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 INFLUENCE OF PORTOCAVAL ANASTOMOSIS ON THE METABOLISM OF LABELLED OCTANOATE, BUTYRATE AND LEUCINE IN RAT BRAIN

Rats were given a portocaval anastomosis and 3 weeks later, when the only ultrastructural change in the CNS is watery swelling of astrocytes, several aspects of brain metabolism were studied. The uptake of leucine by the brain, its incorporation into protein and its oxidation were followed after the simultaneous injection of a mixture of L-[1¹⁴C]leucine and L-[4,5-³H]leucine. The concentration of leucine in blood was lowered in the operated animals whereas in brain it was increased. The specific radioactivity of leucine in the brain was comparable to values in control animals and there was no evidence of a decrease in incorporation of [1-¹⁴C]leucine into brain proteins over the short experimental time period studied. The only difference from the controls in the oxidation of [4,5-³H]leucine was a greater accumulation in glutamine. The amount of glutamine in the brains of the operated animals had increased 4-fold at the time of the metabolic studies. From dual-labelled experiments in which a mixture containing [1-¹⁴C]butyrate and L-[4,5-³H]leucine was injected intravenously, it was shown that, in both control and operated animals, the pools of brain glutamate and glutamine labelled from butyrate were metabolically distinct from those labelled from leucine. The total radioactivity appearing in brain from [1-¹⁴C]butyrate was markedly reduced in operated animals, but the radioactivity from L-[4,5-³H]leucine was not. The metabolism of [1-¹⁴C]octanoate was compared with that of [1-¹⁴C]butyrate. In control animals the labelling of metabolites was almost identical with either precursor. In operated animals there was no reduction in the uptake of [1-¹⁴C]octanoate into the brain. There was evidence that the size of the glutamine pool labelled, relative to glutamate, was increased but that it had a slower fractional turnover coefficient. A link between astroglial changes and an impairment to the carrier mechanism for transport of short chain monocarboxylic acids across the blood-brain barrier is suggested.     

EFFECTS OF EXCITATION AND ANAESTHESIA ON THE GLUTAMINE CONTENT OF THE RAT BRAIN WITH A REFERENCE TO THE ADMINISTRATION OF GLUTAMINE

Abstract— A study of the factors affecting the ghtamine content in brain of rats showed that it was significantly decreased by intraperitoneal injection of physiological saline and by the stimulus of decapitation. It fell markedly at the stage of delirium (excitement) of pentobarbitone sodium anaesthesia and returned to the original value in light and deep surgical anaesthesia; after pentylenetetrazol injection the glutamine content showed a tendency to decrease but this change was only significant in relation to its post-saline level when the convulsant and 0.9 % NaCl were given to lightly-anaesthetized animals.
The i.p. administration of glutamine temporarily abolished the decrease in its concentration in the brain caused by injection and decapitation but never raised it above the original level. Many rats previously treated with glutamine showed no signs of excitement during the induction period of pentobarbitone sodium anaesthesia, and those which were excited showed a comparatively smaller decrease of brain glutamine content. whereas in the anaesthetic state there was no change in the content of brain glutamine after glutamine had been administered. Pentylenetetrazol given at the dose level of 200 mg/kg, but not at that of 100 mg/kg, resulted in the uptake of the added glutamine by the brain up to its normal concentration. Hence, the added glutamine did not readily enter normal or anaesthetized brain, but did penetrate the hyperactive brain where its level had fallen.
The brain levels of free glutamic and aspartic acids were not affected, in general, by procedures which increased cerebral activity. Glutamate was decreased by deep anaesthesia, whereas aspartate was not significantly altered in any of the conditions which were tested.
The significance of these results in relation to cerebral ammonia metabolism and possible changes of the permeability of the tissue to glutamine in different functional states is discussed.     

