THE METABOLISM OF GABA AND OTHER AMINO ACIDS IN RAT SUBSTANTIA NIGRA SLICES FOLLOWING LESIONS OF THE STRIATO-NIGRAL PATHWAY

The metabolism of GABA and other amino acids from various radioactive precursors has been studied in the rat substantia nigra using a sensitive double isotope dansyl derivative assay. Labelled acetate gave greater labelling of glutamate than of glutamine in substantia nigra slices whereas the reverse was the case for cerebral cortex slices. Unilateral transection of the striato-nigral pathway caused a parallel decrease in the GABA and GAD content of the substantia nigra. It also reduced the total synthesis of GABA from all labelled precursors used, namely acetate, glutamate and glucose. After incubation with [1-¹⁴C]acetate the specific activity of glutamate and aspartate, but not that of GABA, increased on the lesioned side compared with the normal side. The specific activity of glutamate, but not that of GABA or aspartate, decreased after incubation with [U-¹⁴C]glucose on the lesioned side compared with the normal side. The results could be explained by the previously proposed hypothesis concerning differential labelling of metabolic pools by the two precursors. [U-¹⁴C]Glutamate lead to increased labelling of GABA on the lesioned side relative to the normal side. Incubation of slices from substantia nigra with β-mercaptopropionic acid caused a decrease of labelling of GABA from glucose and acetate, probably as the result of GAD inhibition. The labelling pattern of the other amino acids, apart from that of glutamate which showed a decrease when synthesised from acetate, did not change appreciably.     

FATTY ACID COMPOSITION OF CEREBROSIDES, SULPHATIDES AND CERAMIDES IN MURINE SUDANOPHILIC LEUCODYSTROPHY: THE JIMPY MUTANT

The fatty acid composition of cerebrosides, sulphatides and ceramides was determined at 15-16 days post partum in the brain of the Jimpy mutant and in littermate controls. There was a marked deficit in the long chain fatty acids (C₂₂-C₂₄) of cerebrosides and sulphatides of Jimpy brain, with the unsubstituted fatty acids affected more than the alpha-hydroxy fatty acids. A decrease of long chain normal fatty acids was also found in the ceramides of Jimpy brain. The deficit of long chain fatty acids in these sphingolipids of the Jimpy brain was more severe than that found in the Quaking mutant which has a less extensive disorder of myelin formation.     

AMINO ACID METABOLISM IN GLIAL CELLS: HOMEOSTATIC REGULATION OF INTRA- and EXTRACELLULAR MILIEU BY C-6 GLIOMA CELLS

Abstract— The amino acid and carbohydrate metabolism of confluent cultures of C-6 glioma cells has been investigated. It was observed that the presence of glutamine in the incubation fluid was essential to maintain high glutamine levels in the cells during a 2 h incubation. When cells were incubated in a cerebrospinal fluid-like medium glutamate, glutamine, aspartate and γ-aminobutyrate (GABA) levels were comparable to those occurring in whole forebrain of adult rat in vivo. Glucose uptake was high, approx 1 μmol/mg protein/2 h, 50% of which was accounted for by lactate production. Of the remaining glucose uptake a substantial proportion was unaccounted for by known oxygen-coupled citric acid cycle flux, or glycogen or amino acid synthesis. Interestingly, the cells released into the medium significant amounts of the neuroinhibitory amino acids, GABA and glycine, and rapidly cleared the medium of the neuroexcitatory amino acids glutamate and aspartate. Metabolism of [2-¹⁴C]glucose and [³H]acetate by the cells indicated rapid labelling of the glutamate and aspartate pools of the cells by glucose in 1 h, but the relative specific activities of glutamine and GABA were much lower. The metabolism of tracer concentrations of [³H]acetate to glutamate by the cells indicated greater dilution of this isotope compared to that of labelled glucose. However, the ratio of ³H to ¹⁴C radioactivity in glutamate and other amino acids was similar to that in the mixture of glucose and acetate added to the medium. Therefore, some active route of acetate metabolism which communicates metabolically with the route of glucose metabolism to glutamate appears to exist in the cells. Significant acetate activation and fatty acid turnover would explain the present results. Some of the amino acid labelling patterns observed in these studies are not consistent with these glial-like cells behaving as models for the small compartment of amino acid metabolism in brain. Enzyme measurements corroborated the metabolic studies. Glutamate decarboxylase activity was 3–10% of the level found in whole brain. GABA transaminase was also low compared to brain as was glutamine synthetase. Glutamate dehydrogenase was present at levels equal to or higher than those of whole brain.     

