单侧迷路破坏后大鼠前庭神经内侧核区氨基酸含量的变化

本实验用脑部微量透析法和高效液相色谱法观察单侧迷路破坏(unilateral labyrinthectomy,经利多卡因或对氨基苯胂酸盐预处理以阻断单侧外周前庭器官)后大鼠同侧及对侧前庭神经内侧核(medial vestibular nucleus,MVN)区部分氨基酸(天冬氨酸、谷氨酸、谷氨酰胺、甘氨酸、牛磺酸和丙氨酸)含量的变化,以了解前庭代偿的部分神经化学机制.实验观察到,对照组大鼠MVN区天冬氨酸、谷氨酸、谷氨酰胺、甘氨酸、牛磺酸和丙氨酸浓度分别为(6.15±0.59),(18.13±1.21),(33.73±1.67),(9.26±0.65),(9.56±0.77)和(10.07±0.83)pmol/8 μL透析样本.左侧中耳内灌注2%利多卡因后10 min,同侧MVN区天冬氨酸、谷氨酸含量立即减少(P＜0.05),牛磺酸含量增加(P＜0.05);对侧MVN区谷氨酸含量立即增加(P＜0.05),甘氨酸和丙氨酸含量减少;双侧核团间谷氨酸、甘氨酸和丙氨酸含量失衡.而用对氨基苯胂酸盐永久阻断单侧前庭器官2周后,同侧MVN区谷氨酸和丙氨酸含量减少,谷氨酰胺含量增高;对侧MVN区谷氨酸含量也减少;同侧MVN区谷氨酰胺含量明显高于对侧MVN区.结果提示,单侧迷路破坏后双侧MVN区氨基酸含量立即失去平衡,随着前庭代偿的进展,此差异减少,但是在前庭代偿后却出现双侧前庭核区谷氨酰氨的含量失衡,说明在前庭代偿过程中氨基酸含量变化起着重要作用.     

Effect of Ouabain Applied by Intrastriatal Microdialysis on the In Vivo Release of Dopamine, Acetylcholine, and Amino Acids in the Brain of Conscious Rats

In the present study we have applied a brain microdialysis technique to investigate the effects of ouabain infusion on the release of dopamine, acetylcholine, and amino acids from striatal neurons in freely moving rats. Ouabain caused an increase in the dialysate levels of dopamine; its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC); and the amino acids glutamate, aspartate, taurine, glycine, alanine, serine, asparagine, and threonine. The ouabain-induced increase in dopamine was dose dependent and explosive (100-fold at an infusion concentration of 1 mmol/L) and contrasted strongly with the small effect of the glycoside on the output of DOPAC. We investigated the nature of ouabain-induced transmitter release by determining its sensitivity to coinfusion with tetrodotoxin or the calcium antagonist Mg2+. In the case of dopamine two mechanisms of ouabain-induced release could be established. At lower infusion concentrations ouabain induced an exocytotic type of release whereas at higher concentrations the release was probably carrier mediated. In the case of amino acids we noticed a calcium-independent release which was nerve impulse flow dependent in the case of glutamate and aspartate and impulse flow independent in the case of alanine, serine, glycine, threonine, and asparagine. Ouabain induced a decrease in the release of acetylcholine and glutamine.     

Depolarization-Induced Release of Amino Acids From the Vestibular Nuclear Complex

There is evidence from immunohistochemistry, quantitative microchemistry, and pharmacology for several amino acids as neurotransmitters in the vestibular nuclear complex (VNC), including glutamate, γ-aminobutyrate (GABA), and glycine. However, evidence from measurements of release has been limited. The purpose of this study was to measure depolarization-stimulated calcium-dependent release of amino acids from the VNC in brain slices. Coronal slices containing predominantly the VNC were prepared from rats and perfused with artificial cerebrospinal fluid (ACSF) in an interface chamber. Fluid was collected from the chamber just downstream from the VNC using a microsiphon. Depolarization was induced by 50 mM potassium in either control calcium and magnesium concentrations or reduced calcium and elevated magnesium. Amino acid concentrations in effluent fluid were measured by high performance liquid chromatography. Glutamate release increased fivefold during depolarization in control calcium concentration and twofold in low calcium/high magnesium. These same ratios were 6 and 1.5 for GABA, 2 and 1.3 for glycine, and 2 and 1.5 for aspartate. Differences between release in control and low calcium/high magnesium ACSF were statistically significant for glutamate, GABA, and glycine. Glutamine release decreased during and after depolarization, and taurine release slowly increased. No evidence for calcium-dependent release was found for serine, glutamine, alanine, threonine, arginine, taurine, or tyrosine. Our results support glutamate and GABA as major neurotransmitters in the VNC. They also support glycine as a neurotransmitter and some function for taurine.     

