EFFECTS OF DIETARY PROTEINS ON FETAL BRAIN PROTEIN AND GLUTAMIC ACID METABOLISM IN RAT

Abstract— The effects of feeding dietary wheat and Bengal gram proteins to pregnant rats on brain protein and glutamic acid metabolism in 15-, 17- and 19-day fetuses were investigated. Wheat and Bengal gram diets resulted in loss of brain weight with decreased DNA, RNA, protein, free x amino N and deficits in the activities of brain glutamine synthetase, glutaminase I. glutaminase II and glutamate decarboxylase at all the gestational ages studied without any change in glutamine transferase activity. The concentrations of the amino acids alanine, glutamic acid, glutamine and GABA were found to be significantly lower on wheat and Bengal gram diets than the control on a 10% casein diet. The wheat with lysine and Bengal gram with methionine, cystine and tryptophan resulted in similar mean values of all the characteristics studied to the mean values observed in rats on the control diet. However, glutaminase I activity remained significantly low on lysine fortified wheat diet, and aspartic acid content was found to increase on both fortified and unfortified wheat and Bengal gram diets. A 20% casein diet showed increased brain weight, DNA. RNA. protein and free x amino N concentrations as compared with the 10% casein diet, while the other parameters remained unchanged.     

THE EFFECTS OF 4-HYDROXYBUTYRIC ACID ON THE BIOSYNTHESIS OF AMINO ACIDS IN THE CENTRAL NERVOUS SYSTEM

—The effect of 4-hydroxybutyrate (GHB) on cerebral glucose metabolism has been studied. GHB increases the glucose level, decreases the lactate concentration and diminishes the incorporation of glucose carbon into glutamic acid, glutamine, aspartic acid and GABA in the brain of the rat in a state of general anaesthesia. The data reported here suggest that GHB interferes in the metabolism of glucose in brain.     

INTRACELLULAR AMINO ACID CONTENT OF NEURONAL, GLIAL, AND NON-NEURAL CELL CULTURES: THE RELATIONSHIP TO GLUTAMIC ACID COMPARTMENTATION

Abstract— A protocol for the accurate determination of intracellular levels of amino acids in tissue cultured cells has been developed and used in the measurement of intracellular amino acids levels in neuronal, glial, and non-neural cell lines, with the objective of establishing morphological correlates for large and small glutamic acid compartments and of examining hypotheses for the morphological basis of glutamic acid compartmentation. This survey of intracellular amino acid levels has revealed striking differences among the cell lines tested, but these differences did not correlate with cell type, i.e. neuronal vs glial, in contrast to earlier results (R ose , 1968) based on bulk separated neuronal and pial fractions from rat brain. Amino acid levels were found to be dependent upon tissue culture conditions, yet reproducible differences could be observed when growth and experimental conditions were carefully controlled. Glutamic acid levels for various cell lines ranged from 50.8 ± 14.3 to 158 ± 8.5 nmol/mg protein. Intracellular glutamine levels demonstrated even greater difference, with values ranging from 0.8 ± 0.2 to 107 ± 42.4 nmol/mg protein. Statistically significant differences in intracellular amino acid levels between cell lines were also observed for aspartic acid, praline, glycine, alanine, valine, cystathionine, isoleucine, and leucine. A number of cell lines demonstrating highly elevated elevated levels of γ-aminobutyrate and β-alanine were identified. The significance of neuronal and glial levels of glutamic acid, glutamine and γ-aminobutyrate to models for glutamic acid compartmentation is discussed.     

DEVELOPMENTAL CHANGES IN MOUSE BRAIN: WEIGHT, WATER CONTENT AND FREE AMINO ACIDS

Abstract— —Glutamic acid, glutamine, GABA and aspartic acid exhibited postnatal increases in the developing mouse brain at the same time that the other amino acids, both essential and non-essential, of the pool decreased. The most significant decreases were observed in the concentrations of taurine, phosphoethanolamine, glycine and alanine. The period of rapid accumulation of the members of the glutamic acid family in the mouse brain was concurrent with dramatic increases and decreases in brain weight and water content, respectively.     

LEVELS OF AMINO ACIDS AND PROTEINS IN PACINIAN CORPUSCLES OF THE CAT

Abstract— Paper chromatography of extracts from mesenteric Pacinian corpuscles of the cat revealed the presence of glutamic acid, glutamine, aspartic acid and alanine as major amino acids, and glycine, serine and threonine in traces; GABA was not detected. Levels of glutamic acid (0·75 μmol/g ' 0·37, s.d. ), glutamine (1·34 ± 0·55), and aspartic acid (0·32 ± 0·22) of mesenteric and pancreatic samples of Pacinian corpuscles were determined by separation on chromatographic columns. The protein values averaged 5·2 ± 0·66 per cant of the wet weight.
Treatment of the cats with reserpine or pargyline or deafferentation of the Pacinian corpuscles did not significantly alter these values.     