INFLUENCE OF LIPIDS ON THE ACTIVITY OF CEREBROSIDE-SULPHOTRANSFERASE IN MOUSE BRAIN: A COMPARATIVE STUDY OF JIMPY AND NORMAL MOUSE BRAINS 1

Abstract— The effect of lipids other than the substrate cerebroside on the activity of cerebroside-sulphotransferase (CST) in Jimpy and normal mouse brain was investigated. The enzyme activity of an acetone-treated microsomal preparation can be stimulated in the presence of the extracted lipids either with or without addition of exogenous cerebroside as a substrate. The CST activity in the Jimpy mutant compared to that in normal animals differs from 18% in homogenate to approx 80% in solubilized or acetone-extracted microsomes. An addition of total lipid from normal mouse brain to microsomal preparations from which lipid has been removed by acetone results in a stimulation of Jimpy CST activity up to a value of 80% of normal mouse brain microsomes. Both Jimpy and normal mouse brain CST can be also stimulated by the addition of single lipid components such as cholesterol and lecithin by 50% in normal and 100% in Jimpy brain microsomes. These findings lead to the hypothesis that there is a lipid requirement for CST activity other than the substrate cerebroside.     

BIOCHEMTCAL CHANGES IN THE BRAIN OF EXPERIMENTAL ANIMALS IN RESPONSE TO ELEVATED PLASMA HOMOCYSTINE AND METHIONINE

The effects of high plasma concentrations of homocystine and methionine on the free amino acids of brain have been examined. Incorporation of the label from [³⁵S]methionine into the free amino acid pools of rabbit brain was enhanced in response to high plasma homocystine or high plasma homocystine and mcthionine. Under comparable conditions a marked decrease in the incorporation of the label from [¹⁴C]glycine into the free pool was observed. The corresponding incorporation of ³⁵S and ¹⁴C into brain proteins parallelled the results obtained with incorporation into the free pools of amino acids. Amino acid analyses of the free amino acid pools of rabbit brain revealed a general decrease in the concentration of amino acids in response to high plasma homocystine or high plasma homocystine and methionine. Inhibition of protein synthesis which occurs under the above experimental conditions is a general phenomenon. myelin and other brain fractions being equally affected. The decrease in concentration of brain amino acids also results in a diminution in concentration of the neurotransmitters GABA, dopamine and noradrenaline. The possible relationship of the observed changes to homocystinuria is discussed.     

ENZYMIC SYNTHESIS OF PSYCHOSINE SULPHATE

—An enzyme which catalyses the synthesis of psychosine sulphate by the transfer of [³⁵S]sulphate from 3′-phosphoadenosine 5′-phosphosulphate to galactosyl-sphingosine has been demonstrated in the brain of the young mouse. The enzyme activity appears to be bound to a microsomal fraction which is spun down with the synaptosomes. The product of the incubation mixture has been characterized as psychosine sulphate by a variety of TLC separations and other chemical procedures. Several parameters (detergent, cations, substrate and 3′-phosphoadenosine 5′-phosphosulphate concentrations, pH and incubation time), affecting the 3′-phosphoadenosine 5′-phosphosulphate-psychosine sulphotrans-ferase activity, have also been investigated. In the normal mouse brain there is a maximum enzyme activity at 17–19 days after birth, which is the period of most rapid myelin formation. In the brains of Jimpy mice, mutants with myelin deficiency, the activity is reduced and reaches a maximum around the 13th day. The lower activity correlates with the small amounts of sulphatides in Jimpy mouse brains. The results are discussed and related to present knowledge of galactolipid biosynthesis.     