Regional changes in amino acid levels of the neonate rat brain during anoxia and recovery

The aim of this study was to compare the changes in amino acids (alanine, aspartate, GABA, glutamate, glutamine, glycine, serine taurine) that are produced in different regions of the neonate brain (telencephalon, diencephalon cerebellum, brain stem) following a survivable period of anoxia and after the re-establishment of air respiration. Anoxia provoked different responses in the different regions. The changes during the anoxic period were as follows. In the brain stem there was a decrease in aspartate, in the telencephalon there was a significant increase in GABA and alanine and a decrease in aspartate, in the diencephalon, glutamate and GABA increased, and in the cerebellum, glycine and alanine levels were enhanced. The changes during recovery were even more dissimilar. Here the greatest shifts were seen in the brain stem with increases in glutamine, GABA, aspartate, glycine, serine, alanine, and taurine. In the telencephalon glutamate fell and alanine increased, in the diencephalon GABA increased, and in the cerebellum, glutamate fell while glycine and alanine increased. In none of the major brain regions did the pattern of changes in neurotransmitters correspond to that seen in anoxic tolerant species.     

Immunocytochemical and circadian biochemical analysis of neuroactive amino acids in the pineal gland of the rat: effect of superior cervical ganglionectomy

Summary Semiquantitative immunocytochemistry by immuno-gold techniques revealed differences in the spatial distribution of glutamate, glutamine, and taurine within the pineal gland, with greatest labeling over pinealocytes, glia, and endothelia, respectively. At the subcellular level, glutamate labeling tended to be highest over pinealocyte synaptic ribbons and mitochondria, and lowest over lipid inclusions. Pineal levels of glutamate, glutamine and taurine, as measured by high performance liquid chromatography, did not vary over a light: dark cycle. Superior cervical sympathetic denervation, which abolishes pineal melatonin synthesis, resulted in a nearly 50% reduction in pineal glutamate levels, but had no effect on levels of glutamine and taurine. Other amino acids (alanine, arginine, aspartate, serine) were reduced by 23%–33% following sympathectomy. These data suggest an important role for glutamate in pinealocyte function(s) possibly related to the noradrenergic innervation of the gland.     

Release of Endogenous Amino Acids from the Striatum from Developing and Adult Mice in Ischemia

In most other studies the release of amino acid neurotransmitters and modulators in vitro has been studied mostly using labeled preloaded compounds. For several reasons the estimated release may not reliably reflect the release of endogenous compounds. The magnitudes of the release cannot thus be quite correctly estimated using radioactive labels. The basal and K⁺-evoked release of the neuroactive endogenous amino acids γ-aminobutyrate (GABA), glycine, taurine, glutamate and aspartate was now studied in slices from the striatum from 7-day-old to 3-month-old mice under control (normoxic) and ischemic conditions. The release of alanine, threonine and serine was assessed as control. GABA and glutamate release was much greater in 3-month-old than in 7-day-old mice, whereas with taurine the situation was the opposite. Ischemia markedly enhanced the release of all these three amino acids. The release of aspartate and glycine was markedly enhanced as well whereas no effects were discernible in the release of glutamine, alanine, serine and threonine. K⁺ stimulation (50 mM) enhanced the release of GABA, glutamate, taurine, aspartate and glycine in most cases, except with taurine in 3-month-old mice under the ischemic conditions and with aspartate in 7-day-old mice under the control conditions. K⁺ stimulation did not affect the release of glutamine, alanine, serine or threonine. The results on endogenous amino acids are qualitatively similar to those obtained in our earlier experiments with labeled preloaded amino acids. In conclusion, in developing mice only inhibitory taurine is released in such amounts that may counteract the harmful effects of excitatory amino acids in ischemia.     

