phase (intraperitoneal, 45 min): 1.64 μmol/kg and 16.8 μmol/kg, respectively]. We attribute the anticonvulsant action of D-CPPene to its antagonist action at the NMDA receptor. The selective inhibition by L-CPPene of potassium-induced increases in extracellular aspartate and glutamate levels is presumably due to an action on presynaptic glutamate receptors.     

NAAG peptidase inhibitor increases dialysate NAAG and reduces glutamate, aspartate and GABA levels in the dorsal hippocampus following fluid percussion injury in the rat

Traumatic brain injury (TBI) produces a rapid and excessive elevation in extracellular glutamate that induces excitotoxic brain cell death. The peptide neurotransmitter N-acetylaspartylglutamate (NAAG) is reported to suppress neurotransmitter release through selective activation of presynaptic group II metabotropic glutamate receptors. Therefore, strategies to elevate levels of NAAG following brain injury could reduce excessive glutamate release associated with TBI. We hypothesized that the NAAG peptidase inhibitor, ZJ-43 would elevate extracellular NAAG levels and reduce extracellular levels of amino acid neurotransmitters following TBI by a group II metabotropic glutamate receptor (mGluR)-mediated mechanism. Dialysate levels of NAAG, glutamate, aspartate and GABA from the dorsal hippocampus were elevated after TBI as measured by in vivo microdialysis. Dialysate levels of NAAG were higher and remained elevated in the ZJ-43 treated group (50 mg/kg, i.p.) compared with control. ZJ-43 treatment also reduced the rise of dialysate glutamate, aspartate, and GABA levels. Co-administration of the group II mGluR antagonist, LY341495 (1 mg/kg, i.p.) partially blocked the effects of ZJ-43 on dialysate glutamate and GABA, suggesting that NAAG effects are mediated through mGluR activation. The results are consistent with the hypothesis that inhibition of NAAG peptidase may reduce excitotoxic events associated with TBI.     

The importance of myocardial amino acids during ischemia and reperfusion in dilated left ventricle of patients with degenerative mitral valve disease

Taurine, glutamine, glutamate, aspartate, and alanine are the most abundant intracellular free amino acids in human heart. The myocardial concentration of these amino acids changes during ischemia and reperfusion due to alterations in metabolic and ionic homeostasis. We hypothesized that dilated left ventricle secondary to mitral valve disease has different levels of amino acids compared to the right ventricle and that such differences determine the extent of amino acids' changes during ischemia and reperfusion. Myocardial concentration of amino acids was measured in biopsies collected from left and right ventricles before cardioplegic arrest (Custodiol HTK) and 10 min after reperfusion in patients undergoing mitral valve surgery. The dilated left ventricle had markedly higher (P < 0.05) concentrations (nmol/mg wet weight) of taurine (17.0 ± 1.5 vs. 10.9 ± 1.5), glutamine (20.5 ± 2.4 vs. 12.1 ± 1.2), and glutamate (18.3 ± 2.2 vs. 11.4 ± 1.5) when compared to right ventricle. There were no differences in the basal levels of alanine or aspartate. Upon reperfusion, a significant (P < 0.05) fall in taurine and glutamine was seen only in the left ventricle. These changes are likely to be due to transport (taurine) and/or metabolism (glutamine). There was a marked increase in the alanine to glutamate ratio in both ventricles indicative of ischemic stress which was confirmed by global release of lactate during reperfusion. This study shows that in contrast to the right ventricle, the dilated left ventricle had remodeled to accumulate amino acids which are used during ischemia and reperfusion. Whether these changes reflect differences in degree of cardioplegic protection between the two ventricles remain to be investigated.     