THE FATTY ACID COMPOSITION OF PHOSPHOLIPIDS AND GLYCOLIPIDS IN JIMPY MOUSE BRAIN

Abstract— Phospholipids and sphingolipids from brains of normal and Jimpy mice were isolated in a pure form by thin-layer chromatographic procedures. The fatty acid composition of the major phospholipids, i.e. ethanolamine glycerophospholipids, serine glycerophospholipids, choline glycerophospholipids and inositol glycerophospholipids, as well as sphingomyelin, cerebrosides and sulphatides was determined by gas-liquid chromatography. A specific fatty acid pattern for each of the four glycerophospholipids was found. The fatty acid composition of inositol glycerophospholipid, which has not previously been studied in mouse brain, was characterized by a high concentration of arachidonic acid. After 16 days of age, fatty acid analysis showed definite differences between the phospholipids from normal and mutant brains. A small increase of polyunsaturated fatty acids in glycerophospholipids of ethanolamine, serine and choline from the Jimpy central nervous system was found, which has been explained by the myelin deficiency. Sphingomyelin, cerebrosides and sulphatide analyses showed a wide distribution of saturated and mono-unsaturated fatty acids in both normal and mutant mice. A reduction in the amount of long-chain fatty acids was demonstrated in mutant brain sphingolipids; in sulphatides and cerebrosides, the amount of non-hydroxy fatty acids was reduced to a greater extent than in sphingomyelin. The distribution of fatty acids in sphingolipids from the myelin and microsomal fractions was also investigated in both types of mice. Cerebrosides were characterized by a high content of long-chain fatty acids in myelin as well as in microsomes. Sulphatides and sphingomyelin, on the other hand, showed a higher content of medium-chain fatty acids in microsomes than in myelin. In the mutant brain, the amount of long-chain fatty acids was reduced in both subcellular fractions. The deviation from normal in the pattern of fatty acid distribution in Jimpy brain is discussed in relation to the current concepts of glycolipid biosynthesis.     

METABOLIC COMPARTMENTATION IN THE BRAIN: EFFECTS OF A CENTRAL NERVOUS SYSTEM DEPRESSANT, 1-HYDROXY-3-AMINO-PYRROLIDONE-2

—The effect of 1-hydroxy-3-aminopyrrolidone-2(HA-966), a CNS depressant, was studied on the metabolism of [¹⁴C]glucose and [³H]acetate in the brain in mice. HA-966 had a marked effect on glucose metabolism. The conversion of glucose carbon into amino acids associated with the tricarboxylic acid cycle (‘cycle’) was severely reduced, while the concentration of brain glucose was approximately doubled. Relative to the specific radioactivity of glucose in the brain, the specific radioactivity of alanine was 60–70 per cent of the control, indicating a reduction in the rate of glycolysis, and those of the‘cycle’amino acids were also lowered. A reduction in‘cycle’flux of 30–35 per cent was estimated. It was established that the depressed glucose utilization flux was not due to either impaired uptake of glucose from blood to brain or to hypothermia. In contrast to [¹⁴C]glucose, there was no change in the labelling of the amino acid fraction from [³H]acetate, which is preferentially metabolized in the 'small’compartment believed to be associated with glia. Thus it seems that CNS depression caused by HA-966 resulted in a selective decrease in energy production in the‘large’metabolic compartment where glucose is oxidized preferentially and which is believed to be associated with neuronal structures. The results also suggested that communication between the metabolic compartments mediated via glutamine and GABA was reduced, since the labelling from [³H]acetate of glutamine was increased and that of GABA decreased by HA-966.     

ANALYSIS OF THE MAJOR AMINO ACIDS OF RAT BRAIN AFTER IN VIVO INHIBITION OF GABA TRANSAMINASE BY ETHANOLAMINE O-SULPHATE

The effect of in vivo inhibition of GABA transaminase by ethanolamine O-sulphate on the content of the free amino acids in rat brain has been studied. Intracisternal injection of 2.0 mg/kg resulted in a progressive increase in GABA levels with time, to reach after 8 h a 100 per cent increase over saline-injected control animals. The effect of injection of 0.5, 1.0 and 2.0 mg/kg was studied 24 h after injection and the results showed that the increased GABA levels were dependent on the dose of inhibitor employed. Apart from the substantial increase in the GABA concentration of the brain there were no significant changes in the content of the other amino acids except for a small but significant decrease in aspartic acid in one experiment. When the extent of inhibition of the transaminase was correlated with the rise in GABA concentration it was shown that no elevation occurred until more than half of the enzymic activity had been inhibited.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN LOCI IN VIVO UNDER NORMAL CONDITIONS