Brain glycogen and its role in supporting glutamate and GABA homeostasis in a type 2 diabetes rat model

The number of people suffering from diabetes is hastily increasing and the condition is associated with altered brain glucose homeostasis. Brain glycogen is located in astrocytes and being a carbohydrate reservoir it contributes to glucose homeostasis. Furthermore, glycogen has been indicated to be important for proper neurotransmission under normal conditions. Previous findings from our laboratory suggested that glucose metabolism was reduced in type 2 diabetes, and thus we wanted to investigate more specifically how brain glycogen metabolism contributes to maintain energy status in the type 2 diabetic state. Also, our objective was to elucidate the contribution of glycogen to support neurotransmitter glutamate and GABA homeostasis. A glycogen phosphorylase (GP) inhibitor was administered to Sprague-Dawley (SprD) and Zucker Diabetic Fatty (ZDF) rats in vivo and after one day of treatment [1-13C]glucose was used to monitor metabolism. Brain levels of 13C labeling in glucose, lactate, alanine, glutamate, GABA, glutamine and aspartate were determined. Our results show that inhibition of brain glycogen metabolism reduced the amounts of glutamate in both the control and type 2 diabetes models. The reduction in glutamate was associated with a decrease in the pyruvate carboxylase/pyruvate dehydrogenase ratio in the control but not the type 2 diabetes model. In the type 2 diabetes model GABA levels were increased suggesting that brain glycogen serves a role in maintaining a proper ratio between excitatory and inhibitory neurotransmitters in type 2 diabetes. Both the control and the type 2 diabetic states had a compensatory increase in glucose-derived 13C processed through the TCA cycle following inhibition of glycogen degradation. Finally, it was indicated that the type 2 diabetes model might have an augmented necessity for compensatory upregulation at the glycolytic level.     

Pathways of glutamine metabolism in Spodoptera frugiperda (Sf9) insect cells: evidence for the presence of the nitrogen assimilation system, and a metabolic switch by 1H/15N NMR

1H/15N and 13C NMR were used to investigate metabolism in Spodoptera frugiperda (Sf9) cells. Labelled substrates ([2-15N]glutamine, [5-15N]glutamine, [2-15N]glutamate, 15NH4Cl, [2-15N]alanine, and [1-13C]glucose) were added to batch cultures and the concentration of labelled excreted metabolites (alanine, NH4+, glutamine, glycerol, and lactate) were quantified. Cultures with excess glucose and glutamine produce alanine as the main metabolic by-product while no ammonium ions are released. 1H/15N NMR data showed that both the amide and amine-nitrogen of glutamine was incorporated into alanine in these cultures. The amide-nitrogen of glutamine was not transferred to the amine-position in glutamate (for further transamination to alanine) via free NH4+ but directly via an azaserine inhibitable amido-transfer reaction. In glutamine-free media 15NH4+ was consumed and incorporated into alanine. 15NH4+ was also incorporated into the amide-position of glutamine synthesised by the cells. These data suggest that the nitrogen assimilation system, glutamine synthetase/glutamate synthase (NADH-GOGAT), is active in glutamine-deprived cells. In cultures devoid of glucose, ammonium is the main metabolic by-product while no alanine is formed. The ammonium ions stem both from the amide and amine-nitrogen of glutamine, most likely via glutaminase and glutamate dehydrogenase. 13C NMR revealed that the [1-13C] label from glucose appeared in glycerol, alanine, lactate, and in extracellular glutamine. Labelling data also showed that intermediates of the tricarboxylic acid cycle were recycled to glycolysis and that carbon sources, other than glucose-derived acetylCoA, entered the cycle. Furthermore, Sf9 cell cultures excreted significant amounts glycerol (1.9-3.2 mM) and ethanol (6 mM), thus highlighting the importance of sinks for reducing equivalents in maintaining the cytosolic redox balance.     