THE PURIFICATION AND PROPERTIES OF RAT BRAIN GLUTAMATE DEHYDROGENASE

—A method is described for the preparation of glutamate dehydrogenase in a highly purified form from rat brain. Only one protein band was detected when the enzyme was subjected to electrophoresis on SDS polyacrylamide gels. The rat brain enzyme was essentially identical to the rat liver enzyme with respect to electrophoresis on SDS polyacrylamide gels, immunochemical properties and most kinetic parameters. However, the brain enzyme was much less reactive with glutamate, was more sensitive to inhibition by haloperidol, and was considerably more stable than the liver enzyme.     

THE SYNTHESIS OF GLUTAMATE BY RAT BRAIN MITOCHONDRIA

The synthesis of glutamate from 2-oxoglutarate generated by the citric acid cycle and ammonium acetate has been studied in brain mitochondria of synaptic or non synaptic origin. Non synaptic brain mitochondria synthesise glutamate at twice the rate (1.3 nmol. min^?1. mg protein^?1) of synaptic mitochondria (0.65 nmol. min^?1. mg protein^?1) when pyruvate is the precursor for 2-oxoglutarate, but at a similar rate (0.9 and 0.7 nmol. min^?1, mg protein^?1) when 3 hydroxybutyrate is the precursor. Glutamate synthesis from ammonium acetate and extramitochondrially addcd 2-oxoglutarate (5 mM) by both synaptic and nonsynaptic mitochondria was 5-fold higher (5-6nmol. min^?1. mg protein^?1) than glutamate synthesis from endogenously produced 2-oxoglutarate. In the uncoupled state (or un-coupler + oligomycin) the rate was reduced by half. (2.5-3 nmol. min^?1. mg protein^?1) as compared to mitochondria synthesising glutamate in states 3 or 4 (± oligomycin). The changes in brain mitochondrial nicotinamide nucleotide redox state have been monitored by fluorimetric, spectrophotometric and enzymatic techniques during glutamate synthesis and compared with liver mitochondria under similar conditions. On the instigation of glutamate synthesis by NH⁺₄ addition a significant NAD(P)H oxidation occurs with liver mitochondria but no detectable change occurs with brain mitochondria. Leucine (2 mM) causes a doubling of glutamate synthesis by both synaptic and non synaptic brain mitochondria with no detectable change in the NAD(P)H redox state. The results are discussed with respect to the control of glutamate synthesis by mitochondrial redox potential and the possible intramitochondrial compartmentation of this process.     

A STUDY OF PROPOSED DETERMINANTS OF BRAIN TRYPTOPHAN CONCENTRATION IN RATS AFTER PORTOCAVAL ANASTOMOSIS OR SHAM OPERATION

Liver dysfunction was produced in rats by surgical portocaval anastomosis (PCA), and the time-course of changes in brain tryptophan and 5-HT metabolism studied in relation to plasma changes possibly influencing brain tryptophan concentration. Brain tryptophan and 5-hydroxyindolylacetic acid (5-HIAA) levels were increased greatly and maximally on the day after PCA and remained high. 5-HT changes were less marked but had a similar time-course. Plasma total tryptophan was little changed but plasma free tryptophan was raised. The latter change showed a similar time-course to that of brain tryptophan but was not large enough to account completely for it. Sham operation was followed by significant but transient increases in plasma free tryptophan, brain tryptophan and 5-HIAA but these were much smaller than after PCA. Brain tryptophan did not correlate with plasma total tryptophan either in control or PCA rats but it correlated significantly with plasma free tryptophan in both groups. However brain levels were much higher in PCA rats than in controls with similar plasma free tryptophan levels at all times from the first day after operation. The increase of brain tryptophan in anastomosed rats not accounted for by plasma free tryptophan was explained neither by insulin changes nor by an increase of the insulin/glucagon ratio nor by changes in plasma concentrations of those amino acids which compete with tryptophan for entry into brain. The results therefore indicate an unknown influence on brain tryptophan concentration in PCA rats. As tyrosine changes in brain and plasma after PCA were very similar to those of tryptophan this influence may not be specific to tryptophan. Results suggest that under the conditions used brain tryptophan concentrations of both PCA and control rats are more influenced by changes of plasma free tryptophan concentration than by changes of plasma concentrations of competing amino acids.     

