GLUCOSE AND AMINO ACID METABOLISM IN ISOLATED NEURONAL AND GLIAL CELL FRACTIONS IN VITRO

Abstract—

• 1 The metabolism of three substrates, [U-¹⁴C]glucose, [U-¹⁴C]pyruvate and [U-¹⁴C]glutamate has been studied in vitro in neuronal and glial cell fractions obtained from rat cerebral cortex by a density gradient technique.
• 2 The mixed cell suspension, after washing, metabolized glucose and glutamate in a manner essentially similar to the tissue slice. Exceptions were a reduced ability to generate lactate from glucose and alanine from glutamate, and a lowered effect of added glucose in suppressing the production of aspartate from glutamate.
• 3 After 2 hr incubation with [U-¹⁴C]glucose, the concentration of the amino acids glutamate, glutamine, GABA, aspartate and alanine were raised in the neuronal, compared to the glial fraction to 234 per cent, 176 per cent, 202 per cent, 167 per cent and 230 per cent respectively although both were lower than in the tissue slice. Incorporation of radio-activity was absolutely lower in the neuronal fraction, however, and the specific activities of the amino acids were: glutamate 12 per cent, GABA 18 per cent, aspartate 34 per cent, and alanine 33 per cent of those in the glial fraction.
• 4 After the incubation with [U-¹⁴C]pyruvate, the pool size of the amino acids were higher than after incubation with glucose, except for GABA, which was reduced to one-third. The concentrations of the amino acids glutamate, glutamine, GABA, aspartate, and alanine in the neuronal fraction were respectively 46 per cent, 143 per cent, 105 per cent, 97 per cent, and 57 per cent of those in the glial. Thus, with the exception of alanine, the specific activity of the neuronal amino acids compared to the glial was little increased when pyruvate replaced glucose as substrate.
• 5 After 2 hr incubation with [U-¹⁴C]glutamate in the presence of non-radioactive glucose, the pool sizes of all the amino acids were increased in both neuronal and glial fractions, with the exception of neuronal alanine and glial glutamine. The concentrations of the amino acids glutamine, GABA, aspartate and alanine were raised in the neuronal fraction, compared to the glial, to 425 per cent, 187 per cent, 222 per cent, and 133 per cent respectively. The specific activities of all the amino acids were higher than with glucose alone with the exception of alanine, and neuronal GABA. Neuronal glutamine and aspartate had specific activities respectively 102 per cent and 84 per cent of glial.
• 6 An unidentified amino acid, with R_F comparable to that of alanine and specific activity close to that of glutamate, was also present after incubation. It was relatively concentrated in the neuronal fraction.
• 7 The distribution of the enzymes glutamate dehydrogenase, aspartate aminotransferase, glutamate decarboxylase and glutamine synthetase between the cell fractions was studied. With the exception of glutamine synthetase, none of the enzymes was lost from the cell fractions during their preparation. Only 14 per cent of the glutamine synthetase, compared with 75 per cent of total protein, was recovered in the fractions. Of the enzymes, glutamate dehydrogenase activity was 406 per cent, and glutamate synthetase activity 177 per cent in the neuronal fraction compared to the glial in the absence of detergent. In the presence of detergent, glutamate dehydrogenase control was 261 per cent, aspartate aminotransferase activity 237 per cent is the neuronal as compared to the glial fraction.
• 8 Incorporation of radioactivity into acid-insoluble material from either glutamate or pyruvate was twice as high into the neuronal as the glial fraction.
• 9 The extent to which these differences may be extrapolated back to the intact tissue is considered, and certain correction factors calculated. The significance of the observations for an understanding of the compartmentation of amino acid pools and metabolism in the brain, and the possible identification of such compartments, is discussed.

     

GLUCOSE AND AMINO ACID METABOLISM IN SOME INVERTEBRATE NERVOUS SYSTEMS

(1) The in vitro metabolism of [U-¹⁴C]glucose and [U-¹⁴C]glutamate was compared in snail, octopus and locust ganglia, and in rat cerebral cortex. (2) The metabolic patterns are quantitatively similar. The major labelled metabolites formed from glucose or glutamate by rat cortex and the invertebrate systems were CO₂, aspartate, glutamate, glutamine and alanine. γ-Aminobutyric acid (GABA) was formed in substantial amounts only by locust and rat. (3) A much larger proportion of labelled glucose and glutamate was converted to alanine by the invertebrates compared with rat cortex, although ¹⁴CO₂ production was lower. (4) The effect of glucose in reducing aspartate formation and stimulating glutamine formation from [U-¹⁴C]glutamate in mammalian cortex was observed in the locust but not in the molluscs. (5) Labelled citric acid cycle intermediates were formed in substantial quantities from glucose and glutamate only by snail and locust.     