Effects of Methylprednisolone on Extracellular Lactic Acidosis and Amino Acids After Severe Compression Injury of Rat Spinal Cord

Abstract: We evaluated in rats with severe spinal cord compression at T8–9 the influence of methylprednisolone (MP) on lactic acidosis and extracellular amino acids, which may cause secondary, perifocal injuries of the cord. MP (30 mg/kg) was given intravenously 30 min before compression and hourly thereafter (15 mg/kg). Other rats with compression, given saline, served as controls. Samples from the extracellular fluid of one dorsal horn were collected by microdialysis and analyzed by HPLC. Microdialysis was performed for 1.5 h to establish basal levels. Samples were collected for 3 h after compression. MP-treated rats showed a reduction of dialysate lactic acid and arginine levels during the first 1–2 h after trauma. The mean dialysate levels of glutamate in MP-treated rats were lower than those of the controls, but the difference was not statistically significant. MP treatment did not influence dialysate levels of aspartate, glutamine, histidine, glycine, threonine, taurine, alanine, GABA, and tyrosine. Our study shows that MP has several effects, including reduced lactic acid formation, reduced levels of arginine (the substrate for nitric oxide production), and a trend toward decreased extracellular accumulation of the excitotoxic amino acid glutamate. We conclude that MP has the capacity to change the composition of the extracellular edema fluid after trauma to the spinal cord. These changes may counteract free radical formation and may be important mechanisms by which MP exerts its beneficial actions.     

Effect of the composition of branched chain amino acids, taurine, and tryptophan on the amino acid metabolism in experimental models of alcoholism

Ethanol withdrawal after forced alcoholization of rats according to Majchrowicz led to the development of amino acid imbalance in the pool of free amino acids in the liver (increasing levels of alanine, aspartate, glutamate, glutamine and histidine, decreasing levels of glycine, lysine, threonine and taurine) and blood plasma (increasing levels of tyrosine and alanine, decreasing levels of most glycogen aminoacids, branched-chain aminoacids and Lys). Less profound changes were observed after prolonged alcohol intoxication (decreasing levels of alanine, ornitine, citrulline and increasing level of Glu in liver, increasing levels of sulfur-containing compounds, Asp and Lys in blood plasma). Amino acid mixture which contained branched-chain amino acids, taurine and tryptophan administered intragastrically was found to correct levels of sulfur-containing amino acids, threonine, lysine and isoleucine after ethanol withdrawal and to eliminate disorders in urea cycle, exchange of threonine, glycine and phenylalanine after prolonged alcohol intoxication.     

Fetal hepatic and umbilical uptakes of glucogenic substrates during a glucagon-somatostatin infusion

To test the hypothesis that fetal hepatic glutamate output diverts the products of hepatic amino acid metabolism from hepatic gluconeogenesis, ovine fetal hepatic and umbilical uptakes of glucose and glucogenic substrates were measured before and during fetal glucagon-somatostatin (GS) infusion and during the combined infusion of GS, alanine, glutamine, and arginine. Before the infusions, hepatic uptake of lactate, alanine, glutamine, arginine, and other substrates was accompanied by hepatic output of pyruvate, aspartate, serine, glutamate, and ornithine. The GS infusion induced hepatic output of 1.00 +/- 0.07 mol glucose carbon/mol O(2) uptake, an equivalent reduction in hepatic output of pyruvate and glutamate carbon, a decrease in umbilical glucose uptake and placental uptake of fetal glutamate, an increase in hepatic alanine and arginine clearances, and a decrease in umbilical alanine, glutamine, and arginine uptakes. The latter result suggests that glucagon inhibits umbilical amino acid uptake. We conclude that fetal hepatic pyruvate and glutamate output is part of an adaptation to placental function that requires the fetal liver to maintain both a high rate of catabolism of glucogenic substrates and a low rate of gluconeogenesis.     

Studies on Amino Acid Metabolism in the Brain Using 15N-Labeled Precursors

The transfer of label from ¹⁵N-alanine and ¹⁵N-glutamate into amino acids in incubated brain slices has been followed using gas chromatography/mass spectrometry (GC/MS). ¹⁵N from alanine appeared in both amino and amide groups of glutamine more rapidly than into aspartate, glutamate and GABA, which were all labeled at similar rates. Maximum labelling of approx. 50% enrichment of these three metabolites was achieved in 3 hr. The ¹⁵N present in doubly-labeled glutamine exceeded that in the singly-labelled after 30 min. ¹⁵N from glutamate was rapidly transferred to aspartate and to alanine, with slower incorporation into glutamine and GABA. As was seen with labeling from alanine, doubly-labeled glutamine was higher than the singly-labeled species, also reaching some 50% enrichment in 3 hr. Depolarisation with 40 mM extracellular K⁺ caused a considerable reversal of the ratio of doubly- to singly-labeled glutamine species from both alanine and glutamate. The results are discussed in terms of the effects of depolarization on the glutamate/glutamine cycle.     

