METABOLISM OF GLUTAMATE AND RELATED AMINO ACIDS IN THE 10-DAY-OLD MOUSE BRAIN: EXPERIMENTS WITH LABELLED ACETATE AND β-HYDROXYBUTYRATE

Abstract– The pattern of incorporation of [³H, 1-¹⁴C]- and [³H. 2-¹⁴C]acetate into glutamate and related amino acids was studied in the brain of 10-day-old mice. A comparison of these patterns with those obtained for the adult brain led to the suggestion that the glutamate pool labelled directly by acetate is a much larger fraction of the total glutamate pool in the 10-day-old brain than it is in the adult brain.
Some data on the pattern of labelling of brain amino acids by 3-hydroxybutyrate. glucose and acetate support the hypothesis that direct carboxylation of pyruvate is somewhat more active in the immature than in the mature brain.
Differences in the labelling patterns of free and protein-bound brain amino acids by acetate, do indicate that the free amino acid pool labelled by acetate is not the precursor pool for protein synthesis.     

14C-LABELLED AMINO ACIDS AND GLUCOSE IN RAT BRAIN AND LIVER AFTER INJECTION OF [2-14C]PROPIONATE

Abstract— [2-¹⁴C]Propionate injected into rats was metabolized into [¹⁴C]glucose and ¹⁴C-labelled aspartate, glutamate, glutamine and alanine. The results are consistent with the conversion of propionate into succinate and the oxidation of succinate into oxaloacetate, the precursor of labelled amino acids and the substrate for gluconeogenesis.
The ratio of the specific radioactivity of glutamine to glutamate was greater than 1 during the 30 min period in the brain, indicating that propionate taken up by the brain was metabolized mainly in the 'small glutamate compartment' in the brain. The results, therefore, support the previous conclusion (G aitonde , 1975) that the labelling of amino acids by [¹⁴C]propionate formed from [U-¹⁴C>]-threonine in thiamin-deficient rats was metabolized in the 'large glutamate compartment' of the brain.
The specific radioactivity ratio of glutamine to glutamate in the liver was less than 1 during the 10 min period but greater than 1 at 30min. These findings which gave evidence against metabolic compartments of glutamate in the liver, were interpreted as indicative of the entry of blood-borne [¹⁴C]glutamine synthesized in other tissues, e.g. brain. The labelling of amino acids when compared to that after injection of [U-¹⁴C]glucose showed that [2-¹⁴C]propionate was quantitatively a better source of amino acids in the liver. The concentration of some amino acids in the brain and liver was less in the adult than in the young rats, except for alanine and glutathione, where the liver content was more than double that in the adult.     

METABOLISM OF GLUCOSE AND FREE AMINO ACIDS IN BRAIN, STUDIED WITH 14C-LABELLED GLUCOSE AND BUTYRATE IN RATS INTOXICATED WITH CARBON DISULPHIDE

Abstract— The effect of 15 h continuous exposure to CS₂ on the metaboliam of glucose and free amino acids in the brain of rats was studied. CS₂ caused a moderate hypoglycaemia. There were also changes in the amounts of some amino acids in the brain. Glutamate and γ-aminobutyrate were lower whereas glutamine was markedly increased. Comparative studies in vivo of the metabolism of [2-¹⁴C]glucose and [1-¹⁴C]butyrate indicated that CS₂ did not affect glycolysis or the incorporation of ¹⁴C from glucose into amino acids except into γ-aminobutyrate which was reduced. Contrary to the findings with [¹⁴C]glucose, CS₂ provoked distinct changes in the labelling of amino acids when [¹⁴C]butyrate was the precursor. The most notable change was a markedly increased incorporation of ¹⁴C into glutamine. Based on the two-compartment model of brain glutamate the experimental findings indicated that CS₂ affected metabolism associated with the 'small' pool of glutamate but had a minimal effect on metabolism associated with the 'large' glutamate pool. The possibility is suggested that the changes observed involved an increased rate of ammonia removal. The low incorporation of ¹⁴C into γ-aminobutyrate from either precursor is consistent with other evidence showing that CS₂ interferes with pyridoxal phosphate-dependent enzymes.     

