ISOACCEPTOR tRNAs FOR GLUTAMATE, GLUTAMINE, ASPARTATE AND ASPARAGINE IN CALF BRAIN

Abstract— Glutamyl, glutaminyl. aspartyl and asparaginyl tRNAs of calf brain were analysed by reverse phase chromatography for isoacceptor tRNAs. The radioactivity profiles revealed two peaks for gluta-mate. three for glutamine, two for aspartate and one for asparagine. Comparison of brain tRNAs with tRNAs from other sources showed that glutamate and aspartate tRNAs of brain closely resembled a majority of other tRNAs in the number and relative abundance of isoacceptors. Glutamine and asparagine tRNAs from different sources exhibited more marked differences.     

THE DISTRIBUTION OF GLYCINE, GABA, GLUTAMATE AND ASPARTATE IN RABBIT SPINAL CORD, CEREBELLUM AND HIPPOCAMPUS

The distribution of glycine, GABA, glutamate and aspartate was measured among about 60 subdivisions of rabbit spinal cord, and among the discrete layers of cerebellum, hippocampus and area dentata. A more detailed mapping for GABA was made within the tip of the dorsal horn of the spinal cord. Spinal ventral horn and dorsal root ganglion cell bodies were analyzed for the amino acids and for total lipid. The distribution of lipid and lipid-free dry weight per unit volume was also determined in spinal cord. Calculated on the basis of tissue water, glycine in the cord is highest in lateral and ventral white matter immediately adjacent to the ventral grey. The distribution of GABA is almost the inverse of that of glycine with highest level in the tip of dorsal horn. It is most highly concentrated in the central 75% of Rexed layers III and IV. Aspartate in the tip of ventral horn is 4-fold higher than in the tip of the dorsal horn and 3 times the average concentration in brain. Glutamate was much more evenly distributed and is relatively low in concentration with slightly higher levels in dorsal than in ventral grey matter. Large cell bodies in both ventral horn and dorsal root ganglion contained high levels of glycine. As reported by others, GABA was found to be high in cerebellar grey layers, area dentata, and regio inferior of hippocampus. Glycine was moderately high in cerebellar layers but moderate to low in hippocampus and area dentata.     

THE EFFECTS OF HYPERKETONEMIA ON GLUTAMATE AND GLUTAMINE METABOLISM IN DEVELOPING RAT BRAIN

Abstract— The effect of increased exposure to ketone bodies in the developing rat brain suggest that intrauterine and postnatal hyperketonemia lead to an altered metabolism of glutamine and glutamate. It is postulated that this effect is related to the delayed development of glutaminase ( l -glutamine amido-hydrolase EC 3.5.1.2) and glutamate dehydrogenase ( l -glutamate: NAD oxidoreductase EC 1.4.1.2).
The specific activities of glutamate dehydrogenase (GDH), glutaminase and glutamine synthetase ( l -glutamate: ammonia ligase EC 6.3.1.2) in the brains of newborn rats increased during early development. A positive correlation was observed between the specific activity of glutaminase and the concentration of glutamate in the brain as well as between the concentrations of blood and brain glutamine and glutamate in both control and hyperketonemic pups. This indicates a different degree of permeability and metabolism for glutamine and glutamate in the brain during the neonatal period, as compared to adulthood.
In hyperketonemic pups, glutamine and glutamate metabolism were found to differ from that in control animals. The concentrations of glutamate were higher, and glutamine lower, in both the blood and brain as compared to that in controls. The concentrations of α-ketoglutarate were also lower in their brain. In the brains of hyperketonemic and control pups, the concentration of malate was the same. During the first 3 weeks of life the increase of spec. act. of GDH and glutaminase was found to be suppressed in the brains of hyperketonemic pups. However, the spec. act. of glutamine synthetase was similar to that of the control pups.     