Valproate Increases Glutaminase and Decreases Glutamine Synthetase Activities in Primary Cultures of Rat Brain Astrocytes

Abstract: It has been proposed that hyperammonemia may be associated with valproate therapy. As astrocytes are the primary site of ammonia detoxification in brain, the effects of valproate on glutamate and glutamine metabolism in astrocytes were studied. It is well established that, because of compartmentation of glutamine synthetase, astrocytes are the site of synthesis of glutamine from glutamate and ammonia. The reverse reaction is catalyzed by the ubiquitous enzyme glutaminase, which is present in both neurons and astrocytes. In astrocytes exposed to 1.2 mM valproate, glutaminase activity increased 80% by day 2 and remained elevated at day 4; glutamine synthetase activity was decreased 30%. Direct addition of valproate to assay tubes with enzyme extracts from untreated astrocytes had significant effects only at concentrations of 10 and 20 mM, When astrocytes were exposed for 4 days to 0.3, 0.6, or 1.2 mM valproate and subsequently incubated with l -[U-¹⁴C]glutamate, label incorporation into [¹⁴C]glutamine was decreased by 11, 25, and 48%, respectively, and is consistent with a reduction in glutamine synthetase activity. Label incorporation from l -[U-¹⁴C]glutamate into [¹⁴C]aspartate also decreased with increasing concentrations of valproate. Following a 4-day exposure to 0.6 mM valproate, the glutamine levels increased 40% and the glutamate levels 100%. These effects were not directly proportional to valproate concentration, because exposure to 1.2 mM valproate resulted in a 15% decrease in glutamine levels and a 25% increase in glutamate levels compared with control cultures. Intracellular aspartate was inversely proportional to all concentrations of extracellular valproate, decreasing 60% with exposure to 1.2 mM valproate. These results indicate that valproate increases glutaminase activity, decreases glutamine synthetase activity, and alters Krebs-cycle activity in astrocytes, suggesting a possible mechanism for hyperammonemia in brain during valproate therapy.     

重复经颅磁刺激对创伤性颅脑损伤患者脑脊液中兴奋性氨基酸 含量的影响

目的:探讨重复经颅磁刺激(rTMS)对急性颅脑损伤患者脑脊液中兴奋性氨基酸(EAA)含量的影响。方法:30例创伤性颅脑损伤(TBI)病人按格拉斯哥昏迷评分分为轻型组(rTMS3)、中型组(rTMS2)、重型组(rTMS1),每组10例,各组病人分别随机分为rTMS对照亚组(A组)及治疗亚组(B组),每亚组5例。于TBI后第15天行腰椎穿刺采集脑脊液,采用高效液相色谱法测定脑脊液中谷氨酸(ASP)及门冬氨酸(GLU)含量。结果:脑脊液ASP和GLU水平随着脑损伤程度的加重而升高,各rTMS治疗组与相应各对照组的EAA相比,rTMS治疗组EAA的水平均低于相应对照组。结论:rTMS可通过降低TBI后脑脊液EAA水平发挥脑保护作用。脑脊液EAA的含量变化可作为TBI严重程度的生化指标。     

Involvement of metabotropic glutamate receptors in excitatory amino acid and GABA release following spinal cord injury in rat

Spinal cord injury (SCI) leads to an increase in extracellular excitatory amino acid (EAA) concentrations resulting in glutamate receptor-mediated excitotoxic events. The glutamate receptors include ionotropic (iGluRs) and metabotropic (mGluR) receptors. Of the three groups of mGluRs, group-I activation can initiate intracellular pathways that lead to further transmitter release. Groups II and III mGluRs function mainly as autoreceptors to regulate neurotransmitter release. In an effort to examine the role of mGluRs in the increase in EAAs following SCI, we administered AIDA, a potent group-I mGluR antagonist immediately after injury. To determine subtype specific roles of the group-I mGluRs, we evaluated EAA release following LY 367385 (mGluR1 antagonist) and MPEP (mGluR5 antagonist) administration. To evaluate group-II and -III mGluRs we administered APDC (group-II agonist) and L-AP4 (group-III agonist) immediately following injury; additionally, we initiated treatment with CPPG (group-II/-III antagonist) and LY 341495 (group-II antagonist) 5 min prior to injury. Subjects were adult male Sprague-Dawley rats (225-250 g), impact injured at T10 with an NYU impactor (12.5 mm drop). Agents were injected into the epicenter of injury, amino acids where collected by microdialysis fibers inserted 0.5 mm caudal from the edge of the impact region and quantified by HPLC. Treatment with AIDA significantly decreased extracellular EAA and GABA concentrations. MPEP reduced EAA concentrations without affecting GABA. Combining LY 367385 and MPEP resulted in a decrease in EAA and GABA concentrations greater than either agent alone. L-AP4 decreased EAA levels, while treatment with LY 341495 increased EAA levels. These results suggest that mGluRs play an important role in EAA toxicity following SCI.     

