Mass Fragmentographic Determination of γ-Aminobutyric Acid and Glutamic Acid in Discrete Amygdaloid Nuclei of Rat Brain

A mass fragmentographic method for the simultaneous quantification of gamma-aminobutyric acid (GABA) and glutamic acid is described. In a convenient one-step reaction, the two amino acids were derivatized with pentafluoropropionic anhydride and pentafluoropropanol. The derivatization products were stable for several days. The technique has been applied to the assay of GABA and Glu in five amygdaloid nuclei of the rat brain. The GABA level was high in the central and medial nuclei, whereas the Glu level was high in the lateral and basal nuclei. The regional distribution of GABA was different from that of Glu within the amygdaloid nuclei.     

The Relationship Between Plasma and Brain Quinolinic Acid Levels and the Severity of Hepatic Encephalopathy in Animal Models of Fulminant Hepatic Failure

Abstract: Quinolinic acid is an excitatory, neurotoxic tryptophan metabolite proposed to play a role in the pathogenesis of hepatic encephalopathy. This involvement was investigated in rat and rabbit models of fulminant hepatic failure at different stages of hepatic encephalopathy. Although plasma and brain tryptophan levels were significantly increased in all stages of hepatic encephalopathy, quinolinic acid levels increased three- to sevenfold only in the plasma, CSF, and brain regions of animals in stage IV hepatic encephalopathy. Plasma-CSF and plasma-brain quinolinic acid levels in rats and rabbits with fulminant hepatic failure were strongly correlated, with CSF and brain concentrations ∼10% those of plasma levels. Moreover, there was no significant regional difference in brain quinolinic acid concentrations in either model. Extrahepatic indoleamine-2,3-dioxygenase activity was not altered in rats in stage IV hepatic encephalopathy, but hepatic l -tryptophan-2,3-dioxygenase activity was increased. These results suggest that quinolinic acid synthesized in the liver enters the plasma and then accumulates in the CNS after crossing a permeabilized blood-brain barrier in the end stages of liver failure. Furthermore, the observation of low brain concentrations of quinolinic acid only in stage IV encephalopathy suggests that the contribution of quinolinic acid to the pathogenesis of hepatic encephalopathy in these animal models is minor.     

Content of Quinolinic Acid and of Other Tryptophan Metabolites Increases in Brain Regions of Rats Used as Experimental Models of Hepatic Encephalopathy

The content of the tryptophan metabolites quinolinic acid (QUIN), 5-hydroxytryptamine (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) was measured in various brain areas of rats bearing a portocaval anastomosis (PCA) for 4 weeks, using mass fragmentography or HPLC. In these animals, the content of the excitotoxic compound QUIN increased by 75% in the cortex and 125% in the cerebellum. The content of 5-HT increased by 27% in the brainstem. No changes occurred in other brain areas. On the other hand, the content of 5-HIAA increased by 66% in the cortex, 65% in the caudate, 64% in the hippocampus, 120% in the diencephalon, and 185% in the brainstem. Probenecid administration caused a larger increase of 5-HIAA accumulation in various brain areas of PCA-bearing rats than in those of sham-operated controls. The cortical content of QUIN and 5-HIAA increased after administration of ammonium acetate (7 mmol/kg), whereas an equimolar amount of sodium acetate was inactive. These results confirm that profound changes in the disposition of tryptophan occur in the brains of experimental animals used as models of hepatic encephalopathy. Furthermore, this study adds the excitotoxic compound QUIN to the list of molecules possibly involved in the pathogenesis of this brain disorder.     

Electrical Stimulation of the Stratum Radiatum Increases the Release and Neosynthesis of Aspartate, Glutamate, and γ-Aminobutyric Acid in Rat Hippocampal Slices

The release of endogenous aspartic, glutamic, and gamma-aminobutyric acids (Asp, Glu, GABA, respectively) was measured in the effluent from superfused hippocampal slices using a new and sensitive mass spectrometric method. The stimulation of the stratum radiatum of the rat dorsal hippocampus caused a Ca2+-dependent increase in the release of these amino acids. This release was accompanied by an increase in the incorporation of [13C2] from [13C]glucose into Asp, Glu, and GABA, suggesting an increase in their neosynthesis. The removal of Ca2+ from the superfusion fluid brought about a marked decrease in Asp and Glu release at rest, and prevented their stimulation-evoked release and the appearance of population spikes. The results support the hypothesis that Asp and Glu are excitatory neurotransmitters in intrinsic hippocampal circuits and are possibly released from the Schaffer collaterals and commissural fibres. The increase in GABA release and neosynthesis during stimulation of the stratum radiatum could be related to recurrent inhibition evoked by transsynaptic stimulation of the pyramidal cells.     