THE GLUTAMATE AND GLUTAMINE CONTENT OF RAT BRAIN AFTER PORTOCAVAL ANASTOMOSIS

Abstract— Rats have been subjected to portocaval anastomosis and the ammonium ion in plasma and the glutamate and glutamine levels in plasma, red cells and brain have been estimated up to 6 weeks after operation. The glutamine, but not the glutamate, levels in brain were consistently raised, being about 2.5 times greater than normal and the level can be correlated with the level of plasma ammonium ion. Consideration is given to the possibility that the glutamine may be in the greatly enlarged neuroglial compartment in this abnormal metabolic state.     

谷氨酰胺对大鼠海马脑片谷氨酸递质释放的影响

在以前的工作中我们观察到 ,饲料中补充谷氨酰胺 (Ｇｌｎ)可使大鼠脑组织中Ｇｌｎ和谷氨酸 (Ｇｌｕ)含量升高 ,并引起一系列代谢和功能的改变。当脑组织处于丰富的Ｇｌｎ环境中时 ,Ｇｌｕ等兴奋性氨基酸的释放是否会受到影响呢 ?由于条件所限 ,在整体无法观察这一过程 ,但离体脑片为我们提供了一个较为理想的研究方法。本实验通过对离体海马脑片进行孵育 ,观察Ｇｌｎ对Ｇｌｕ递质释放的影响 ,从而进一步探讨Ｇｌｎ的中枢作用机制。1　材料与方法(1)人工脑脊液 (ＡＣＳＦ)的配制　所用标准ＡＣＳＦ的配方为 (ｍｍｏｌ/Ｌ) :ＮａＣｌ 12 4,Ｋ…     

ÈVIDENCE FOR THE COMPARTMENTATION OF GLUTAMATE METABOLISM IN ISOLATED RAT RETINA

—Glucose is a major precursor of glutamate and related amino acids in the retina of adult rats. ¹⁴C from labelled glucose appears to gain access to a large glutamate pool, and the resulting specific activity of glutamate labelled from glucose is always higher than that of glutamine or the other amino acids. Radioactive acetate appeared to label a small glutamate pool. The specific activity of glutamine labelled from acetate relative to that of glutamate was always greater than 1.0. Other precursors of the small glutamate pool were found to include glutamate, aspartate, GABA, serine, leucine and sodium bicarbonate. The level of radioactivity present in retinae incubated with [U-¹⁴C]glucose or [1-¹⁴C]sodium acetate was reduced in the presence of 10^?5m -ouabain. Under these conditions, the relative specific activity of glutamine labelled from [1-¹⁴C]sodium acetate was lowered, but it was raised when [U-¹⁴C]glucose was used as substrate. Ouabain also considerably reduced the synthesis of GABA from [1-¹⁴C]sodium acetate. In all cases ouabain caused a fall in the tissue levels of the amino acids. Aminooxyacetic acid (10^?4m ) almost completely abolished the labelling of GABA from both [U-¹⁴C]glucose and [1-¹⁴C]sodium acetate, while the RSA of glutamine labelled from the latter substrate was significantly increased. Aminooxyacetic acid raised the tissue concentration of glutamate, but caused a fall in the tissue concentrations of glutamine, aspartate and GABA. The results suggest that there are separate compartments for the metabolism of glutamate in retina and that these can be modified in different ways by different drugs.     

大鼠视皮质—外侧膝状体HRP示踪结合谷氨酸及GABA免疫组化研究

采用HRP逆行追踪观察了大鼠视皮质锥体细胞至外侧膝状体背核的投射,结合谷氨酸及GABA免疫组化方法探测了该类细胞的化学递质。光镜下HRP标记细胞与谷氨酸和GABA免疫阳性细胞清晰可辨。谷氨酸免疫阳性神经元主要分布在视皮质第Ⅱ～Ⅵ层。在第Ⅵ层可见HRP和谷氨酸双标记的锥体细胞,双标细胞的数量约占HRP标记细胞总数的42.7％。GABA免疫阳性神经元分布在皮质各层。但未见有GABA和HRP的双标记神经元。因而有充分理由推测:谷氨酸可能是视皮质投射至外侧膝状体背核的锥体细胞的神经递质之一。     