Free amino acids in the dog spinal cord during prolonged ischaemia

The authors studied the effect of 40 and 80 minutes' occlusion of the abdominal aorta on the aspartic acid, glutamic acid, glutamine, glycine and gamma-aminobutyric acid concentration in the sacral, lumbar, thoracic and cervical segments of the dog spinal cord. The most important changes were displayed by the glutamine concentration, which rose significantly after ischaemia in all the regions studied. The alanine and GABA concentration rose in the sacral and lumbar segments respectively, while the glutamic and aspartic acid concentrations in the sacral region fell. At the same time, enzyme activities degrading the synthetic substrates Leu-p-nitroanilide and Gly-p-nitroanilide increased. The significance of the findings is discussed with reference to nerve cell catabolism and function.     

乙酰胆碱,谷氨酸与GABA对丘脑腹内侧核神经元活动的影响

本文采用微电泳方法观察到在大鼠丘脑腹内侧核（ＶＭ）,微电泳给予乙酰胆碱（ＡＣＨ）使所有受试神经元自发放电频率加快,谷氨酸（ＧＬＵ）使大多数神经元放电加快,它们的作用依赖于电流强度;而γ氨基丁酸（ＧＡＢＡ）和氯苯氨丁酸则抑制大多数神经元的放电活动,但前者的作用快速而暂短,而后者的作用相对缓慢而持久。在微电泳ＡＣＨ或ＧＬＵ的过程中,给予ＧＡＢＡ可拮抗它们的兴奋作用。双钴碱使大多数神经元的自发放电频率加快,而阿托品和ＭＫ８０１对自发放电的影响较小。这些结果表明ＧＡＢＡ,ＡＣＨ和ＧＬＵ等递质活动在同一ＶＭ神经元有重要的会聚作用;ＧＡＢＡ对ＶＭ神经元有紧张性抑制作用。     

THE INFLUENCE OF EXCESS METHIONINE ON THE FREE AMINO ACIDS OF BRAIN AND LIVER OF THE WEANLING RAT*

Abstract— —High circulating levels of l -methionine produced by inclusion in the diet or parenteral injection of the amino acid caused alterations in the free amino acid pattern of liver and brain tissues. Acute effects following l -methionine injection were more pronounced than those following long term feeding where adaptation played a role. The net effect following parenteral injection was to increase the total free amino acids of liver while decreasing those of brain. Individually, hepatic levels of aspartic acid, threonine, serine, glutamine, glutamic acid, glycine, and alanine were depressed while levels of taurine, cystathionine, methionine, lysine, and ornithine were markedly elevated. Brain levels of aspartic acid, threonine, serine, glutamic acid, glycine, alanine, and γ-aminobutyric acid were markedly depressed and increased levels of cystathionine, methionine, lysine, and glutamine were observed. A generalized aminoaciduria occurred shortly after excessive methionine intake. Disruption of the free amino acid pools was of two kinds. The first depended on the continued presence of excess l -methionine, the second did not.     

PROPERTIES OF THE UPTAKE AND RELEASE OF GLUTAMIC ACID BY SYNAPTOSOMES FROM RAT CEREBRAL CORTEX

Abstract– (1) The uptake and release of glutamic acid by guinea-pig cerebral cortex slices and rat synaptosomal fractions were studied, comparing the naturally occurring l - and non-natural d -isomers. Negligible metabolism of d -glutamic acid was observed in the slices. (2) Whereas in the cerebral slices the accumulation of glutamic acid was almost the same for the two isomers, d -glutamic acid was accumulated into the synaptosomal fraction at a markedly lower rate than was the L-isomer. (3) The uptake systems for d -isomer into the slices and synaptosomal fraction were found to be of single component, in contrast with the two component systems, high and low affinity components, for the uptake of l -glutamic acid. The apparent K_m values for the uptake of d -glutamic acid into the slices and synaptosomal fraction were comparable with those reported for the low affinity components for l -isomer. The uptake systems for d -glutamic acid were dependent on the presence of Na⁺ ions in the medium, like those for l -glutamic acid and GABA. (4) The evoked release of radioactive preloaded d -glutamic acid was observed both from the slices and synaptosomal fraction following stimulation by high K⁺ ions in the medium. From these observations, it is evident that the evoked release of an amino acid by depolarization in vitro is not necessarily accompanied by a high affinity uptake process. (5) The uptake of l -glutamic acid, expecially into the synaptosomal fraction, was highly resistant to ouabain. On the other hand, the uptake rate of d -glutamic acid and GABA into the synaptosomal fraction was inhibited by varying concentrations of ouabain in accordance with the inhibition for brain Na-K ATPase. (6) The uptake of l -glutamic acid into subfractions of the P₂ fraction was studied in relation to the distribution of the ‘synaptosomal marker enzymes’. An attempt to correlate the activities of enzymes of glutamic acid metabolism with the uptake of l -glutamic acid into the synaptosomal fraction from various parts of brain was unsuccessful. The high affinity uptake of l -glutamic acid was found to be very active in the synaptosomal fraction from any part of brain examined.     

