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.     

THE EFFECT OF DEFICIENCY OF THIAMINE ON THE METABOLISM OF [U-14C]GLUCOSE AND [U-14C]RIBOSE AND THE LEVELS OF AMINO ACIDS IN RAT BRAIN

Abstract— The incorporation of ¹⁴C into amino acids of the brain was determined at different times after injection of [U-¹⁴C]glucose and [U-¹⁴C]ribose to rats maintained on thiamine-supplemented and thiamine-deficient diets for 22 days.
The ¹⁴C-content of amino acids in the brain of thiamine-deficient rats decreased at times 2–10 min after injection of [U-¹⁴C]glucose. but it increased at 2 min and decreased at times 5–10 min after injection of [U-¹⁴C]ribose.
The results of labelling of amino acids indicated that the activities in vivo of the thiamine pyrophosphate requiring enzymes, pyruvate oxidase, a-oxoglutarate dehydrogenase and transketolase were similar in the two groups. It was suggested that the observed decrease in the labelling of amino acids was due to one or more of the following factors: (i) a decrease in the activities of glycolytic enzymes catalysing the conversion of glucose into triose phosphate; (ii) a decrease in the transport of substrate to the active site of the enzymes; or (iii) altered neurohistopathology of the brain.
Thiamine deficiency in rats showed a 5% decrease in glutamate ( P < 0–05), 46% decrease in threonine (P < 0001) and 16% increase in glycine ( P < 0–01) content of the brain.     

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.     

COMPARTMENTATION AND LABELLING OF AMINO ACIDS IN THE DEVELOPING RAT BRAIN AFTER INJECTION OF [U-14C]RIBOSE

Abstract— [U-¹⁴C]Ribose was given by subcutaneous injection to young rats aged 2–56 days. During the first week after birth ¹⁴C in the brain was found mainly combined in glucose, fructose and sedoheptulose which contained 46–57 per cent of the ¹⁴C in the acid soluble metabolites in the rat brain. In contrast, during the critical period (10–15 days after birth) the ¹⁴C in the free sugars decreased from 24 to 3 per cent, while the ¹⁴C content of amino acids in the brain increased from 11 to 44 per cent of the total perchloric acid-soluble ¹⁴C. The increase in labelling of amino acids during the critical period was attributed to increased glycolysis and increased oxidation of pyruvate. The relative specific radioactivity of y -aminobutyrate and aspartate in the rat brain at 28 days after birth was equal to or greater than the relative specific radioactivity of glutamate. Assuming that the increase in amino acid content following the cessation of cell proliferation in the brain is located mainly in cell processes (cytoplasm of axons, dendrites, glial processes and nerve terminals), tentative values were estimated for the pool sizes of glutamate, glutamine, aspartate and y -amino butyrate.     

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.     

IN VIVO FORMATION AND CATABOLISM OF [14C]HISTAMINE IN MOUSE BRAIN

Abstract— The formation of histamine in brain was studied in mice injected with l -[¹⁴C]-histidine (ring 2-¹⁴C) intravenously (i.v.) or intracerebrally; [¹⁴C]histamine appeared rapidly and exhibited a rapid rate of turnover. Drugs known to block various pathways of histamine catabolism were tested for effects on brain–[¹⁴C]histamine and [¹⁴C]-methyl-histamine in mice given (1) [¹⁴C]histamine i.v., (2) [¹⁴C]histamine intracerebrally, and (3) l -[¹⁴C]histidine i.v. Blood-borne histamine did not enter brain; brain histamine was formed locally by decarboxylation of histidine Methylhistamine did cross the blood-brain barrier. Methylation was the major route of histamine catabolism in mouse brain and some of the methylhistamine formed was destroyed by monoamine oxidase. No evidence for catabolism by the action of diamine oxidase was found.     