CHANGES IN GLUTAMIC ACID AND GLUTAMINE METABOLISM IN THE RAT BRAIN AFTER WHOLE BODY X-IRRADIATION

After whole body irradiation with X-rays, an increase in the free ammonia concentration in the rat brain was observed. Parallel to this increase, evidence was found of a strong activation of glutaminase. Incubation increased the endogenous ammonia-forming capacity of brain homogenates to a much greater extent in irradiated rats than in normal rats. Glutamine synthetase activity decreased within the first 2 h after irradiation but remained unchanged at 24 and 96 h after irradiation. On the other hand, at 48 h after irradiation, glutamate dehydrogenase activity in the brain had fallen by 75 per cent in comparison with the initial activity. It is concluded that metabolic systems other than the glutamine-glutamic acid system contribute to the ammonia formation in the brain after irradiation.     

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…     

THE FORMATION OF GLUTAMATE, ASPARTATE AND GABA IN THE RAT RETINA; GLUCOSE AND GLUTAMINE AS PRECURSORS

Abstract— The distribution of the neuroactive amino acids taurine, GABA, glycine, glutamate and aspartate, together with glutamine, have been studied in the rat retina. Peak levels of taurine were found in photoreceptor cells and of GABA and glycine in a retinal fraction enriched in amacrine cells and, synaptic terminals. In vitro , GABA formation from [³H]glutamine and [¹⁴C]glucose was also most prominent in this fraction; at 500 μ m [³H]glutamine was the better precursor.
Observations on metabolism in the photoreceptor cell layer of the tissue suggest an active turnover of glutamate, aspartate and GABA, and show that glutamine may serve as an alternative substrate to glucose here, perhaps via the GABA bypath.     

THE EFFECT OF PORTA-CAVAL ANASTOMOSIS UPON THE ENERGY STATE AND UPON ACID-BASE PARAMETERS OF THE RAT BRAIN

Abstract— In order to evaluate the influence of porta-caval anastomosis upon the energy state of the brain, lightly anaesthetized rats were studied either 1 or 5 weeks after the shunting procedure and the brains (frontal lobe, cerebellum and brainstem) were analysed for carbohydrate substrates and organic phosphates. The ammonia contents of arterial blood, cerebrospinal fluid (CSF) and tissue increased progressively in the shunted groups and at 5 weeks the increases were three- to six-fold. In all brain structures studied there were decreases in the glucose and in the aspartate contents but regional differences existed for glucose-6-phosphate, α-ketoglutaratc and glutamate. In the brainstem the tissue contents of glucose-6-phosphate and α-ketoglutarate fell while glutamate was unchanged. Calculation of the cytoplasmatic redox state from the lactate dehydrogenase (LDH) and the malate dehydrogenase (MDH) equilibria indicated that the NADH/NAD⁺ ratio increased in the shunted groups. However, since there was no significant fall in the calculated adenylate energy charge, it is concluded that porta-caval anastomosis, and the accompanying hyperammonemia, do not disrupt the balance between production and utilization of energy in the brain.     

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.     