METABOLISM OF γ-HYDROXY-[1-14C] BUTYRATE BY RAT BRAIN: RELATIONSHIP TO THE KREBS CYCLE AND METABOLIC COMPARTMENTATION OF AMINO ACIDS

Abstract— Ninhydrin decarboxylation experiments were carried out on the labelled amino acids produced following intraventricular injection of either γ-hydroxy-[1-¹⁴C]butyric acid (GHB) or [1-¹⁴C] succinate. The loss of isotope (as ¹⁴CO₂) was similar for both substances. The [1-¹⁴C]GHB metabolites lost 75% of the label and the [1-¹⁴C] succinate metabolites lost 68%. This observation gives support to the hypothesis that the rat brain has the enzymatic capacity to metabolize [1-¹⁴C]GHB to succinate and to amino acids that have the isotope in the carboxylic acid group adjacent to the a-amino group. These results also indicate that the label from [1-¹⁴C]GHB does not enter the Krebs cycle as acetate. The specific activity ratio of radiolabelled glutamine to glutamic acid was determined in order to evaluate which of the two major metabolic compartments preferentially metabolize GHB. It was found that for [1-¹⁴C]GHB this ratio was 4.20 ± 0.18 (S.E. for n = 7) and for [l-¹⁴C]succinate this ratio was 7.71 (average of two trials, 7.74 and 7.69). These results suggest that the compartment thought to be associated with glial cells and synaptosomal structures is largely responsible for the metabolism of GHB. Metabolism as it might relate to the neuropharmacological action of GHB is discussed.     

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.     

Glutamine and Glucose as Precursors of Transmitter Amino Acids: Ex Vivo Studies

Abstract: Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo . Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-¹⁴C]glucose, [U-¹⁴C]glutamine, or [³H]glutamine administered alone or together in doublelabel experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 μ M ); tetrodotoxin (1 μ M ) was present in the control medium. Single-label experiments showed that [U-¹⁴C]- glutamine was more effective than [U-¹⁴C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [³H]glutamine and [U-¹⁴C]- glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment empbasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.     

GAMMA-HYDROXYBUTYRATE DEGRADATION IN THE BRAIN IN VIVO: NEGLIGIBLE DIRECT CONVERSION TO GABA

Abstract— The metabolism of γ-hydroxybutyrate (GHB) was studied by following the fate of [1-¹⁴C]GHB in mouse brain after an intravenous injection. Cerebral uptake of GHB was rapid and this substance disappeared from brain tissue with a half-life of approx 5 min. Degradation of [1-¹⁴C]GHB took place in the brain since ¹⁴C was incorporated in amino acids associated with the tricarboxylic acid cycle: the labelling pattern was consistent with the oxidation of GHB via succinate through the cycle, rather than with β-oxidation of GHB. Conversion of [¹⁴C]GHB into [¹⁴C]GABA prior to oxidation was negligible, thus it is unlikely that the pharmacological action of GHB would be mediated through GABA formation. [¹⁴C]GHB oxidation also elicited the signs of metabolic compartmentation of the tricarboxylic acid cycle in the brain (glutamine/glutamate specific radioactivity ratio was about 4).     

[2-3H]GLYCEROL AS A PRECURSOR OF PHOSPHOLIPIDS IN RAT BRAIN: EVIDENCE FOR LACK OF RECYCLING

Abstract— When [2-³H]glycerol was injected intracranially into young rats, it was presented as a pulse label, leaving the brain rapidly and giving up much of its labelled hydrogen to water. [2-³H]glycerol was efficiently incorporated into brain lipids, especially into choline and ethanolamine phospholipids. Following injection of a mixture of [³H]- and [¹⁴C]-labelled glycerol, the ratio of ³H to ¹⁴C in the phospholipids of both whole brain and the microsomal fraction decreased as a function of time after injection. This finding indicated less recycling of the tritium label. This lack of recycling was further indicated by the finding that 94 per cent of the tritium label of phosphatidyl choline was in the glycerol portion of the molecule rather than in the fatty acids. At 2 weeks following injection with [³H]glycerol, 93 per cent of the total radioactivity in brain appeared in the lipid fraction. In contrast, following injection with [¹⁴C]glycerol, only 57 per cent of the radioactivity appeared in lipid, with about 20 per cent in protein.     