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.     

METABOLISM OF GLUCOSE, AMINO ACIDS, AND SOME RELATED METABOLITES IN THE BRAIN OF TOADS (BUFO BOREAS) ADAPTED TO FRESH WATER OR HYPEROSMOTIC ENVIRONMENTS

The levels and specific radioactivities (SA) of glucose, lactate, pyruvate, α-oxoglutarate and seven amino acids in the brain of toads adapted to fresh water or to an hyperosmotic environment were analysed at various times (5 min–4 h) after an injection of [U-¹⁴C]glucose into the bloodstream. The concentrations and SA of glucose, lactate and five amino acids in blood plasma also were measured. In addition, the SA of glutamine, glutamate, aspartate and GABA in brain were determined 30 min after an injection of [1,5-¹⁴C]citrate into the cisterna magna. The flow of labelled carbon atoms from glucose to amino acids and related metabolites in the toad brain was qualitatively similar to that in the mammalian brain, but quantitatively less than one-tenth of the rate in the brain of rats. Hyperosmotic adaptation induced a large increase in the levels of glucose and amino acids in the brain without affecting the rate of glucose utilization. The SA of several amino acids relative to the SA of glucose were initially lower in hyperosmotically-adapted toads than in toads adapted to fresh water, presumably because of a greater dilution of isotope by the larger amino acid pools in the hyperosmotically-adapted toads. The rates of synthesis of alanine and glutamine from pyruvate and glutamate, respectively, appeared to increase with hyperosmotic adaptation, but the rate of GABA synthesis from glutamate was unaltered. The SA of α-oxoglutarate and glutamate were similar at all time periods in both groups of toads, an indication that these compounds were interconverted much more rapidly than the rate at which α-oxoglutarate was formed from isocitrate. The SA of lactate in comparison to that of glucose varied but was always considerably lower, even at 4 h after the [¹⁴C]glucose injection. After[U-¹⁴C]glucose, glutamine had a SA lower than that of glutamate, whereas after the injection of [1⁴C]citrate, glutamine was formed with a SA much higher than that of glutamate. Hence, glutamate in the toad brain exhibited metabolic compartmentation similar to that in rat brain.     

Some neurochemical aspects of fluorocitrate intoxication

Abstract— Some metabolic and biochemical effects of fluorocitrate were studied in vivo in rat brain and cat spinal cord. During the preconvulsant and convulsant phases of fluorocitrate poisoning the contents of free glutamate, glutamine and aspartate declined progressively, while that of alanine increased. Incorporation of ¹⁴C from [U-¹⁴C]glucose into these amino acids also decreased, although somewhat more gradually. GABA exhibited a biphasic change, its content rising after an initial decrease while its relative specific activity rose initially and subsequently diminished. Incorporation of ¹⁴C from [U-¹⁴C]glucose and [U-¹⁴C]lysine into neural protein declined sharply. The citric acid content rose markedly in rat brain and cat spinal cord. In rat brain the glycogen content declined but ATP and ammonia contents were unchanged. The significance of these results with respect to energy metabolism and the possible mechanism of the convulsions during fluorocitrate poisoning is discussed.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

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.     

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.     

The effect of hexafluorodiethyl ether (flurothyl) on the metabolism of rat brain amino acids labelled by (U-14C)glucose

The incorporation of radioactivity into the Krebs cycle related amino acids from [U-¹⁴C]glucose has been studied in rat brain during the development of a seizure induced by Flurothyl at (1) the time of the first myoclonic jerk, (2) 40 seconds later, and (3) at the onset of the seizure. Under the experimental conditions employed no change was observed in the levels of the amino acids or in the specific radioactivity of glutamate, aspartate, glutamine, GABA or alanine at any of these times.     