THE TOXIC EFFECT OF SODIUM GLUTAMATE ON RAT RETINA: CHANGES IN PUTATIVE TRANSMITTERS AND THEIR CORRESPONDING ENZYMES

Abstract– Subcutaneous administration of high doses of sodium glutamate to rats during their first week after birth produced an almost total loss of choline acetyltransferase, a 90% reduction in glutamate decarboxylase and 70% reductions in acetylcholinesterase and DOPA decarboxylase activities in the adult retina. In addition there was a 70% decrease in GABA and 35-55% decrease in aspartate, glutamate, glycine, alanine and glutamine. No reduction in taurine was observed. The results support the view that the enzymes are mainly localized in the interneurons of retina and that taurine is present in the photoreceptor cells.
Glutamate treatment was also followed by a small reduction in choline acetyltransferase and glutamate decarboxylase of the superior colliculus and in choline acetyltransferase of hippocampus, whereas no changes could be detected in the lateral geniculate body of the adult rat. Unilateral enucleation performed on 1-day-old animals did not alter choline acetyltransferase, acetylcholinesterase, glutamate decarboxylase and DOPA decarboxylase activities in the superior colliculus and in the lateral geniculate body of the adult rat.     

鸡视网膜谷氨酸受体和GABA受体在两栖类卵母细胞中的表达

本工作利用两栖类卵母细胞作为功能表达系统,对鸡视网膜中的谷氨酸受体和ＧＡＢＡ受体的类型和基本性质进行了研究。在注射鸡视网膜ｍＲＮＡ的卵母细胞上,谷氨酸受体有明显的表达。Ｌ－Ｇｌｕ及其类似物ＫＡ,ＡＭＰＡ,ＱＡ都毫无例外地能诱导卵母细胞产生快速平滑的去极化电流,而ＮＭＤＡ,Ｌ－ＡＰ４,ＡＣＰＤ以及天冬氨酸不能诱导明显的电流反应。并且ＡＭＰＡ,ＱＡ对ＫＡ反应存在一定的抑制作用,提示ＡＭＰＡ,ＱＡ可能与ＫＡ作用于同一受体。抑制性氨基酸ＧＡＢＡ的受体被证明大部分为ＧＡＢＡＡ亚型,但有小部分的ＧＡＢＡ反应不能为荷包牡丹碱（ｂｉｃｕｃｕｌｌｉｎｅ）所阻断。     

A METHOD FOR THE ISOLATION AND ESTIMATION OF GABA, GLUTAMATE AND GLUTAMINE FROM COMPLEX MIXTURES BY A COMBINATION OF ENZYMIC INTERCONVERSION AND FORMATION OF 1-DIMETHYLAMINONAPHTHALENE-5-SULPHONYLBUTYROLACTAM

Abstract— A method is presented which allows the isolation and estimation of GABA, glutamate and glutamine in extracts of nervous tissue. It depends on the enzymic conversion of the last two compounds to GABA, before isolating the GABA as DNS-γ-butyrolactam. The low polarity of this compound makes this possible without the use of chromatography. Furthermore, the specific radioactivities of these 3 compounds in a complex mixture may be found without elaborate separation procedures. Details are given of optimum reaction conditions, and comparisons made with other methods of estimation of these amino acids.     

鲫鱼视网膜谷氨酸受体和GABA受体在爪蟾卵母细胞中的表达

我们以两栖类卵母细胞为功能表达系统,通过注射鲫鱼（Ｃａｒａｓｓｉｕｓｃａｒａｓｓｉｕｓ）视网膜ｍＲＮＡ,利用电压箝及药物灌流手段,系统地研究了鲫鱼视网膜内氨基酸受体的类型和特征,结果如下：（１）Ｇｌｕ受体：ＫＡ可以诱发明显的去极化电流,而且Ｄｉａｚｏｘｉｄｅ能增强ＫＡ诱导的反应,这提示鲫鱼视网膜内某些Ｃｌｕ受体是ＡＭＰＡ选择性亚型（ＡＭＰＡ－ｐｒｅｆｅｒｒｉｎｇｓｕｂｔｙｐｅ）。（２）ＣＡＢＡ受体：ＧＡＢＡ能诱发一个快速、光滑的内向电流,绝大部分对ＧＡＢＡ的反应可被ｂｉｃｕｃｕｌｌｉｎｅ所压抑,而ＧＡＢＡ＿Ｂ受体的激动剂ｂａｃｌｏｆｅｎ则无任何作用,这提示,鲫鱼视网膜内大部分是ＧＡＢＡ＿Ａ受体。     