SYNTHESIS OF GLYCOPROTEINS AND GANGLIO-SIDES IN DEVELOPING RAT BRAIN

Abstract— Intracerebral injections of radioactive fucose into developing rats resulted in specific labelling of the brain glycoproteins in their fucose moieties. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate revealed that the radioactive glycoproteins were very heterogeneous with regard to molecular weight. A procedure utilizing [³H]fucose and [¹⁴C]fucose together with double-label counting techniques was developed for comparing the electrophoretic patterns of newly synthesized glycoproteins from different samples of tissue. By the use of this procedure we showed that the incorporation of radioactive fucose into the glycoproteins of high mol. wt. was relatively greater in the brains of 5-day-old rats than in those of 25-day-old rats. Intracerebral injection of N -[ Ac -³H]acetyl- d -mannosamine resulted in a high degree of specificity for the labelling of sialic acid moieties in glycoproteins and gangliosides. The ratio of the d.p.m. of N -[³H]acetylmannosamine incorporated into glycoproteins to the d.p.m. incorporated into gangliosides was higher in 5-day-old rats than in 15- or 25-day-old rats. Experiments in which 15-day-old rats were injected with a mixture of [¹⁴C]fucose and N -[³H]acetylmannosamine showed that there were differences in the relative degrees of incorporation of the two radioactive precursors into the various glycoproteins. The greatest incorporation of [¹⁴C]fucose relative to that of N- [³H]acetylmannosamine occurred in some of the glycoproteins of smaller mol. wt.     

[14C]AMINO ACID FORMATION FROM LABELLED GLUCOSE AND/OR ACETATE IN BRAIN CORTEX SLICES WITH EXPERIMENTALLY ELICITED PROLIFERATION OF ASTROGLIA. CORRELATION OF BIOCHEMICAL AND MORPHOLOGICAL CHANGES

Abstract— Changes in morphology and in transformations of [U-¹⁴C]glucose and [1-¹⁴C]acetate into amino acids of the brain cortex were followed on the Sth, 10th and 21st days after production of mechanical lesions and compared with control tissue. In the experimental tissue, proliferation of astroglia and reduction of the number of neurons had taken place. On the 10th day, accumulation of mitochondria and of some gliofilaments in the cytoplasm of astroglia was observed. On the 21st day, the gliofilaments occupied a substantial portion of the astroglial cytoplasm and the mitochondria were reduced in number and compressed to the cell membrane. Incorporation of ¹⁴C from acetate into amino acids was substantially increased on the 10th day (up to 240% with respect to controls) and normalized again on the 21st day. Incorporation of [¹⁴C]glucose into amino acids decreased somewhat during the experimental period. It has been proposed that the proliferation of astrocytes and their ultrastructural changes may account for the increased transformation of [¹⁴C]acetate into amino acids, in particular into glutamine which is formed from the small glutamate pool.     

THE RELEASE OF AMINO ACIDS FROM THE HEMISECTED SPINAL CORD DURING STIMULATION

Abstract— Isolated frog or toad hemicords were incubated for 40 min with either [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate. l -[¹⁴C]aspartate, l -[¹⁴C]serine, l [¹⁴C]threonine or l -[³H]leucine, and the release of these compounds from the cord was measured under resting conditions and during electrical stimulation. Stimulation of spinal roots produced no significant change in the efflux of any of the compounds tested. Direct stimulation of the rostral cord however, produced a large increase in the efflux of [¹⁴C]glycine, [³H]GABA, l -[¹⁴C]glutamate and l -[¹⁴C]aspartate. These increased effluxes were calcium dependent, the effects of stimulation being reduced in a calcium-free, or magnesium-supplemented (10 mM) medium. Stimulation failed to produce an increase in the efflux of l -[¹⁴C]serine, l -[¹⁴C]threonine, l -[¹⁴H]leucine, [¹⁴C]mannitol or [¹⁴C]urea. These results are consistent with the suggestions that glycine, GABA, glutamate and aspartate may be synaptic transmitters in the spinal cord.     