Selective Stimulation of Neurotransmitter Release from Chick Retina by Kainic and Glutamic Acids

The excitatory action of kainic and glutamic acids in chick whole retina was demonstrated as an immediate stimulation of the release of labeled gamma-aminobutyric acid (GABA) and glycine in a superfusion system. This stimulatory effect was 3-10 times greater than that produced by a depolarizing K+ concentration; in addition, it was independent of Ca2+ in the medium, but notably inhibited when Na+ was omitted from the medium. Under identical experimental conditions, neither kainic nor glutamic acid had any effect on the release of labeled dopamine or alpha-aminoisobutyric acid, thus indicating that their effect is not unspecific or due to cell damage. Similar although less marked stimulation of labeled GABA and glycine release by kainic acid was obtained in subcellular retinal fractions, particularly in fraction P1, which contained photoreceptor terminals and outer segments. This stimulation was also Ca2+ independent and greatly reduced when Na+ was omitted from the medium. It is suggested that the stimulation of GABA release by kainic and glutamic acids is probably due to a Na+-dependent, carrier-mediated mechanism that responds to the entry of Na+ produced by the interaction of glutamic and kainic acids with retinal membranes. In cortical or striatal slices from mouse brain, these acids had a negligible stimulatory effect on GABA and dopamine release.     

氨基酸代谢与肝性脑病

肝性脑病 (HepaticEncephalopathy)又称肝昏迷 ,即由于严重肝病引起的中枢神经系统功能紊乱 ,患者出现一系列神经精神病状 ,直至进入昏迷。在此仅从氨基酸代谢异常的角度叙述与肝性脑病的关系。     

Enhanced Potassium-Stimulated γ-Aminobutyric Acid Release by Astrocytes Derived from Rats with Early Hepatogenic Encephalopathy

Bulk-isolated astrocytes from rats with early hepatogenic encephalopathy (HE) induced with thioacetamide responded to the increase of potassium in the incubation medium from 5 mM to 75 mM with a markedly enhanced release of previously taken up [14C]gamma-aminobutyric acid ([14C]GABA). The process was not affected by omission of calcium and/or addition of EGTA to the incubation medium. Only a slight stimulation of GABA release by high potassium was observed in astrocytes from control rats. In contrast, histamine and histidine were vigorously released from control astrocytes in high-potassium medium, and their release was not enhanced by HE, indicating that the observed phenomenon is specific for GABA.     

肝性脑病患者脑脊液Tau 蛋白水平测定及其临床意义

目的:探讨肝性脑病患者脑脊液中总Tau蛋白(t-Tau)和磷酸化Tau蛋白(p-Tau)水平的变化及其与疾病严重程度的相关性。方法:采集26例肝性脑病患者及31例健康对照的脑脊液标本,采用酶联免疫吸附法(Elisa)检测脑脊液中的t-Tau和p-Tau水平,分析其与Child-Pugh评分和West Haven分级的相关性。结果:(1)肝性脑病患者脑脊液t-Tau和p-Tau水平显著高于健康对照组(P0.01)。(2)肝性脑病患者脑脊液t-Tau(γ=0.876,P0.01;γ=0.952,P0.01)、p-Tau(γ=0.808,P0.01;γ=0.808,P0.01)水平与Child-Pugh评分及West-Haven分级呈正相关。结论:由于脑脊液t-Tau和p-Tau水平为神经元损害的标志物,同时反应脑内应激状况,本研究证实肝性脑病患者脑内处于应激状态并有神经元损伤。     

Glutamic Acid as a Putative Transmitter of the Interhemispheric Corticocortical Connections in the Rat