Exogenous Glutamate Inhibits the Root Growth and Increases the Glutamine Content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Previously, our work with ginseng hairy root shows that the tissue of low-branching and slow-growing phenotype contains high level of glutamine. In order to check if the high glutamine concentration inhibits the root growth, we applied exogenous glutamine or glutamate into growth medium and check the root growth of Arabidopsis. While glutamine did not affect root growth, over 0.1 mM glutamate inhibited severe root growth. However, when the amino acid solution was adjusted to pH 5.7 and added into medium, Arabidopsis seedlings show normal growth pattern on medium containing glutamate or aspartate. These results demonstrated that inhibition of the root growth by high concentration of exogenous glutamate was a result of the low pH toxicity caused by acidic amino acid, although low concentration (0.05 mM) of glutamate has an inhibitory effect on the primary root growth. The application of exogenous glutamine or glutamate increases glutamine concentration within root tissue about 3- to 4-fold. However, concentration of glutamate is not significantly increased. The KO mutant on each of the Gln1_1, Gln1_2, or Glu2 gene was little effective on the root growth. These results indicate that high concentration of endogenous glutamine observed in root tissue does not affect root growth.     

Low Temperature Effects on Soybean (Glycine max [L.] Merr. cv. Wells) Free Amino Acid Pools during Germination

The free amino acid concentrations in cotyledons and axes of soybean (Glycine max [L.] Merr. cv. Wells) seedlings were determined by automated single column analysis after germination at 10 and 23 C. After 5 days germination at 10 C, glutamate and aspartate were in high concentration in both cotyledons and axes (38 and 24% of total free amino acids recovered, respectively), whereas the concentrations of their amide derivatives, asparagine and glutamine, were low in cotyledons (4.4%) and high in axes (21%). In contrast, after 5 days germination at 23 C, asparagine and glutamine accounted for 22 and 45% of total free amino acids in cotyledons and axes respectively, and aspartate and glutamate concentrations were low. The activities of glutamine synthetase and asparagine synthetase were considerably lower in tissues from the 10 C treatment than those from the 23 C treatment.

Aspartate and glutamate concentrations were nearly equal in all but one sample. Both glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were much higher in axis tissues at 23 C as compared to 10 C. Arrhenius plots of axis glutamate oxaloacetate transaminase and glutamate dehydrogenase activities were biphasic and triphasic, respectively, with energies of activation for both increasing with low temperature. Energies of activation were identical for glutamate oxaloacetate transaminase from 10 and 23 C treatments but much higher for glutamate dehydrogenase from 23 C-treated axes. This indicates a difference in enzyme complement for glutamate dehydrogenase with the two treatments.

Hydrolysis of free amino acid sample (basic fraction) aliquots showed large quantities of peptides in 23 C-treated axes at 2 days, while few or no peptides were found in the 10 C treatment. Amino acid residues most prevalent in peptides were aspartate, threonine, serine, glutamate, and glycine.