By macroautoradiography and by GLC separation, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, hippocampus, thalamus and hypothalamus were investigated. (1) The autoradiographical densities in the thalamus, cerebral neocortex and hippocampus measured with a microdensitometer were higher than that in the hypothalamus at 5 min after subcutaneous injection. At 180 min, densities in the cerebral neocortex, hippocampus and hypothalamus were higher than that in thalamus. (2) The free amino acid levels determined by GLC varied with each brain region. (3) The specific radioactivity (d.p.m./μmol) of alanine in each brain region was higher than that of the other amino acids at 5 min after the injection. The specific radioactivity of GABA in the brain regions was clearly higher than that of (glutamate + glutamine), (aspartate + asparagine) and glycine at 5 and 15 min. (4) The autoradiographical data were in good agreement with the chemical data at 5 min but were different at 180 min. (5) Variations in specific radioactivity of each free amino acid among brain regions at 5 min were influenced greatly by existing free amino acid concentrations in each region.     

CHANGES WITHIN METABOLIC COMPARTMENTS IN THE BRAINS OF YOUNG RATS INGESTING LEAD

—[2-¹⁴C]Glucose and [³H]acetate were injected simultaneously into 19-day-old rats suckling from mothers fed either a normal diet or a diet containing 4·5% lead acetate. Changes in the rate of conversion of both precursors into amino acids associated with the tricarboxylic acid cycle were observed. [^I4C]Glucose. In the brain of young rats ingesting lead, the specific radioactivity of glutamate, aspartate, γ-aminobutyrate and glutamine were all significantly lowered relative to that of glucose. Glutamine labelling was the most affected. [³H]Acetate. In comparison with controls, the total amount of ³H in either water or acid-soluble constituents of the brain was the same, but the ³H content of the amino acids was significantly reduced in the lead-treated rats. In both groups, glutamine had the highest specific radioactivity but the time courses of the labelling of glutamine were different. In the control the peak incorporation was reached during the first 5 min, whereas in the experimental animals this occurred at about 10 min after the injection of the precursor, and the specific radioactivity even at that time was less than in controls. When compared with controls, the depression in the labelling of glutamine was accompanied at 5 min by an increase in the specific radioactivity of aspartate. In the lead-treated rats the labelling of GABA was also slowed and the time course seemed to follow that of glutamine rather than glutamate. In spite of the differences in the metabolism of [³H]acetate, metabolic compartmentation of glutamate, assessed by a glutamine : glutamate specific radioactivity ratio higher than 1, was evident even in the brain of the lead-treated animals, although the values of the ratio at 5 and 10 min were less than in controls. There was no evidence of a diminished supply of substrates to the brain in lead intoxication. The overall changes would be consistent with a retardation in the biochemical maturation of the brain in terms of development of glucose metabolism and metabolic compartmentation.     

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'¹⁴C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [¹⁴C]succinate was rapid, as shown by the incorporation of ¹⁴C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [¹⁴C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-¹⁴C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of ¹⁴C into GABA was very low (< 1 per cent of the amino acid -¹⁴C) after the injection of [1.4-¹⁴C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-¹⁴C]succinate 2 min beforehand. Since the oxidation of [l,4-¹⁴C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce ¹⁴C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD⁺ ratios and [¹⁴C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.     

INTERACTION OF CATECHOLAMINE AND AMINO ACID METABOLISM IN BRAIN: EFFECT OF PARGYLINE AND l-DOPA

Abstract— The combination of l -DOPA and pargyline caused a decrease in level of aspartate and an increase in that of glutamine in vivo in cerebral cortex, cerebellum, brain stem, hypothalamus, neostriatum and cervical cord of rat. There was also a decreased incorporation of radioactivity from [1-¹⁴C]acetate into amino acids in vivo , most notably in cerebellum and brain stem. The labelling of glutamine was especially affected. In addition, cortical slices were prepared from guinea pigs which had been pretreated with pargyline. These slices were incubated with and without 1 m m l -DOPA in media containing [1-¹⁴C]acetate. Pargyline alone caused a stimulation of the labelling of glutamate and aspartate but not glutamine and GABA; the levels of aspartate and GABA were greater than in control slices. The addition of l -DOPA to slices from pargylinized animals caused a severe decrease in glutamine labelling but not in that of glutamate or aspartate; the level of glutamine was increased while that of glutamate was decreased. The results are discussed in terms of the known biochemical and morphological compartmentation of amino acids in brain. It is suggested that catecholamines, in the process of functioning as transmitters, may also function as metabolic regulators of other transmitters, e.g. amino acids, as well as of the energy required for balanced neuronal function.     