INCORPORATION OF [14C]N-ACETYL NEURAMINIC ACID INTO BRAIN GLYCOPROTEINS AND GANGLIOSIDES IN VIVO1

—Intracerebrally administered [¹⁴C]N-acetyl neuraminic acid was incorporated into brain glycoproteins and gangliosides. Incorporation into both classes of compounds was markedly inhibited by acetoxycycloheximide but incorporation into the soluble glycoproteins of the nerve-ending fraction was inhibited least of all. In contrast to glucosamine and fucose, a relatively small proportion of the injected [¹⁴C]NANA was incorporated.     

INCORPORATION OF [1-14C]OLEIC ACID AND [1-14C]ARACHIDONIC ACID INTO LIPIDS IN THE SUBCELLULAR FRACTIONS OF MOUSE BRAIN

Abstract— The distribution of radioactivity among lipids of subcellular membrane fractions was examined after intracerebral injections of [1-¹⁴C]oleic and [1-¹⁴C]arachidonic acids. Labelled free fatty acids were distributed among the synaptosomal-rich, microsomal, myelin and cytosol fractions at 1 min after injection. However, incorporation of the fatty acids into phospholipids and trïacylglycerols after pulse labelling occurred mainly in the microsomal and synaptosomal-rich fractions. With both types of labelled precursors, there was a higher percentage of radioactivity of diacyl-glycerophosphoryl-inositols in the synaptosomal-rich fraction as compared to the microsomal fraction. Radioactivity of [1-¹⁴C]oleic acid was effectively incorporated into the triacylglycerols in the microsomal fraction whereas radioactivity of the [1-¹⁴C]arachidonic acid was preferentially incorporated into the diacyl-glycerophosphorylinositols in the synaptosomal-rich fraction. Result of the study indicates that synaptosomal-rich fraction in brain is able to metabolize long chain free fatty acids in vivo and to incorporate these precursors into the membrane phosphoglycerides.     

IN VIVO LABELLING OF ACETYL-ASPARTYL PEPTIDES IN MOUSE BRAIN FROM INTRACRANIALLY AND INTRAPERITONEALLY ADMINISTERED ACETYL-l-[U-14C] ASPARTATE

Abstract— Following intracranial and intraperitoneal injection of acetyl- l -[U-¹⁴C]aspartate into mice about 5% and 0.7% of the radioactivity, respectively, was recovered from the brain after 30 min.
On chromatographic separation of the cationic and anionic compounds on a Dowex 50 column, the former fraction contained about 60% of the radioactivity, predominantly as labelled aspartate and glutamate. The anionic compounds, containing 20% of the labelled compounds, were fractionated in several chromatographic systems and resolved into a great variety of labelled peptidic compounds of which five acetyl-[U¹⁴-C]aspartyl peptides, containing two to four amino acids, were purified. One of these, acetyl-aspartyl glutamine, has not previously been found in brain.     

STUDY OF RNA IN SUBCELLULAR FRACTIONS FROM RAT BRAIN: SIMULTANEOUS INCORPORATION OF [14C]URIDINE AND [3H]METHYL-l-METHIONINE

Abstract— By using a combination of subcutaneous and intraventricular injections of [¹⁴C]uridine and [³H]methyl- l -methionine we have obtained maximum incorporation in about 40 min of both radioactive precursors into nuclear RNA from rat brain. In this nuclear fraction we found at least two different types of RNA that were rapidly labelled. One of them incorporated both [¹⁴C]uridine and [³H]methyl groups and seemed to correspond to species of rRNA and their precursors. The other RNA fraction was less methylated or non-methylated and exhibited sedimentation coefficients distributed along a continuous 8–30 % sucrose density gradient. At least part of the latter type of RNA very probably was mRNA, but much of it must conespond to a different RNA similar to that recently described in HeLa cells by P enman , V esco and P enman (1968).
We also found that labelled 185 and 285 rRNA components began leaving the nucleus for the cytoplasm within 24 to 33 min after the radioactive precursors had been injected, and, in the cytoplasmic fraction, the patterns of incorporation for [¹⁴C]uridine and [³H]-methyl groups were similar for the 18S and 28S rRNA components. We estimate that in this fraction of rat brain the 18S rRNA component was 1·4 times more methylated than the 28S component. We also detected a lower sedimentation coefficient for the non- or slightly methylated, species of soluble RNA found in the cytoplasmic fraction.     