THE INFLUENCE OF INTRAVENTRICULARLY INJECTED AMINO ACID EXCITANTS ON THE LABELLING OF ENDOGENOUS BRAIN AMINO ACIDS FROM [U-14C]ACETATE IN NEMBUTALIZED MICE

Mice were anaesthetized with nembutal and the effects of intraventricularly injected excitant amino acids on [U-¹⁴C]acetate metabolism were investigated. The natural excitant amino acids, l -glutamate and l -aspartate, reduced the incorporation of ¹⁴C from [U-¹⁴C]acetate into glutamine, GAB A and possibly alanine. The synthetic excitant amino acid, N-methyl-d -aspartate caused a reduction in the incorporation of ¹⁴C from intraventricularly injected [U-¹⁴C]acetate into all of the brain amino acids labelled by [U-¹⁴C]acetate within 5 min. It is suggested that these effects may be due to changes in pool sizes of tricarboxylic cycle intermediates, to inhibition of acetyl-CoA formation, or both. Differences in the metabolic effects of the synthetic and natural excitants are interpreted in terms of the uptake of the natural amino acids into glutamine-forming pool(s) of glutamate metabolism.     

Stimulatory effect of gamma-aminobutyric acid upon animo acid incorporation into protein by a ribosomal system from immature rat brain

Abstract—

• 1 GABAstimulated the incorporation of L-[U-¹⁴C]leucine, primarily into the particulate protein of a ribosomal system from immature rat brain, but not from immature rat liver.
• 2 The GABA effect required the presence of Na⁺ and occurred at GABA concentrations which are thought to be physiological (1–5 mM).
• 3 Of all other amino acids tested at tissue extract concentrations in the system, only glycine had a similar effect. No analogues of GABA tested had a significant stimulatory effect upon leucine incorporation into protein, with the exception of homocarnosine which was mildly stimulatory.
• 4 The effect of GABA upon the incorporation of L-[U-¹⁴C]leucine was examined in the presence of added amino acid substrates, both individually and as mixtures. Also, the incorporation of L-[U-¹⁴C]leucine was compared with incorporation of L-[U-¹⁴C]Iysine and L-[U-¹⁴C]phenylalanine. The results are discussed in terms of GABA interaction with activating, transfer and transport mechanisms of other amino acids, inhibition of proteinase activity, and the possibility that GABA is stimulating the synthesis or turnover of specific proteins in the brain ribosomal system.
• 5 The results illustrate the fact that studies of ‘protein synthesis’ in immature rat brain ribosomes, as measured by amino acid incorporation, will yield answers which depend heavily upon substrate conditions and upon the labelled amino acid used as the marker for protein synthesis or turnover.

     

GLUTAMINE UPTAKE AND METABOLISM BY THE ISOLATED TOAD BRAIN: EVIDENCE PERTAINING TO ITS PROPOSED ROLE AS A TRANSMITTER PRECURSOR

Abstract— Hemisections of toad brains, when incubated in a physiological medium containing no glutamine. released considerable amounts of this amino acid into the medium. When glutamine was included in the medium at a concentration of 0.2 mm the net efflux from the tissue was reduced but not totally prevented. Although there was no net uptake of glutamine, the tissue did accumulate [U-¹⁴C]glu-tamine and some of this labelled glutamine was rapidly metabolized to glutamate, GABA and aspartate. The precursor-product relationship for the metabolism of glutamine to glutamate differed from the classic single compartment model in that the specific radioactivity of glutamate rose very quickly to approx one-tenth that of glutamine, but increased slowly thereafter. These data suggest that the [¹⁴C]glutamine was taken up into two metabolically distinct compartments and/or that some of the [¹⁴C]glutamine was converted to [¹⁴C]glutamate during the uptake process. The uptake of [¹⁴C]glutamine was diminished when the tissue was incubated in a non-oxygenated medium or when Na⁺ was omitted (substituted with sucrose) and K⁺ was concomitantly elevated. However, on a relative basis, the incorporation of radioactivity into glutamate and GABA was increased by these incubation conditions. The metabolism of glutamine to aspartate was greatly depressed when the tissue was not oxygenated. The glutamate formed from [U-¹⁴C]glutamine taken up by the tissue was converted to GABA at a faster rate than was glutamate derived from [U-¹⁴C]glucose. [U-¹⁴C]gly-cerol or exogenous [U-¹⁴C]glutamate. This suggests that glutamine was metabolized to GABA selectively; i.e. on a relative basis, glutamine served as a better source of carbon for the synthesis of GABA than did glucose, glycerol or exogenous glutamate. When the brain hemisections were incubated in the normal physiological medium with or without glutamine. there was very little efflux of glutamate, GABA or aspartate from the tissue. However when NaCl was omitted from the medium (substituted with sucrose) and K⁺ was elevated to 29 miu. a marked efflux of these three amino acids into the medium did occur, and over a period of 160min, the content of each amino acid in the tissue was depleted considerably. When glutamine (0.2 mm ) was included in the Na⁺ deficient-high K.⁺ medium, the average amount of glutamate, GABA and aspartate in the tissue plus the medium was greater than when glutamine was not included in the medium. Such data indicate that CNS tissues can utilize glutamine for a net synthesis of glutamate, GABA and aspartate. The results of this study provide further evidence in support of the concept that the functional (transmitter) pools of glutamate and GABA are maintained and regulated in part via biosynthesis from glutamine. One specific mechanism instrumental in regulating the content of glutamate in nerve terminals may be a process of glutamine uptake coupled to deamidation.     