Development of a protocol for the automated analysis of amino acids in brain tissue samples and microdialysates

An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN⁻). The resulting highly stable N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2–12.5 μM). The limit of detection for CBI derivatives of amino acids was in the range 5–20 fmol (S/N=2) using a 5 μl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain.     

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.     

INCORPORATION OF 14C FROM [U-14C]GLUCOSE INTO FREE AMINO ACIDS IN MOUSE BRAIN REGIONS UNDER CYANIDE INTOXICATION

—During anoxia induced by the administration of potassium cyanide, [U-¹⁴C]glucose was injected intraperitoneally into adult mice and they were decapitated at 5, 15 and 30 min after the injection. After freeze-drying in vacuo, differences in the uptake of radioactive carbon from [U-¹⁴C]glucose into free amino acids (glutamate + glutamine, aspartate + asparagine, GABA, alanine and glycine) in mouse cerebral neocortex, cerebellar hemisphere, caudate nucleus, thalamus, hypothalamus and medulla oblongata were investigated (by macroautoradiography and GLC separation) and compared with those obtained under normal conditions. (1) During anoxia, autoradiographical densities in the thalamus and medulla oblongata were higher than that in the cerebral neocortex and caudate nucleus. (2) Among specific radioactivities (d.p.m./μmol) of free amino acids, alanine gave the highest value during anoxia, except in the cerebellar hemisphere and hypothalamus at 5 min and the medulla oblongata at 30 min. (3) During anoxia, the specific radioactivities of alanine and glycine in each brain region did not significantly decrease at 15 and 30 min compared with those under normal conditions. During anoxia, the specific radioactivity of glutamate + glutamine in the cerebellar hemisphere and hypothalamus did not significantly decrease compared with the normal conditions, while that of GABA, aspartate + asparagine and glutamate + glutamine in the cerebral neocortex, caudate nucleus, thalamus and medulla oblongata showed an increase. (4) The percentage decrease of glutamate + glutamine and aspartate + asparagine at 5 and 15 min was highly significant in the cerebral neocortex and caudate nucleus.     

STUDIES OF THE INTER-RELATIONSHIP BETWEEN CEREBROSPINAL FLUID AND PLASMA AMINO ACID CONCENTRATIONS IN NORMAL INDIVIDUALS

Abstract— —The concentration of free amino acids has been determined in lumbar CSF in 37 fasting normal subjects. The values obtained have been compared with the concentration of the same amino acids measured in venous plasma collected simultaneously and with ventricular CSF amino acid concentrations. Twenty-three amino acids have been identified and quantitated in CSF and plasma. Trace quantities of eight other amino acids have been also detected.
The concentration of 13 amino acids in CSF has been shown to be directly related to the plasma concentration. No such relationship was noted for the other 7 amino acids. Significant variations in the concentration of individual amino acids relating to both age and sex have been noted. A large number of unidentified ninhydrin positive compounds have been found in CSF. Preliminary studies have identified one of these as ɛ-aminobutyric acid (GABA).     

Perinatal changes of transport systems for amino acids in slices of mouse brain

Perinatal changes in the uptake of amino acids were measured in slices of fetal (15- and 19-day) and newborn (4-, 24-, and 48-hr-old) mouse brain. Uptake increased with age; smaller changes occurred with basic and neutral amino acid transport systems, and the largest changes occurred in fetal brain with amino acids of putative neurotransmitter function (taurine, glycine, GABA, and the acidic amino acids). The pattern of increase in uptake was similar at high and at low external amino acid concentrations. Developmental changes in tissue content of Na⁺, K⁺, or ATP were small during this period, and so are unlikely to be responsible for the observed changes in uptake. It appears that by the 15th day of fetal life, the transport systems for essential amino acids are fairly well developed in the brain, and the transport systems for neurotransmitter amino acids are not so well developed, but undergo a rapid increase in the 15–19-day period. From birth to adulthood, the concentrative capacity of slices of mouse brain for nonessential (putative neurotransmitter) amino acids is much greater than for essential amino acids.This research was supported in part by NIH Grant No. RR05707.     

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.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the brain.