REGIONAL DISTRIBUTION OF GLUTAMATE DECARBOXYLASE AND GABA WITHIN THE AMYGDALOID COMPLEX AND STRIA TERMINALIS SYSTEM OF THE RAT

The distribution of glutamate decarboxylase (GAD) and δ-amino butyric acid have been studied in the amygdaloid complex and in the stria terminalis system of the rat. The central and medial nuclei of the amygdala had significantly higher activities of GAD than the lateral olfactory tract nucleus, anterior amygdala, anterior lateral nucleus, posterior lateral nucleus, cortical nucleus, basomedial nucleus, basolateral nucleus, and pyriform cortex. The enzyme activity was about two and a half times higher in the central and medial nuclei than in the pyriform cortex. GABA was also significantly more concentratcd in these nuclei than in the pyriform cortex although this was not true for four other amino acids studied–glutamic acid, aspartic acid, taurine and glycine. GAD activity was also measured in the stria terminalis (the major afferent and efferent pathway of the amygdala) and in its bed nucleus. The enzyme activity was higher in the stria terminalis than in four other fibre tracts studied–the optic tract, anterior commissure, corpus callosum, and fimbria. GAD activity was exceptionally high in the bed nucleus of the stria terminalis particularly in its ventral part. The significance of the results are discussed in terms of what is known about the evolution and anatomy of the amygdala.     

THE TOXIC EFFECT OF SODIUM GLUTAMATE AND DL-α-AMINOADIPIC ACID ON RAT RETINA: CHANGES IN HIGH AFFINITY UPTAKE OF PUTATIVE TRANSMITTERS

Subcutaneous administration of high doses of sodium glutamate to new born rats was used to destroy retinal interneurons and ganglion cells. Such treatment was accompanied by 90% reduction in the high affinity uptake of choline, 60–70% reductions in the uptakes of GABA, diamino-n-butyric acid and glycine and 30–40% reductions in the uptakes of asparatate and glutamate measured on retinal homogenates from 30-day-old rats. The high affinity uptakes of β-alanine and taurine were unchanged. Preincubation of retinal homogenates with 1 mM β-alanine or 100 μM diamino-n-butyric acid severely reduced the high affinity GABA uptake in control and experimental animals. In intact retinae, however, the glutamate treatment increased the high affinity uptake of β-alanine by 70%, whereas that of diamino-n-butyric acid was reduced by 40% and the high affinity uptakes of GABA and glutamate were unchanged. Four hours after injection of the gliotoxic compound DL-α-aminoadipic acid into the vitreous body of 30-day-old rats, the Müller cells could no longer be identified. This lesion was accompanied by 55% reduction in the high affinity uptake of β-alanine and 25% reduction in the uptakes of GABA and glutamate on intact retinae. The high affinity uptakes of diamino-n-butyric acid, choline and the enzyme activities of choline acetyltransferase and glutamate decarboxylase were unchanged under these conditions. After 24 h, however, the Müller cells could be recognized again, and the β-alanine uptake had normalized.     