KINETIC STUDY OF GLUTAMATE TRANSPORT IN RAT BRAIN MITOCHONDRIA

Abstract— The experiments reported here confirm that glutamate can penetrate the inner membrane of isolated rat brain non-synaptosomal mitochondria, either on a glutamate-hydroxyl antiporter or on a glutamate-aspartate antiporter. An inhibition of respiratory activity of mitochondria with glutamate as substrate was obtained in the presence of avenaciolide or N-ethylmaleimide. Swelling of the mitochondria in iso-osmotic NH4⁺-l -glutamate was inhibited in the presence of avenaciolide and N-ethylmaleimide, but mersalyl, kainic acid, glisoxepide and amino-oxyacetic acid had no effect on the glutamate-hydroxyl exchange. Glutamate induced the reduction of intramitochondrial NAD(P), as estimated by double-beam spectrophotometry, and this reduction was inhibited on the one hand by N-ethylmaleimide, avenaciolide or fuscine, on the other hand by aminooxyacetic acid. A direct estimation of the penetration of l -[¹⁴C]glutamate into brain mitochondria was performed by using the centrifugation-stop procedure. This penetration followed saturation kinetics, with a mean apparent K_m of 1.56 M^M at pH 7.4 and at 20°C, the value of Knax was 4.34 nmol per min per mg protein in the same conditions. IV-Ethylmaleimide slowed down the initial rate of glutamate penetration, and this inhibition appeared to be non-competitive with a K_i of 0.7 Mm -at pH 7.4 and at 20°C. The entry of glutamate was pH-dependent and it increased 2-fold in the pH range of 7.4 to 6.4. A temperature-dependence of glutamate transport was also shown between 2 and 25°C; the Arrhenius plot was a straight line, with a calculated E_A of 12.8 kCal per mol of glutamate and a Q₁₀ of 2.16. The activity of γ-glutamyl transpeptidase was practically absent in these rat brain mitochondria. Oxidation of extramitochondrial NADH by the‘malate-aspartate shuttle’reconstituted in vitro was followed in rat brain non-synaptosomal mitochondria. In the absence of extramitochondrial malate or glutamate the ‘shuttle’ did not function, and in the absence of extramitochondrial aspartate the rate of NADH oxidation was low. Glutamine or γ-aminobutyrate did not replace glutamate efficiently. A high inhibition of the‘malate-aspartate shuttle’occurred in the presence of avenaciolide or mersalyl, and a moderate one in the presence of n-ethylmaleimide, glisoxepide or n-butylmalonate. Glutaminase activity in intact brain mitochondria was inhibited in the presence of extramitochondrial glutamate.     

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.     

ELEVATION IN RAT BRAIN HISTAMINE CONTENT AFTER FOCUSED MICROWAVE IRRADIATION1

Abstract— —Microwave irradiation focused on the head of small rodents is now widely used as a means of more accurately measuring acetylcholine, choline, cyclic AMP, and several other important brain constituents. Because of its probable neurotransmitter role and rapid turnover, a similar approach was taken to study brain histamine. Histamine was measured by a modified radio-enzymatic method and was found to be nearly tripled in brains from microwave treated rats, compared to decapitation controls (124 vs 42 ng/g). Possible explanations include a microwave-induced inactivation of histamine breakdown, a microwave-induced redistribution of previously unmeasured histamine, and microwave-induced histidine decarboxylation. Brain histamine remained unchanged up to 30 min after decapitation and microwave heated brains from decapitated rats also had elevated histamine levels, indicating that brain histamine levels in decapitated rats do not represent the remainder of a rapidly depleting pool. No evidence for previously unmeasured histamine was found. Furthermore, microwave irradiation did not enhance the formation of [³H]histamine after intraventricular [³H]histidine administration, indicating a lack of microwave-induced histidine decarboxylation. It is concluded that the elevation in rat brain histamine after focused microwave irradiation is probably not artifactual, although the mechanism responsible for this phenomenon remains obscure.     

EFFECTS OF UNDER NUTRITION AND PROTEIN DEFICIENCY ON GLUTAMATE DEHYDROGENASE AND DECARBOXYLASE IN RAT BRAIN

Abstract— Studies were made on the effects of undernutrition at different ages during the neonatal period and of the comparative effects of postweaning protein and calorie deficiencies in neonatally undernourished or normally reared animals. Neonatal undernutrition resulted in deficits in body wt, brain wt and the activities of brain glutamate dehydrogenase and glutamate decarboxylase. Percentage deficits in brain wt were maximum in the first week of life but those in brain enzymes were greater in the second week. Rehabilitation of neonatally undernourished animals reversed the deficits in brain wt and brain enzymes. Post-weaning protein deficiency produced similar deficits in brain enzymes in both neonatally undernourished and normally reared animals. With post-weaning undernutrition, however, these deficits were found only in animals subjected to neonatal undernutrition as well.