Effects of ketone bodies on amino acid metabolism in isolated rat diaphragm.

1. Diaphragms from 48h-starved rats were incubated in Krebs-Ringer bicarbonate medium at 37degreesC for 30min and then transferred into new medium and incubated for 1, 2 and 3 h. 2. The amount of free amino acids found at the end of each time of incubation was larger than the amount at the beginning of incubation, indicating that in this system proteolysis is prevailing. 3. The diaphragms was releasing mainly alanine and glutamine into the incubation medium. 4. Within the periods of incubation the release and metabolism of free amino acids was proceeding at a constant rate. 5. Addition of sodium DL-3-hydroxybutyrate decreased the tissue content of several amino acids, among which were tyrosine and phenylalanine, suggesting that proteolysis was decreased by ketone bodies. 6. In the presence of glucose (10mM) and branched-chain amino acids (0.5mM), sodium DL-3-hydroxybutyrate at concentrations of 4 or 6 mM resulted in 30% decrease in tissue alanine content and a 20% decline in alanine release. Release of taurine and glutamine was decreased by 19 and 16% respectively with 6 mM-sodium DL-3-hydroxybutyrate. Addition of sodium acetoacetate (1-3mM) also resulted in a 20-35% decrease in tissue content of alanine, glutamine and taurine and in a 15-24% decrease of alanine and glutamine release. Smaller decreases (less than 15%) in the release of glycine, threonine, proline, serine and aspartate were also observed in the presence of sodium DL-3-hydroxybutyrate or sodium acetoacetate. 7. Substitution of pyruvate (1.0mM) for glucose in the presence of acetoacetate restored alanine and glutamine production to control values. In the presence of acetoacetate, pyruvate also increased the tissue content of aspartate by 77% and decreased the tissue content of glutamate by 30%. 8. It is suggested that in diaphragms from starved rats, ketone bodies (a) in the absence of other substrates inhibit protein catabolism and (b) in the presence of glucose and branched-chain amino acids decrease alanine and glutamine production, by inhibiting glycolysis.     

Nitrogen metabolism in tumor bearing mice

In experiments with whole animals infested with a highly malignant strain of Ehrlich ascites tumor cells, serial concentrations of amino acids were determined for host plasma, ascitic fluid, and tumor cells, throughout tumor development. Concentration gradients of glutamine, asparagine, valine, leucine, isoleucine, phenylalanine, tyrosine, histidine, tryptophan, arginine, serine, methionine, and taurine from the host plasma toward the ascitic liquid were established; while on the other hand, concentration gradients from the ascitic liquid toward the plasma were established for glutamate, aspartate, glycine, alanine, proline, and threonine. With the exception of aspartate the concentrations of these amino acids were highest inside the cells. Arginine was the only amino acid not detected in tumor cells. In vitro incubations of tumor cells in the presence of glutamine and/or glucose, as the energy and nitrogen sources, confirmed the amino acid fluxes previously deduced from the observed relative concentrations of amino acids in plasma, ascitic liquid, and tumor cells, suggesting that glutamate, alanine, aspartate, glycine, and serine can be produced by tumors. These findings support that changes in amino acid patterns occurring in the host system are related to tumor development.     

Changes in plasma amino acid concentrations with increasing age in patients with propionic acidemia

The objective of the study is to analyze plasma amino acid concentrations in propionic acidemia (PA) for the purpose of elucidating possible correlations between propionyl-CoA carboxylase deficiency and distinct amino acid behavior. Plasma concentrations of 19 amino acids were measured in 240 random samples from 11 patients (6 families) with enzymatically and/or genetically proven propionic acidemia (sampling period, January 2001–December 2007). They were compared with reference values from the literature and correlated with age using the Pearson correlation coefficient test. Decreased plasma concentrations were observed for glutamine, histidine, threonine, valine, isoleucine, leucine, phenylalanine and arginine. Levels of glycine, alanine and aspartate were elevated, while values of serine, asparagine, ornithine and glutamate were normal. For lysine, proline and methionine a clear association was not possible. Significant correlations with age were observed for 13 amino acids (positive correlation: asparagine, glutamine, proline, alanine, histidine, threonine, methionine, arginine; negative correlation: leucine, phenylalanine, ornithine, glutamate and aspartate). This study gives new insight over long-term changes in plasma amino acid concentrations and may provide options for future therapies (e.g., substitution of anaplerotic substances) in PA patients.     