Intrauterine Malnutrition and the Brain: Effects on Enzymes and Free Amino Acids Related to Glutamate Metabolism

Abstract: The effect of feeding pregnant rats with wheat and Bengal gram (black chick pea) diets during the later part of pregnancy on brain growth, enzymes, and free amino acids of glutamate metabolism in 1-day-old rats was investigated. These diets did not induce growth dissociation, and the body and brain weights were equally affected. The concentrations of DNA, RNA, protein, and free α-amino nitrogen in brain decreased significantly and the activities of glutamine synthetase, glutamine transferase, glutaminase 1, glutaminase 11, and glutamate decarboxylase and the concentrations of free amino acids, glutamic acid, glutamine, alanine, and GABA were also decreased. The concentration of aspartic acid, however, was increased. Wheat and Bengal gram diets fortified with lysine and with methionine, cystine, and tryptophan respectively showed various beneficial effects on the changes observed in the brain. A 20% casein diet induced higher body and brain weights and better brain protein and free α-amino nitrogen concentrations than those observed on a 10% casein diet.     

POSTNATAL CHANGES IN FREE AMINO ACID,DNA, RNA AND PROTEIN CONCENTRATIONS OF MINIATURE SWINE BRAIN1

Postnatal developmental characteristics of miniature swine brain were evaluated through the first 9 weeks of age. Differential growth rates of cerebrum, cerebellum and brain stem were defined in terms of DNA, RNA, protein and free amino acid concentrations at ages 5, 21, 35 and 63 days. Within the experimental conditions provided, hyperplasia ceased just prior to ages 21, 35 and 63 days for cerebellum, brain stem and cerebrum, respectively. An additional cerebral growth spurt, observed between weaning at age 35 days and sacrifice at age 63 days, may be indicative of impaired brain development due to inadequate nutrition provided by the dam's milk. Developmental changes in mean concentrations of brain free amino acids varied with anatomical area and differed somewhat from those of other species previously reported. For example, mean cerebral concentrations of aspartic acid, γ-aminobutyric acid and asparagine + glutamine decreased significantly (P < 0·05) with age and mean glutamic acid concentration was 5 times that of taurine.     

MOUSE BRAIN TYROSINE HYDROXYLASE AND GLUTAMIC ACID DECARBOXYLASE FOLLOWING TREATMENT WITH ADRENOCORTICOTROPHIC HORMONE, VASOPRESSIN OR CORTICOSTERONE

The activities of tyrosine hydroxylase (TH) and glutamic acid decarboxylase (GAD) from several mouse brain regions were assayed following repeated administration of adrenocorticotrophic hormone (ACTH), lysine vasopressin (LVP) or corticosterone. Although similar treatments with ACTH have been shown to result in changes of catecholamine turnover and GABA content, no changes in the activity of either TH or GAD were observed in any brain region. Likewise LVP had no effect on either enzyme. Since the assays for TH were performed with concentrations of tyrosine and tetrahydrobiopterin cofactor below their respective Michaelis constants, this suggests that the changes of catecholamine turnover are not mediated by changes of TH activity. Twice daily corticosterone adrninistration for four days increased TH activity in the hypothalamus but not in any other brain region.     

ORNITHINE AND GLUTAMIC ACID DECARBOXYLASE ACTIVITIES IN THE DEVELOPING CHICK RETINA

Abstract— γ-Aminobutyric acid was found in 6-day-old chick embryo retina; approx 20% was formed from putrescine and the remainder from glutamic acid. However, at the 18th embryonic day only 1% of the γ-aminobutyric acid synthesized in retina was derived from putrescine. These results show that γ-aminobutyric acid is synthesized in chick embryo retina prior to synaptogenesis, and that γ-aminobutyric acid is synthesized via two pathways. The first pathway involves the conversion of putrescine to γ-aminobutyric acid; the second path is dependent upon the conversion of glutamic acid to γ-aminobutyric acid, catalyzed by glutamic acid decarboxylase.     