POSTNATAL ALTERATIONS IN EFFECTS OF POTASSIUM ON UPTAKE AND RELEASE OF GLUTAMATE AND GABA IN RAT BRAIN CORTEX SLICES

The uptake and release of glutamate and of GABA, as well as the effect of high potassium concentrations (35 or 80 mM) hereupon, were studied by aid of ¹⁴C-labelled amino acids in brain cortex slices from rats of different ages between birth and adulthood. Both the extent of the uptake (i.e. the tissue/medium ratio of ¹⁴C at, or close to, equilibrium) and the rate of uptake (i.e. the tissue/ medium ratio of ¹⁴C after short (5 min) incubation periods) increased with age. Differences were, however, found between glutamate and GABA, and the extent of the GABA uptake had a distinct maximum during the second postnatal week. At all ages, high concentrations of potassium caused a decrease in the rate of GABA uptake but were without effect on the rate with which glutamate was taken up. The release of the two amino acids occurred with approximately the same half-time (50 min) in slices from animals of at least 14 days of age. Before that time the release of glutamate was somewhat faster, whereas that of GABA was much slower, especially during the first postnatal week (half-time 90 min). The ontogenetic alterations in the effect of excess potassium were complex and varied both between the two potassium concentrations used and between the two amino acids. The results are thus compatible with the existence of different transport systems for the two amino acids, They also suggest that glutamate may exert other functions in addition to its role as a putative transmitter.     

REACTIONS OF FREE AND tRNA BOUND GLUTAMATE AND GLUTAMINE

—[¹⁴C]-Glutamate and [¹⁴C]-glutamine were incorporated into calf brain tRNA in the presence of homologous aminoacyl-tRNA synthetases. When the tRNAs were then deaminoacylated and chromatographed, a number of radioactive products were found in addition to the original amino acids. One of the products of glutamate transformation was identified to be glutamine. Formation of the radioactive products of glutamate in the presence and absence of tRNA indicated that glutamine was produced from glutamate at the level of the free amino acid followed by the incorporation of both substances into tRNA. Examination of the products of deaminoacylation of glutaminyl-tRNA showed that glutamine underwent structural alterations at the level of the aminoacyl-tRNAs to give rise to a cyclic derivative of glutarimide. This reaction was specific for glutamine, and constituted approximately 15 per cent of the total radioactivity in the deaminoacylation products of glutaminyl-tRNA.     

BIOCHEMICAL CORRELATES OF TRANSMISSION MEDIATED BY GLUTAMATE AND ASPARTATE

Abstract— Glutamate and aspartate probably serve as transmitters of hippocampal perforant path and commissural afferents, respectively. We therefore used slices of hippocampal regions to evaluate certain biochemical properties as markers for sites of transmission mediated by these amino acids. In these studies content and accumulation of glutamate and aspartate were compared with their Ca²⁺-dependent effluxes.
Hippocampal regions varied little in their contents of glutamate and aspartate, but slices of regio superior and dentate gyrus accumulated and released more of each than slices of regio inferior. A commissurotomy or bilateral entorhinal lesion altered Ca²⁺-dependent efflux and accumulation in the same direction, but did not affect the glutamate or aspartate content of any hippocampal region. Elimination of hippocampal mossy fibers reduced the Ca²⁺-dependent efflux of glutamate and probably aspartate from slices of dentate gyrus, but not of regio inferior, where most mossy fiber synapses are located. The mossy fibers appeared relatively deficient in aspartate in both strains tested, but only in Purdue-Wistar rats were they enriched in glutamate. Removal of the perforant path input to the fascia dentata did not significantly change the activity of any of the enzymes most actively involved in glutamate synthesis.
These results suggest that accumulation or high affinity transport of glutamate or aspartate can be employed to localize afferents which use these amino acids as transmitters, although it is not so reliable or selective a marker as Ca²⁺-dependent efflux. Enrichment in either glutamate or aspartate content or in the activity of enzymes which synthesize them is not a reliable marker. Neither amino acid is likely to be used as a transmitter by the hippocampal mossy fibers.     

THE UTILIZATION OF GLUTAMINE BY THE RETINA: AN AUTORADIOGRAPHIC AND METABOLIC STUDY

The cells able to accumulate exogenously applied [³H]glutamine in rat, cat, frog, pigeon and guinea pig retinas have been located by autoradiography, and the fate of the labelled glutamine, as regards its incorporation into aspartic, glutamic and γ-amino-butyric acids, followed for 60min. The results support the notion of glutamine as a precursor of transmitter amino acids in some neurones. In particular, it would appear to be a source of a relatively stable pool of GABA which may be located, with species variation, in amacrine or ganglion cells. In the pigeon retina a glutamate pool incorporates and retains a major percentage of the label, and perikarya in the middle of the inner nuclear layer of the tissue are predominantly labelled.