The effect of hemidecortication on the endogenous levels of amino acids in medial, sulcal, and dorsal frontal cortex as well as in parietal, temporal, and occipital cortex of the rat was investigated. Under aseptic conditions, the right cerebral cortex was aspirated by suction. Then, 21 days later, the content of glutamic acid, aspartic acid, gamma-aminobutyric acid, glycine, serine, threonine, and alanine was analyzed in six areas of the intact contralateral cortex using GLC. The results demonstrated a specific decrease in the endogenous levels of glutamic acid in both parietal and temporal cortex after hemidecortication of the contralateral side. This finding suggests that glutamic acid may serve as a neurotransmitter for some of the interhemispheric corticoparietal and corticotemporal fibers. In a follow-up experiment, the effect of a frontal lesion on the endogenous levels of the same amino acids in the striatum was also examined. In this case, the glutamic acid content exhibited a decrease of 31% relative to the control value. This observation confirms the earlier finding of a glutamate-containing pathway from the frontal cortex to the striatum.     

Mass Fragmentographic Method for the Determination of Trace Amounts of Putative Amino Acid Neurotransmitters and Related Compounds from Brain Perfusates Collected In Vivo

Abstract: A mass fragmentographic method for the determination of trace amounts of amino acid neurotransmitter candidates from brain perfusates is described. The analytical procedure includes the measurements of glycine, β-alanine, γ-aminobutyric acid, proline, aspartic acid, and glutamic acid; αalanine, leucine, and sarcosine, undergoing gas chromatographic coelution, are detected simultaneously. Amino acids extracted from dried perfusate residues are converted to the corresponding N -pentafluoropropionyl hexafluoroisopropyl esters by a single-step procedure. Gas chromatographic separation of the amino acid derivatives is achieved on a packed glass column filled with trifluoropropylsilicone as stationary phase. The limit of detection for the different derivatives (signal-to-noise, 3:1) ranges from 50 femtomol to 1 picomol. Deuterium-labeled amino acid analogues are used as internal standards for quantitative measurements. The mass spectral characteristics of the derivatives are compared and discussed. The technique has been applied to the assay of amino acids released in vivo within the pigeon optic tectum, demonstrating the capabilities of the present analytical approach.     

Metabolism and Brain Uptake of γ-Aminobutyric Acid in Galactosamine-Induced Hepatic Encephalopathy in Rats

Abstract: Kinetic studies of [³H]γ-aminobutyric acid ([³H]GABA) after an intravenous injection were performed in normal rats and in rats with severe degree of hepatic encephalopathy due to fulminant hepatic failure induced by galactosamine. Moreover, plasma and brain GABA levels, and GABA and glutamic acid decarboxylase activity were studied in some brain areas. After intravenous injection, [3H]GABA disappeared very rapidly in the blood of normal rats, with a prompt increase of ³H metabolites. In comatose rats, a delayed disappearance of [3H]GABA.as parallelled by a lower amount of metabolites, indirectly indicating a peripheral decrease of GABA-transaminase activity. The amount of [³H]GABA in brain was lightly but constantly lower in comatose rats than in controls, indicating that the change in permeability of the blood-brain barrier in hepatic encephalopathy does not affect the [3H]GABA uptake of the brain. Furthermore, the assay of endogenous GABA in blood, whole brain, and brain areas did not show any significant difference in any of the two groups. The finding that glutamic acid decarboxylase activity in brain was reduced, together with the indirect evidence of a reduction in GABA-transaminase, may account for the steady state of GABA in hepatic encephalopathy. However, the reduction in glutamic acid decarboxylase activity is in favor of a functional derangement at the GABA-ergic nerve terminals in this pathological condition.     

Endogenous Benzodiazepine Receptor Ligands in Human and Animal Hepatic Encephalopathy

The role of endogenous benzodiazepine receptor ligands in the pathogenesis of hepatic encephalopathy was studied in humans and in rat models of hepatic encephalopathy. Endogenous benzodiazepine ligands were extracted from rat brain and human CSF by acid treatment and purification by HPLC. Detection and partial characterization of these endogenous benzodiazepine ligands were carried out using both radioreceptor binding assays and radioimmunoassays with anti-benzodiazepine antibodies. Four different benzodiazepine receptor ligands were identified in human and rat tissue, two of which may be diazepam and desmethyldiazepam, based on elution profiles and anti-benzo-diazepine antibody reactivity. Human CSF and serum from patients with hepatic encephalopathy contained approximately 10 times more endogenous benzodiazepine receptor ligand than CSF from controls or nonencephalopathic patients with liver disease. The levels of brain benzodiazepine receptor ligand compounds were also increased approximately 10-fold in rats suffering from fulminant hepatic failure, but not in rats with portacaval shunts, a model of chronic hepatic disease. The increased concentrations of these substances could be behaviorally significant and may contribute to the pathogenesis of hepatic encephalopathy.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