DECREASED METABOLISM IN VIVO OF GLUCOSE INTO AMINO ACIDS OF THE BRAIN OF THIAMINE-DEFICIENT RATS AFTER TREATMENT WITH PYRITHIAMINE

Abstract— Thiamine deficiency produced by administration of pyrithiamine to rats maintained on a thiamine-deficient diet resulted in a marked disturbance in amino acid and glucose levels of the brain. In the two pyrithiamine-treated groups of rats (Expt. A and Expt. B) there was a significant decrease in the levels of glutamate (23%, 9%) and aspartate (42%, 57%), and an increase in the levels of glycine (26%, 27%) in the brain, irrespective of whether the animals showed signs of paralysis (Expt. A) or not (Expt. B). as a result of thiamine deficiency. A significant decrease in the levels of γ-aminobutyrate (22%) and serine (28%) in the brain was also observed in those pyrithiamine-treated rats which showed signs of paralysis (Expt. A). Threonine content increased by 57% in Expt. A and 40% in Expt. B in the brain of pyrithiamine-treated rats, but these changes were not statistically significant. The utilization of [U-¹⁴C]glucose into amino acids decreased and accumulation of glucose and [U-¹⁴C]glucose increased significantly in the brain after injection of [U-¹⁴C]glucose to pyrithiamine-treated rats which showed abnormal neurological symptoms (Expt. A). The decrease in ¹⁴C-content of amino acids was due to decreased conversion of [U-¹⁴C]glucose into alanine, glutamate, glutamine, aspartate and γ-aminobutyrate. The flux of [¹⁴C]glutamate into glutamine and γ-aminobutyrate also decreased significantly only in the brain of animals paralysed on treatment with pyrithiamine. The decrease in the labelling of, amino acids was attributed to a decrease in the activities of pyruvate dehydrogenase and α-oxoglutarate dehydrogenase in the brain of pyrithiamine-treated rats. The measurement of specific radioactivity of glucose, glucose-6-phosphate and lactate also indicated a decrease in the activities of glycolytic enzymes in the brain of pyrithiamine-treated animals in Expt. A only. It was suggested that an alteration in the rate of oxidation in vivo of pyruvate in the brain of thiamine-deficient rats is controlled by the glycolytic enzymes, probably at the hexokinase level. The lack of neurotoxic effect and absence of significant decrease in the metabolism of [U-¹⁴C]glucose in the brain of pyrithiamine-treated animals in Expt. B were probably due to the fact that animals in Expt. B were older and weighed more than those in Expt. A, both at the start and the termination of the experiments.     

Compartmentation of [14C]Glutamate and [14C]Glutamine Oxidative Metabolism in the Rat Hippocampus as Determined by Microdialysis

Abstract: Metabolic compartmentation of amino acid metabolism in brain is exemplified by the differential synthesis of glutamate and glutamine from the identical precursor and by the localization of the enzyme glutamine synthetase in glial cells. In the current study, we determined if the oxidative metabolism of glutamate and glutamine was also compartmentalized. The relative oxidation rates of glutamate and glutamine in the hippocampus of free-moving rats was determined by using microdialysis both to infuse the radioactive substrate and to collect ¹⁴CO₂ generated during their oxidation. At the end of the oxidation experiment, the radioactive substrate was replaced by artificial CSF, 2 min-fractions were collected, and the specific activities of glutamate and glutamine were determined. Extrapolation of the specific activity back to the time that artificial CSF replaced ¹⁴C-amino acids in the microdialysis probe yielded an approximation of the interstitial specific activity during the oxidation. The extrapolated interstitial specific activities for [¹⁴C]glutamate and [¹⁴C]glutamine were 59 ± 18 and 2.1 ± 0.5 dpm/pmol, respectively. The initial infused specific activities for [U-¹⁴C]glutamate and [U-¹⁴C]glutamine were 408 ± 8 and 387 ± 1 dpm/pmol, respectively. The dilution of glutamine was greater than that of glutamate, consistent with the difference in concentrations of these amino acids in the interstitial space. Based on the extrapolated interstitial specific activities, the rate of glutamine oxidation exceeds that of glutamate oxidation by a factor of 5.3. These data indicate compartmentation of either uptake and/or oxidative metabolism of these two amino acids. The presence of [¹⁴C]glutamine in the interstitial space when [¹⁴C]glutamate was perfused into the brain provided further evidence for the glutamate/glutamine cycle in brain.     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.     