Free amino acids in the spinal cord, ganglia and ischiadic nerve of the dog after ligature of the abdominal aorta

The effect of occlusion of the abdominal aorta for 10, 20 and 40 minutes on the concentration of aspartic and glutamic acids, glutamine, glycine, alanine and gamma-amino butyric acid in the anterior and posterior horns of the lumbosacral spinal cord was studied in the dog, further, concentration of amino acids (except GABA) in lumbosacral spinal ganglia and in the ischiadic nerve following 40 minutes of occlusion. The changes were most marked after 40 minutes of occlusion with a rise in concentration of alanine, glutamine and glutamic acid in the dorsal part of grey matter. Striking was also the simultaneous elevated concentration of Glu and Gln in spinal ganglia. The significance of these changes is discussed from the aspect of metabolism and function of nerve cells. Under physiological conditions the free amino acid pool in the central nervous system remains essentially constant. Under pathological conditions, however, like ischemic-hypoxic states, various changes occur.     

AMINO ACID METABOLISM AND AMMONIA FORMATION IN BRAIN SLICES

The formation of ammonia and changes in the contents of free amino acids have been investigated in slices of guinea pig cerebral cortex incubated under the following conditions: (1) aerobically in glucose-free saline; (2) aerobically in glucose-free saline containing 10 mM-bromofuroic acid, an inhibitor of glutamate dehydrogenase (EC 1.4.1.2); (3) aerobically in saline containing 11-1 mM-glucose and (4) anaerobically in glucose-free saline. Ammonia was formed at a steady rate aerobically in glucose-free medium. The formation of ammonia was largely suppressed in the absence of oxygen or in the presence of glucose whereas the inhibitor of glutamate dehydrogenase produced about 50 per cent inhibition. Other inhibitors of glutamate dehydrogenase exerted a similar effect. Ammonia formation was also inhibited by some inhibitors of aminotransferases but not by others. Inhibition was generally more pronounced during the second and third hour of incubation. With the exception of glutamine which decreased slightly, the contents of all amino acids increased markedly during the anaerobic incubation. During aerobic incubation in a glucose-free medium, there was an almost complete disappearance of glutamic acid and GABA. Glutamine also decreased, but to a relatively smaller extent. The content of all other amino acids increased during aerobic incubation in glucose-free medium, although to a lesser extent than under anaerobic conditions. The greater increase of amino acids appearing anaerobically in comparison to the increase or decrease occurring under aerobic conditions corresponded closely to the greater amount of ammonia formed aerobically over that formed anaerobically. This finding is interpreted as indicating a similar degree of proteolysis under anaerobic and aerobic conditions; aerobically, the amino acids are partly metabolized with the concomitant liberation of ammonia. In glucose-supplemented medium, the content of glutamine was markedly increased. The content of glutamate and aspartate remained unchanged, whereas that of some other amino acids increased but to a lesser extent than in the absence of glucose. Proteolysis in the presence of glucose was estimated at about 65 per cent of that in its absence. In the presence of bromofuroate the rate of disappearance of glutamate was unchanged, but there was a larger increase in the content of aspartate and a smaller decrease of GABA and glutamine. Other changes did not differ significantly from those observed in the absence of bromofuroate. We conclude that the metabolism of amino acids in general and of glutamic acid in particular differs according to whether they are already present within the brain slice or are added to the incubation medium. Only the endogenous amino acids appear to be able to serve as precursors of ammonia and as substrates for energy production.     

Hormone mediated changes of brain glutamic acid system in amino acid imbalance

The effects of adrenal cortical hormone and thyroxine on brain glutamic acid, gamma-amino butyric acid (GABA) and glutamine were studied in rats fed on the amino acid imbalanced diet (8% casein diet supplemented with 0.3% L-threonine). The studies revealed that the decrease in brain glutamic acid and GABA levels in threonine imbalance was recovered by hydrocortisone supplementation. The increased level of brain glutamine in threonine imbalance could not, however, be reversed by hydrocortisone supplementation. Thyroxine supplementation was found to have no impact on any of the members of glutamic acid family in the brain of rats receiving the threonine-imbalanced diet. It was suggested that the decreased levels of brain glutamic acid and GABA in threonine imbalance were caused by diminished adrenal cortical function and the influence of adrenal cortical hormone could be suggested to reside at the level of formation of both glutamic acid and GABA.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING AMINO ACIDS. STEREOSELECTIVITY OF THE BINDING OF L-[14C]GLUTAMIC ACID IN CEREBRAL CORTEX