Oligodendroglial Glycerophospholipid Synthesis: Incorporation of Radioactive Precursors into Ethanolamine Glycerophospholipids by Calf Oligodendroglia Prepared by a Percoll Procedure and Maintained in Suspension Culture

Abstract: Oligodendroglia prepared from minced calf cerebral white matter by trypsinization at pH 7.4, screening, and isosmotic Percoll (polyvinylpyr-rolidone-coated silica gel) density gradient centrifugation survived in culture on polylysine-coated glass, extending processes and maintaining phenotypic characteristics of oligodendroglia. In the present study, ethanolamine glycerophospholipid (EGP) metabolism of the freshly isolated cells was examined during short-term suspension culture by dual label time course and substrate concentration dependence experiments with [2-³H]glycerol and either [1,2-¹⁴C]ethanolamine or L-[U-¹⁴C]serine. Rates of incorporation of ³H from the glycerol and of ¹⁴C from the ethanolamine into EGP were constant for 14 h. In medium containing 3 mM-[1,2-¹⁴C]ethanolamine and 4.8 mM-[2-³H]glycerol, rates of incorporation of ¹⁴C and ³H into diacyl glycerophosphoethanolamine (diacyl GPE) were similar. Under the same conditions, ³H specific activities of alkylacyl GPE and alkenylacyl GPE were much lower than ¹⁴C specific activities, likely as a result of the loss of tritium during synthesis of these forms of EGP via dihydroxyacetone phosphate. L-[U-¹⁴C]serine was incorporated into serine glycerophospholipid (SGP) by base exchange rather than de novo synthesis. ¹⁴C from L-[U-¹⁴C]serine also appeared in EGP after an initial lag period of several hours. Methylation of oligodendroglial EGP to choline glycerophospholipid (CGP) was not detected.     

Effect of Chronic Lead Ingestion by Rats on Glucose Metabolism and Acetylcholine Synthesis in Cerebral Cortex Slices

Abstract: The effect of chronic low-level lead (Pb²⁺) ingestion on the metabolic pathways leading to the acetyl moiety of acetylcholine (ACh) was examined. Cerebral cortex slices, prepared from untreated or Pb²⁺-exposed rats (600 ppm lead acetate in the drinking water for 20 days), were incubated in Krebs-Ringer bicarbonate buffer with 10 m M glucose and tracer amounts of [6-³H]glucose and either [6-¹⁴C]glucose or [3-¹⁴C] β -hydroxybutyrate. Altering the concentration of Pb²⁺ in the drinking water produced a dose-related increase in blood and brain lead levels. When tissue from Pb²⁺-exposed rats was incubated with mixed-labeled glucose, incorporation into lacate, citrate, and ACh was considerably decreased, although no changes occurred in the ³H/¹⁴C ratios. Similar effects of Pb²⁺ were found when ¹⁴C-labeled β -hydroxy-butyrate was substituted for the [¹⁴C]glucose. It appears from these data that Pb²⁺ exerts a generalized effect on energy metabolism and not on a specific step in glucose metabolism. The impairment of glucose metabolism may explain partially the Pb²⁺-induced changes observed in cholinergic function.     

THE BIOSYNTHESIS OF CHOLESTEROL AND OTHER STEROLS BY BRAIN TISSUE

Abstract— The distribution of [¹⁴C]labelled material into subcellular fractions of 30-day-old rat brain was studied as a function of time, following intracerebral injection of [2-¹⁴C] mevalonic acid. As in the adult and 15-day-old brain, the microsomal fraction was indicated as the site of sterol synthesis. Unlike the 15-day-old animal, the myelin fraction from the 30-day-old rat was the predominately labelled fraction at 2 weeks after injection of the animal. Significant amounts of [¹⁴C]cholesterol were not present until about 4 h after injection. In order to ascertain whether different populations of cholesterol were being labelled, depending on the age of the animal injected, we compared the labelling of myelin and non-myelin components in animals injected at 15 or at 30 days of age, and sacrificed, respectively, from 14 to 29 days or from 1 to 28 days after injection. Our results indicated that there was an apparent shift of labelled sterol from non-myelin to myelin fractions at about 37–44 days of age.     