Acute metabolic effects of taurine on the enzymes metabolizing glutamate and gaba

100 mg of taurine per kg body weight had been administered intraperitoneally and 30 min after the administration the animals were sacrificed. Glutamate dehydrogenase, aspartate aminotransferase, alanine aminotransferase, glutaminase, glutamine synthetase, glutamate decarboxylase and GABA aminotransferase along with the content of glutamate and GABA in cerebral cortex, cerebellum and brain stem were studied and compared with the same obtained in the rats treated with normal saline in place of taurine. The results indicated a significant decrease in the activity of glutamate dehydrogenase in cerebral cortex and cerebellum and a significant increase in brain stem. Glutaminase and glutamine synthetase were found to increase significantly both in cerebral cortex and cerebellum. The activities of glutamate decarboxylase was found to increase in all the three regions along with a significant decrease in GABA aminotransferase while the content of glutamate showed a decrease in all the three brain regions, the content of GABA was observed to increase significantly. The above effects of taurine on the metabolism of glutamate and GABA are discussed in relation to the functional role of GABA and glutamate. The results indicate that taurine administration would result in a state of inhibition in brain.     

Age related profiles of free amino acids in human skeletal muscle

Sarcopenia describes the involuntary decline in muscle mass with aging, coupled with fatigue, and loss of force and function. We investigated 113 human muscle biopsy specimens obtained from patients with neuromuscular diseases and controls. We measured 21 amino acids in these muscle biopsies. Age emerged as a significant negative predictor of cytosolic concentration ratio of glutamine to total branched chain amino acids and of glutamine to total aromatic amino acids using stepwise multiple linear regression analysis. This pattern of alteration corresponds well to documented alterations in skeletal muscle of critically ill patients and after immobilization. Additionally, in myositis, citrulline was significantly elevated, while glutamate, lysine and taurine were significantly reduced. Furthermore, in sporadic amyotrophic lateral sclerosis (sALS) the total aromatic amino acids, arginine, glutamate, threonine, and tyrosine were significantly elevated. This study provides evidence, that alteration of glutamine is correlated to aging and might reflect increased proteolysis in aged and diseased human skeletal muscle.     

高分辨魔角旋转核磁共振和主成分分析研究人类低级星形细胞瘤和脑膜瘤的代谢组特征

采用高分辨魔角旋转核磁共振（HRMAS ^1H NMR）技术结合主成分分析（PCA）方法研究了39例人体脑肿瘤组织的代谢组特征．39例肿瘤样本分别来自39个脑肿瘤患者,包括15例低级星形细胞瘤,13例纤维型脑膜瘤和11例过渡型脑膜瘤．核磁共振波谱分析结果表明,脑肿瘤组织的代谢组中丰要含有脂肪酸、乳酸、胆碱代谢物（如胆碱、磷酸胆碱和甘油磷酸胆碱）、氯基酸（如丙氨酸、谷氨酸、谷氮酰胺、牛磺酸）、N-乙酰天门冬氨酸（NAA）和谷胱甘肽等代谢物．通过对核磁共振谱进行主成分分析（PCA）,发现低级星形细胞瘤和脑膜瘤的代谢组之间具有明显的差异,而在过渡型和纤维型两个亚类脑膜瘤之间该差别相对较小．与脑膜瘤相比,低级星形细胞瘤中甘油磷酸胆碱、磷酸胆碱、肌醇与肌酸的含量较高,而丙氨酸、谷氨酸、谷氨酰胺、谷胱甘肽和牛磺酸的含量较低．NAA的含量在低级星形细胞瘤中尽管较低但能观察到,而脑膜瘤中却未发现NAA的信号．结果衷明,HRMAS ^1H NMR和多变量统计分析相结合的组织代谢组学方法,不仅能有效区分不同类型的脑肿瘤,而且还可以为脑肿瘤提供丰富的代谢组信息,这些信息对研究肿瘤发生发展的机制具有潜在的意义．     