THE EFFECTS OF CARBAMYLCHOLINE ON INCORPORATION IN VIVO OF [1-14C]ARACHIDONIC ACID INTO GLYCEROLIPIDS OF MOUSE BRAIN

Abstract— The effects of carbamylcholine on incorporation of [1-¹⁴C]arachidonate into the glycerolipids in mouse brain synaptosome-rich and microsomal fractions were examined at 1, 3 and 10 min after intracerebral injection of the labeled precursor. When carbamylcholine was included with the labeled arachidonate, there was a decrease in the proportion of labeled fatty acid incorporated into the phospholipids. Among the phospholipids in the synaptosome-rich fraction, a decrease in incorporation of radioactivity into diacyl-glycerophosphoinositols and diacyl-glycerophosphocholines was observed at 1 and 3 min after injection. A decrease in labeling of diacyl-glycerophosphoethanolamines and diacyl-glycerophosphocholines in the microsomal fraction was observed at 3 and 10 min after injection. The decrease in phospholipid labeling was marked by an increase in labeling of diacylglycerols which was observed initially in the synaptosome-rich fraction, but also in the microsomal fraction at later time periods. Other lipid changes included an increase in triacylglycerol labeling which was found in the synaptosome-rich fraction and an increase in phosphatidic acid labeling which was found in the microsomal fraction. Results of the in vivo study have demonstrated changes in brain lipid metabolism during carbamylcholine stimulation. Furthermore, these changes appear to be initiated mainly in the synaptosome-rich fraction.     

EFFECTS OF SPREADING CORTICAL DEPRESSION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEINS OF RAT BRAIN

Abstract— Incorporation of dl -[1-¹⁴C]leucine into proteins of the cerebral cortex of the rat was measured during spreading cortical depression (CSD) evoked by a single topical application of 25% (w/v) KCI. Maximal inhibition (42 per cent) of the rate of incorporation occurred 1 hr after application of KCI. Spreading depression of 2–3 hr duration was associated with 22 per cent and 13 per cent decreases, respectively, of incorporation of labelled leucine. Specific activity of the free pool leucine was not decreased during CSD but appeared to be higher than controls at 20 min after initiation of CSD. The specific activity of the total free pool amino acids was also increased at 10, 20, 60 and 120 min after application of KCI.
The inhibitory effect of CSD on incorporation of leucine into proteins was uniformly distributed among the crude mitochondrial, microsomal and soluble subcellular fractions from brains of adult animals, while in fractions from 25-day old animals there appeared to be relatively more inhibition in the crude mitochondrial fraction.     

THE EFFECT OF MORPHINE ADMINISTRATION ON THE INCORPORATION OF [14C]LEUCINE INTO PROTEIN IN CELL-FREE SYSTEMS FROM RAT BRAIN AND LIVER

—Ribosomes isolated from the brains of rats treated with morphine in vivo were less active in promoting the incorporation of [¹⁴C]leucine into protein than ribosomes isolated from untreated rats. This inhibitory phenomenon was studied in relation to dose of morphine, time after drug administration and the pharmacological responses of hypothermia and analgesia. The inhibition of [¹⁴C]leucine incorporation into brain proteins in vitro was transient after a single injection of morphine and dose-dependent, and related to the hypothermic response, but not prevented by keeping the rats at an ambient temperature which prevented hypothermia. The incorporation of [¹⁴C]leucine into protein by liver ribosomes was also inhibited in preparations from morphine treated rats.     

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.