[14C]GLUTAMATE UPTAKE AND COMPARTMENTATION IN GLIA OF RAT DORSAL SENSORY GANGLION

Abstract— The characteristics of the uptake of l -[U-¹⁴C] glutamate into rat dorsal sensory ganglia were investigated. The uptake was mediated by two distinct kinetic systems, with apparent K_m values of the order of 10⁻³ M (low affinity) and 10⁻⁵ m (high affinity). The high affinity uptake system was strongly dependent upon temperature and sodium ion concn, and was depressed by a number of metabolic inhibitors. Following uptake, [¹⁴C] glutamate was extensively metabolized, primarily to glutamine, although this was not so with cultured ganglia, where in addition to an increased uptake of [¹⁴C] glutamate, the specific radioactivity of glutamate was increased and that of glutamine decreased. The labelled substrates [U-¹⁴C]pyruvate and [U-¹⁴C] acetate were used to investigate this phenomenon and the results are discussed in relation to current knowledge of metabolic compartmentation in nervous tissue.     

Glucose Metabolism in the Developing Cerebral Cortex as Detected by 1H{13C} Nuclear Magnetic Resonance Spectroscopy Ex Vivo

Abstract: Metabolism of [1-¹³C]glucose was monitored in superfused cerebral cortex slice preparations from 1-, 2-, and 5-week-old rats using ¹H-observed/¹³C-edited (¹H{¹³C}) NMR spectroscopy. The rate of label incorporation into glutamate C-4 did not differ among the three age groups: 0.52–0.67% of total ¹H NMR-detected glutamate/min. This was rather unexpected, as oxygen uptake proceeded at 1.1 ± 0.1, 1.9 ± 0.1, and 2.0 ± 0.1 µmol/min/g wet weight in brain slices prepared from 1-, 2-, and 5-week-old animals, respectively. Steady-state glutamate C-4 fractional enrichments in the slice preparations were ∼23% in all age groups. In the acid extracts of slices glutamate C-4 enrichments were smaller, however, in 1- and 2-week-old (17.8 ± 1.7 and 16.8 ± 0.8%, respectively) than in 5-week-old rats (22.7 ± 0.7%) after 75 min of incubation with 5 m M [1-¹³C]glucose. We add a new assignment to the ¹H{¹³C} NMR spectroscopy, as acetate C-2 was detected in slice preparations from 5-week-old animals. In the acid extracts of slice preparations acetate C-2 was labeled by ∼30% in 5-week-old rats but by 15% in both 1- and 2-week-old animals, showing that the turnover rate was increased in 5-week-old animals. In the extracts 3–4% of the C-6 of N -acetyl-aspartate (NAA; CH₃ of the acetyl group) contained label as determined by both NMR and mass spectrometry, which indicated that there was no significant labeling to other carbons in NAA. NAA accumulated label from [1-¹³C]glucose but not from [2-¹³C]acetate, and the rate of label incorporation increased by threefold on cerebral maturation.     

Portacaval Anastomosis Results in Altered Neuron-Astrocytic Metabolic Trafficking of Amino Acids: Evidence from 13C-NMR Studies

Abstract: ¹³C-NMR spectroscopy was used to evaluate the dynamic consequences of portacaval anastomosis on neuronal and astrocytic metabolism and metabolic trafficking between neurons and astrocytes. Glutamate is predominantly labeled from [1-¹³C]glucose, whereas [2-¹³C]acetate is more efficient in labeling glutamine, in accordance with its primary metabolism in astrocytes. Alanine and succinate labeling was only observed with [1-¹³C]glucose as precursor. Brain [1-¹³C]glucose metabolism in portacaval-shunted rats was similar to that in sham-operated controls with the exception of labeled glutamine and succinate formation, which was increased in shunted rats. The ¹³C enrichment was, however, decreased owing to an increase in total glutamine and succinate. Using [2-¹³C]acetate, on the other hand, flux of astrocytic label to neurons was severely decreased because label incorporation into glutamate, aspartate, and GABA was decreased following portacaval shunting. The latter amino acids are predominantly localized in neurons. These findings demonstrate that metabolic trafficking of amino acids from astrocytes to neurons is impaired in portacaval-shunted rats.     