Astrocyte Heterogeneity: Endogenous Amino Acid Levels and Release Evoked by Non-N-Methyl-D-Aspartate Receptor Agonists and by Potassium-Induced Swelling in Type-1 and Type-2 Astrocytes

The aim of the present study was to determine whether endogenous amino acids are released from type-1 and type-2 astrocytes following non-N-methyl-D-aspartate (NMDA) receptor activation and whether such release is related to cell swelling. Amino acid levels and release were measured by HPLC in secondary cultures from neonatal rat cortex, highly enriched in type-1 or type-2 astrocytes. The following observations were made. (a) The endogenous level of several amino acids (glutamate, alanine, glutamine, asparagine, taurine, serine, and threonine) was substantially higher in type-1 than in type-2 astrocytes. (b) The spontaneous release of glutamine and taurine was higher in type-1 than in type-2 astrocytes; that of other amino acids was similar. (c) Exposure of type-2 astrocyte cultures to 50 microM kainate or quisqualate doubled the release of glutamate and caused a lower, but significant increase in that of aspartate, glycine, taurine, alanine, serine (only in the case of kainate), and glutamine (only in the case of quisqualate). These effects were reversed by the antagonist CNQX. (d) Exposure of type-1 astrocyte cultures to 50-200 microM kainate or 50 microM quisqualate did not affect endogenous amino acid release, even after treating the cultures with dibutyryl cyclic AMP. (e) Exposure of type-1 or type-2 astrocyte cultures to 50 mM KCl (replacing an equimolar concentration of NaCl) enhanced the release of taurine greater than glutamate greater than aspartate. The effect was somewhat more pronounced in type-2 than in type-1 astrocytes. Veratridine (50 microM) did not cause any increase in amino acid release. (f) The release of amino acids induced by high [K+] appeared to be related to cell swelling, in both type-1 and type-2 astrocytes. Swelling and K(+)-induced release were somewhat higher in type-2 than in type-1 astrocytes. In contrast, neither kainate nor quisqualate caused any appreciable increase in cell volume. It is concluded that non-NMDA receptor agonists stimulate the release of several endogenous amino acids (some of which are neuroactive) from type-2 but not from type-1 astrocytes. The effect does not seem to be related to cell swelling, which causes a different release profile in both type-1 and type-2 astrocytes. The absence of kainate- and quisqualate-evoked release in type-1 astrocytes suggests that the density of non-NMDA receptors in this cell type is very low.     

Effect of Systemic Administration of N-Methyl-d-Aspartic Acid on Extracellular Taurine Level Measured by Microdialysis in the Hippocampal CA1 Field and Striatum of Rats

Abstract: The extracellular concentrations of amino acids in the hippocampal CA1 field and striatum of conscious freely moving rats were monitored simultaneously by in vivo brain microdialysis using HPLC with electrochemical detection. Under basal conditions, aspartate, glutamate, glutamine, glycine, taurine, and alanine were detected, but γ-aminobutyric acid was undetectable in both regions. In-traperitoneal injection of N -methyl- d -aspartic acid (NMDA; 10 mg/kg) caused a significant increase (three-to fivefold) in the taurine concentration in the dialysate obtained from both the hippocampal CA1 and striatum, whereas other amino acids (aspartate, glutamate, and alanine) did not show significant changes. Local application of NMDA (300 γ) to both regions via the dialysis probes also caused a similar increase (three-to fivefold) in both regions. Under infusion of hypertonic Ringer's solution containing 150 m M sucrose, the effect of NMDA on the level of taurine in both the regional dialysates was not affected. The effect of NMDA was totally reduced by intraperitoneal administration of MK-801 (0.3–1.0 mg/kg), a noncompetitive antagonist of NMDA receptors. Continuous infusion of dl -2-amino-5-phosphonovaleric acid (1.0 mM), a competitive antagonist of NMDA receptors, via the dialysis probes completely inhibited the effect of NMDA. These findings suggest that systemic administration of NMDA is effective as well as local administration into the brain and that NMDA receptors might be involved in the regulation of the extracellular taurine level in the brain without dependence on cell swelling.