From the total lipid extract of ncrve-ending membranes or the homogenate of cerebral cortex a hydrophobic protein fraction binding L-[¹⁴C]glutamic acid was separated by chromatography on Sephadex LH20. This protein could only be partially separated from the [¹⁴C]GABA-binding protein and from the lipids that are present in the fraction; however, it was demonstrated that both amino acids bind to different sites. The saturation of the binding showed a high (Kd₁= 0.3μM), a medium (Kd, = 5 μM) and a low (Kd, = 55 μM) affinity binding site. The high affinity binding site had a binding capacity of 0.53 nmol/mg of protein and was highly stereoselective for the L-enantiomer. The binding of L-[¹⁴C]glutamic acid was not inhibited by GABA, was slightly inhibited by glycine and glutamine and was strongly inhibited in a progressive order by DL-a-methylglutamic acid, L-nuciferine, L-aspartic acid and L-glutamic acid diethyl ester. These results are compared with those previously obtained with the L-glutamic acid-binding protein isolated from crustacean muscle. The stereoselectivity of the binding and the possible role of this protein in synaptic transmission are discussed.     

BIOSYNTHESIS OF ASPARTIC, GLUTAMIC, γ-AMINO-BUTYRIC ACIDS AND GLUTAMINE IN BRAIN OF RATS DEPRIVED OF TOTAL SLEEP OR OF PARADOXICAL SLEEP

Abstract— The [¹⁴C]glucose incorporation rate into brain aspartate, glutamate, GABA and glutamine in rats deprived of total and paradoxical sleep was studied. The specific radioactivities of the isolated metabolites were related to the specific radioactivity of brain glucose. It was expected that the incorporation of radioactivity would reflect subtle changes occurring in brain amino acid metabolism during total and selective paradoxical sleep deprivation. The results show an increased specific radioactivity of glutamine in total sleep-deprived rats. The variation in specific radioactivity of glutamine without a change in its concentration indicates an increased turnover of this compound. The reasons and possible mechanism for the change in glutamine specific radioactivity are discussed.     

CARBOHYDRATE AND AMINO ACID METABOLISM IN RAT CEREBRAL CORTEX IN MODERATE AND EXTREME HYPERCAPNIA

—The time course of changes in glycolytic and citric acid cycle intermediates and in amino acids was studied in acute and steady state hypercapnia. Experiments on unanaesthetized animals exposed to 10% CO₂ for 10, 20 and 60s showed that there was a transient decrease in glycogen concentration, progressive increases in glucose-6-phosphate and fructose-6-phosphate and decreases in pyruvate and lactate. During this time the levels of amino acids and Krebs cycle intermediates did not change, except for a small fall in malate at 60s. The results indicate that there was a decrease in glycolytic flux due to an inhibition of the phosphofructokinase reaction. Since the tissue levels of phosphocreatine, ATP, ADP and AMP were unchanged inhibition of phosphofructokinase was probably due to the fall in pH. Anaesthetized animals were exposed to about 5% CO₂ (for 2, 5, 15, 30 and 60 min) or to about 45% CO₂ (for 5 and 15 min). Except for succinate, which increased, all citric acid cycle metabolites analysed (citrate, α-ketoglutarate, fumarate and malate) decreased with the rise in CO₂-tension. The sum of the amino acids analysed (glutamate, glutamine, aspartate, asparagine, alanine and GABA) decreased at extreme hypercapnia. The results suggest that Krebs cycle intermediates and amino acids are partly used as substrates for energy production when there is reduced pyruvate availability due to hypercapnia. It is proposed that amino acid carbon is made available for oxidation via transamination (aspartate aminotransferase reaction) and deamination (glutamate dehydrogenase reaction) and that citric acid cycle intermediates are metabolized following a reversal of reactions usually leading to CO₂ fixation.     

Elevation of cerebral levels of nonessential amino acids in vivo by administration of large doses

Taurine, aspartic acid, glutamic acid, glycine, and GABA were administered either intragastrically or in liquid diets to mice and rats. This resulted in a great increase in the plasma concentration of the administered amino acid, with plasma levels remaining elevated for several days.The prolonged increase in plasma levels resulted in significant increases in brain levels. Under these experimental conditions, taurine, aspartic acid, and glutamic acid were increased 30–60%; glycine and GABA 100%. During these experiments, plasma levels of taurine, aspartate, and glutamate were below brain levels; those of glycine and GABA were above.The findings show that even slowly penetrating amino acid levels can be increased in brain after parenteral administration of large doses.