Release of [3H]Dopamine from Striatal and Cerebral Cortical Slices from Rats with Thioacetamide-Induced Hepatic Encephalopathy: Different Responses to Stimulation by Potassium Ions and Agonists of Ionotropic Glutamate Receptors

The effects of depolarizing stimuli; high (50 mM) potassium ions and the glutamate receptor agonists N-methyl-D-aspartate, kainate and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) on the release of newly-loaded [³H]dopamine were studied in frontal cortical and striatal slices from control rats and from rats with acute hepatic encephalopathy induced with a hepatotoxin, thioacetamide. Hepatic encephalopathy enhanced the stimulatory effect of potassium ions by 20% in striatal slices and by 34% in frontal cortical slices. In striatal slices the stimulatory effects of N-methyl-D-aspartate and kainate were depressed in hepatic encephalopathy by 46% and 21%, respectively, which may be taken to reflect impaired modulation of striatal dopamine release by glutamate acting at N-methyl-D-aspartate or kainate receptors. In frontal cortical slices, the stimulatory effect of kainate was enhanced by 35% in hepatic encephalopathy but N-methyl-D-aspartate-stimulated release was not affected. The release evoked by 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate was not affected in hepatic encephalopathy in either brain region. Stimulation of dopamine release in the frontal cortex by depolarization or glutamate acting at kainate receptors could inhibit the activity of descending corticostriatal glutamatergic pathways, further impairing regulation of dopamine release by glutamate in the stratum.     

兔实验性肝性脑病1H磁共振波谱初步研究

目的:研究兔实验性肝性脑病1H磁共振波谱(magnetic resonance spectroscopy,MRS)变化。方法:将24只兔子随机分三组:对照组,肝硬化组,肝性脑病组,各8只。肝性脑病组采用四氯化碳(CCl4)联合内毒素方法制作肝性脑病兔子模型,肝硬化组采用CCl4制作肝硬化模型。分别在第4、6、8、10、12周取肝脏病理活检,第12周测量血氨值,并进行兔子脑组织的MRS扫描。计算N-乙酰天门冬氨酸(N-acetyl asparte,NAA)、肌酸(creatine,Cr)、胆碱(choline,Cho)、肌醇(myo-inositol,mI)和谷氨酰胺复合物(glutamine and glutamate,Glx)的峰下面积,计算NAA/Cr、Cho/Cr、mI/Cr、Glx/Cr。结果:与对照组及肝硬化组相比,肝性脑病组兔血氨上升,脑部MRS显示Glx/Cr升高,Cho/Cr降低,差异显著(P0.05)。与对照组相比,肝硬化组血氨以及MRS改变无统计学意义。结论:兔实验性肝性脑病1H磁共振波谱存在变化。     

大鼠脑谷氨酸脱羧酶基因的cDNA克隆及序列分析

胰岛β－细胞自身抗原蛋白之一是脑中谷氨酸脱羧酶（Ｇｌｕｔａｍｉｃａｃｉｄｄｅｃａｒｂｏｘｙｌａｓｅ,ＧＡＤ,ＥＣ４．１．１．１５）同源物。以双链ｃＤＮＡ为模板,用ＰＣＲ方法快速克隆了Ｗｉｓｔａｒ大鼠脑ＧＡＤ基因的ｃＤＮＡ,将此包括编码５９３个氨基酸的全长ＤＮＡ片段重组入ｐＵＣ质粒并用双脱氧末端终止法测定了全部序列,证明其全长为１７７９ｂｐ．经比较发现Ｗｉｓｔａｒ大鼠脑与Ｒｕｓｓｅｌｌ报导的大鼠脑ＧＡＤ基因序列,有一处碱基的差别,但并不涉及氨基酸的改变。同时还对用ＰＣＲ扩增长片段ＤＮＡ进行了方法学上的探讨。     