Synthesis of Acetylcholine from Acetate in a Sympathetic Ganglion

Abstract: The present experiments tested whether acetate plays a role in the provision of acetyl-CoA for acetylcholine synthesis in the cat's superior cervical ganglion. Labeled acetylcholine was identified in extracts of ganglia that had been perfused for 20 min with Krebs solution containing choline (10⁻⁵ M ) and [³H], [1-⁴C], or [2-¹⁴C]acetate (10³ M ); perfusion for 60 min or with [³H]acetate (10⁻² M ) increased the labeling. The acetylcholine synthesized from acetate was available for release by a Ca²⁺-dependent mechanism during subsequent periods of preganglionic nerve stimulation. When ganglia were stimulated via their preganglionic nerves or by exposure to 46 m M K⁺, the labeling of acetylcholine from [³H]acetate was reduced when compared with resting ganglia. The reduced synthesis of acetylcholine from acetate during stimulation was not due to acetate recapture, shunting of acetate into lipid synthesis, or the transmitter release process itself. In ganglia perfused with [2-¹⁴C]glucose, the amount of labeled acetylcholine formed was clearly enhanced during stimulation. An increase in acetylcholine labeling from [³H]acetate was shown during a 15-min resting period following a 60-min period of preganglionic nerve stimulation (20 Hz). It is concluded that acetate is not the main physiological acetyl precursor for acetylcholine synthesis in this sympathetic ganglion, and that during preganglionic nerve stimulation there is enhanced delivery of acetyl-CoA to choline acetyltransferase from a source other than acetate.     

Effects of Ketone Bodies on Astrocyte Amino Acid Metabolism

Abstract: The effects of acetoacetate and 3-hydroxybutyrate on glial amino acid metabolism were studied in primary cultures of astrocytes. The exchange of nitrogen among amino acids was measured with ¹⁵N as a metabolic probe and gas chromatography-mass spectrometry as a tool with which to quantify isotope abundance. Addition of either acetoacetate or 3-hydroxybutyrate (5 m M ) to the incubation medium did not alter the initial rate of appearance of [¹⁵N]glutamate in the glia, but it did inhibit transamination of glutamate to [¹⁵N]aspartate. Addition of acetoacetate also inhibited formation of [2-¹⁵N]glutamine, but 3-hydroxybutyrate had a stimulatory effect. The presence in the medium of sodium acetate (5 m M ) was also associated with diminished production of [¹⁵N]aspartate and [2-¹⁵N]glutamine with [¹⁵N]glutamate as precursor. Studies with [2-¹⁵N]glutamine as precursor indicated that treatment of the astrocytes with ketone bodies did not alter flux through the glutaminase pathway. Nor did the presence of the ketone bodies reduce significantly the flux of nitrogen from [¹⁵N]GABA to [2-¹⁵N]glutamine when the former species served as a metabolic tracer. The concentration of internal citrate increased in the presence of acetoacetate, 3-hydroxybutyrate, and acetate. Studies with purified sheep brain glutamine synthetase showed that citrate inhibited this enzyme. These findings are considered in terms of the known anticonvulsant effect of a ketogenic diet.     

The Synthesis, Storage, and Release of Propionylcholine by the Electric Organ of Torpedo marmorata

Abstract: Little is known about the specificity of the mechanisms involved in the synthesis and release of acetylcholine for the acetyl moiety. To test this, blocks of tissue from the electric organ of Torpedo were incubated with either [1-¹⁴C]acetate or [1-¹⁴C]propionate, and the synthesis, storage, and release of [1-¹⁴C]acetylcholine and [¹⁴C]propionylcholine were compared. To obtain equivalent amounts of the two labeled choline esters, a 50-fold higher concentration of propionate than of acetate was needed. Following subcellular fractionation, similar proportions of [¹⁴C]acetylcholine and [¹⁴C]propionylcholine were recovered with synaptosomes and with synaptic vesicles. Furthermore, both labeled choline esters were protected to a similar extent from degradation during homogenization of tissue in physiological medium, indicating that the two choline esters were equally well incorporated into synaptic vesicles. Yet depolarization of tissue blocks by 50 m M KCI released much less [¹⁴C]propionylcholinc than [¹⁴C]acetylcholine. During field stimulation of the tissue blocks, the difference between the releasibility of the two choline esters was less marked, but acetylcholine was still released in preference to propionylcholine. Evidence for specificity of the release mechanism was also obtained when the release of the two choline esters in response to field stimulation was compared in tissue blocks preincubated with both [³H]choline and [¹⁴C]propionate.