GLUTAMATE METABOLISM IN OCTOPUS BRAIN IN VIVO;ABSENCE OF A WAELSCH EFFECT

Abstract— The metabolism of [U-¹⁴C]glutamate was followed in vivo in the octopus Eledone cirrhosa following intracranial injection, and compared with that in the mammalian brain.
By contrast with the rat brain, the specific activity of glutamine recovered from Eledone optic and vertical lobes was lower than that of glutamate at short time intervals after injection. Thus the Waelsch effect was not apparent in this species. Again, in contrast with the rat brain, radioactivity could be found in alanine but not in GABA following [U-¹⁴C]glutamate injection. This was compatible with observations made previously in vitro.
The significance of these intraspecies differences in metabolism and compartmentation is discussed.     

Decreases in Amino Acid and Acetylcholine Metabolism During Hypoxia

Abstract: Hypoxia impairs brain function by incompletely defined mechanisms. Mild hypoxia, which impairs memory and judgment, decreases acetylcholine (ACh) synthesis, but not the levels of ATP or the adenylate energy charge. However, the effects of mild hypoxia on the synthesis of the glucosederived amino acids [alanine, aspartate, γ-amino butyric acid (GABA), glutamate, glutamine, and serine] have not been characterized. Thus, we examined the incorporation of [U-¹⁴C]glucose into these amino acids and ACh during anemic hypoxia (injection of NaNO₂), hypoxic hypoxia (15 or 10% O₂), and hypoxic hypoxia plus hypercarbia (15 or 10% O₂ with 5% CO₂). In general, the synthesis of the amino acids and of ACh declined in parallel with each type of hypoxia we studied. For example, anemic hypoxia (75 mg/kg of NaNO₂) decreased the incorporation of [U-¹⁴C]glucose into the amino acids and into ACh similarly. [Percent inhibition: ACh (57.4), alanine (34.4), aspartate (49.2), GABA (61.9). glutamine (59.2), glutamate (51.0), and serine (36.7)]. A comparison of several levels (37.5, 75, 150, 225 mg/kg of NaNO₂) of anemic hypoxia showed a parallel decrease in the flux of glucose into ACh and into the amino acids whose synthesis depends on mitochondrial oxidation: GABA (r= 0.98), glutamate (r= 0.99), aspartate (r= 0.96), and glutamine (r= 0.97). The synthesis of the amino acids not dependent on mitochondrial oxidation did not correlate as well with changes in ACh metabolism: serine (r= 0.68) and alanine (r= 0.76). The decreases in glucose incorporation into ACh and into the amino acids with hypoxic hypoxia (15% or 10% O₂) or hypoxic hypoxia with 5% CO₂ were very similar to those with the two lowest levels of anemic hypoxia. Thus, any explanation of the brain's sensitivity to a decrease in oxygen availability must include the alterations in the metabolism of the amino acid neurotransmitters as well as ACh.     

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.     

Age-related changes of glucose metabolism in rat cerebral cortex with reference to glucose-derived amino acids

The parietal cortical slices obtained from 8 week-old (young) and 78 week-old (middle-aged) male Wistar rats were incubated withd-[U-¹⁴C]glucose in oxygensaturated Gey's balanced salt solution. Subsequently, the radioactivities of liberated CO₂ and glucose-derived amino acids (alanine, aspartate, GABA, glutamate and glutamine) obtained from the slices were measured. In the middle-aged rats as compared to the young rats, the amount of radioactivity of CO₂ (P<0.01) and glutamate (P<0.05) showed a significant raduction with glutamine unchanged, while that of alanine (P<0.01), aspartate (P<0.05) and GABA (P<0.05) increased significantly. The results indicate that with advancing age the overall glucose oxidation in the cerebral cortex declines but the metabolic pathway to form amino acids is not uniformly suppressed. Therefore, the above characteristic glucose metabolism could be related to the development of heterogeneous enzyme activities associated with aging in the brain.