Glutamate Dehydrogenase Reaction as a Source of Glutamic Acid in Synaptosomes

The role of the glutamate dehydrogenase reaction as a pathway of glutamate synthesis was studied by incubating synaptosomes with 5 mM 15NH4Cl and then utilizing gas chromatography-mass spectrometry to measure isotopic enrichment in glutamate and aspartate. The rate of formation of [15N]glutamate and [15N]aspartate from 5 mM 15NH4Cl was approximately 0.2 nmol/min/mg of protein, a value much less than flux through glutaminase (4.8 nmol/min/mg of protein) but greater than flux through glutamine synthetase (0.045 nmol/min/mg of protein). Addition of 1 mM 2-oxoglutarate to the medium did not affect the rate of [15N]glutamate formation. O2 consumption and lactate formation were increased in the presence of 5 mM NH3, whereas the intrasynaptosomal concentrations of glutamate and aspartate were unaffected. Treatment of synaptosomes with veratridine stimulated reductive amination of 2-oxoglutarate during the early time points. The production of ([15N]glutamate + [15N]aspartate) was enhanced about twofold in the presence of 5 mM beta-(+/-)-2-aminobicyclo [2.2.1]heptane-2-carboxylic acid, a known effector of glutamate dehydrogenase. Supplementation of the incubation medium with a mixture of unlabelled amino acids at concentrations similar to those present in the extracellular fluid of the brain had little effect on the intrasynaptosomal [glutamate] and [aspartate]. However, the enrichment in these amino acids was consistently greater in the presence of supplementary amino acids, which appeared to stimulate modestly the reductive amination of 2-oxoglutarate. It is concluded: (a) compared with the phosphate-dependent glutaminase reaction, reductive amination is a relatively minor pathway of synaptosomal glutamate synthesis in both the basal state and during depolarization; (b) NH3 toxicity, at least in synaptosomes, is not referable to energy failure caused by a depletion of 2-oxoglutarate in the glutamate dehydrogenase reaction; and (c) transamination is not a major mechanism of glutamate nitrogen production in nerve endings.     

Amino Acid Release from Cerebral Cortex in Experimental Acute Liver Failure, Studied by In Vivo Cerebral Cortex Microdialysis

Both increased gamma-aminobutyric acid (GABA)-ergic and decreased glutamatergic neurotransmission have been suggested relative to the pathophysiology of hepatic encephalopathy. This proposed disturbance in neurotransmitter balance, however, is based mainly on brain tissue analysis. Because the approach of whole tissue analysis is of limited value with regard to in vivo neurotransmission, we have studied the extracellular concentrations in the cerebral cortex of several neuroactive amino acids by application of the in vivo microdialysis technique. During acute hepatic encephalopathy induced in rats by complete liver ischemia, increased extracellular concentrations of the neuroactive amino acids glutamate, taurine, and glycine were observed, whereas extracellular concentrations of aspartate and GABA were unaltered and glutamine decreased. It is therefore suggested that hepatic encephalopathy is associated with glycine potentiated glutamate neurotoxicity rather than with a shortage of the neurotransmitter glutamate. In addition, increased extracellular concentration of taurine might contribute to the disturbed neurotransmitter balance. The observation of decreasing glutamine concentrations, after an initial increase, points to a possible astrocytic dysfunction involved in the pathophysiology of hepatic encephalopathy.     

Capsaicin Does Not Change Tissue Levels of Glutamic Acid, Its Uptake, or Release in the Rat Spinal Cord

Capsaicin treatment (50 mg/kg, subcutaneous) of newborn rats resulted in 1 75% decrease of substance P immunoreactivity in the dorsal spinal cord of the adult animal, but failed to affect levels of the proposed sensory neurotransmitter glutamic acid or to alter high-affinity uptake of [3H]glutamic acid into synaptosomes of the same tissue. Furthermore, capsaicin (30 microM) in vitro had no influence on the release of [3H]glutamic acid from spinal cord P2 fractions of untreated adult rats, but induced a marked release of substance P. The results suggest that, in contrast to substance P fibers, neurons containing glutamic acid are not sensitive to capsaicin. Eleven other neurochemical parameters measured in the spinal cord did not appear to be changed by the treatment with capsaicin, suggesting a considerable neurochemical selectivity of the lesion.