     

Reduced Cortical Glutamine Concentrations in Electrically Kindled Rats

The effect of kindling rats (with an electrical stimulus applied daily to the frontal cortex) on the concentrations of taurine, aspartate, glutamate, glutamine, and gamma-aminobutyric acid has been investigated. When compared with control groups, cortical glutamine concentrations were significantly decreased in kindled rats by approximately 20%. This decrease in glutamine directly correlated with the after-discharge duration (r = 0.84, p = 0.005). The significance of this in relation to glutamate metabolism and kindling is discussed here.     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

Physiological Elevations of Glucocorticoids Potentiate Glutamate Accumulation in the Hippocampus

Abstract: Glucocorticoids (GCs) are secreted during stress and can damage the hippocampus over the course of aging and impair the capacity of hippocampal neurons to survive excitotoxic insults. Using microdialysis, we have previously observed that GCs augment the extracellular accumulation of glutamate and aspartate in the hippocampus following kainic acid-induced seizures. In that study, adrenalectomized rats maintained on minimal GC concentrations were compared with those exposed to GCs elevated to near-pharmacological levels. We wished to gain insight into the physiological relevance of these observations. Thus, we have examined the effects of GCs over the normal physiological range on glutamate and aspartate profiles; this was done by implanting adrenalectomized rats with GC-secreting pellets, which produce stable and controllable circulating GC concentrations. We observe that incremental increases in GC concentrations cause incremental increases in glutamate accumulation before the kainic acid insult, as well as in the magnitude of the glutamate response to kainic acid. Elevating GC concentrations from the circadian trough to peak doubled cumulative glutamate accumulation, whereas a rise into the stress range caused a fourfold increase in accumulation. Similar, although smaller, effects also occurred with aspartate accumulation (as well as with taurine but not glutamine accumulation). These data show that the highly elevated GC concentrations that accompany neurological insults such as seizure or hypoxia-ischemia will greatly exacerbate the glutamate accumulation at that time. Furthermore, stress levels of GCs augmented glutamate accumulation even in the absence of an excitotoxic insult, perhaps explaining how sustained stress itself damages the hippocampus. Finally, even the moderately ?levated basal GC concentrations that typically occur in aged rats augmented glutamate accumulation, perhaps explaining how GCs damage the hippocampus over the course of normal aging.     

Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver

Tracer quantities (in 0.2 ml) of 13N-labeled glutamate, alanine, or glutamine(amide) were administered rapidly (less than or equal to 2 s) via the portal vein of anesthetized adult male rats. Liver content of tracer at 5 s was 57 +/- 6 (n = 6), 24 +/- 1 (n = 3), and 69 +/- 7 (n = 3)% of the injected dose, respectively. Portal-hepatic vein differences for the corresponding amino acids were 17 +/- 6, 26 +/- 8, and 19 +/- 9% (n = 4), respectively, suggesting some export of glutamate and glutamine, but not of alanine, to the hepatic vein. Following L-[13N]glutamate administration, label rapidly appeared in liver alanine and aspartate (within seconds). The data emphasize the rapidity of nitrogen exchange via linked transaminases. By 30 s following administration of either L-[13N]glutamate or L-[13N]alanine, label in liver glutamate was comparable; yet, by 1 min greater than or equal to 9 times as much label was present in liver glutamine(amine) following L-[13N]glutamate administration than following L-[13N]alanine administration. Conversely, label in liver urea at 1 min was more pronounced in the latter case despite: (a) comparable total pool sizes of glutamate and alanine in liver; and (b) label incorporation from alanine into urea must occur via prior transfer of alanine nitrogen to glutamate. The data provide evidence for zonal differences in uptake of alanine and glutamate from the portal vein in vivo. The rate of turnover of L-[amide-13N]glutamine was considerably slower than that of L-[13N]alanine or of L-[13N]glutamate, presumably due in part to the higher concentration of glutamine in that organ. Nevertheless, it was possible to show that despite occasional suggestions to the contrary, glutamine(amide) is a source of urea nitrogen in vivo. The present findings continue to emphasize the rapidity of nitrogen exchange reactions in vivo.     

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.