Differential Transmitter Release from Nerve Terminals Isolated from Basal Ganglia and Substantia Nigra

Abstract: The K⁺-induced release of amino acids and dopamine from synaptosomes of basal ganglia and substantia nigra of sheep was studied. K⁺ (56 mM) caused an increase in the release of GABA from caudate, putamen, globus pallidus, and substantia nigra, the increased release being 227, 171, 198, and 366%, respectively, compared with samples incubated without stimulation. The release of glutamate was also increased by 56 mM-K⁺ (136–183%) from all regions except the globus pallidus, and a significant release of aspartate was only seen in response to K⁺ stimulation of synaptosomes from putamen (50%). Veratrine (75 μM) also stimulated a similar pattern of amino acid release from these regions. Regional correlation was shown between the presence of an uptake system for an amino acid and its evoked release. [¹⁴C]Dopamine formed from L-[U-¹⁴C]tyrosine was released only from caudate and putamen synaptosomes by K⁺ stimulation, the increases being 105% and 74%, respectively. Synthesis of [¹⁴C]dopamine from L-[U-¹⁴C]tyrosine occurred only in synaptosomes prepared from these two regions and was not detected in synaptosomes from substantia nigra or globus pallidus although whole-tissue homogenates of substantia nigra were able to synthesise dopamine.     

CAPABILITY OF TUBULIN AND MICROTUBULES TO INCORPORATE AND TO RELEASE TYROSINE AND PHENYLALANINE AND THE EFFECT OF THE INCORPORATION OF THESE AMINO ACIDS ON TUBULIN ASSEMBLY

Abstract— Incorporation of [¹⁴C]tyrosine into the C-terminal position of α-tubulin of rat brain cytosol was 10-fold higher for non-assembled than for assembled tubulin. The incorporation into tubulin from disassembled microtubules was higher than into non-assembled tubulin; therefore, the low incorporation into microtubules was not due to a lower acceptor capacity of their tubulin constituent.
[¹⁴C]Tyrosine was released from assembled and non-assembled [¹⁴C]tyrosinated tubulin by the action of an endogenous carboxypeptidase. Release from non-assembled tubulin was shown by incubating a tubulinyl-[¹⁴C]tyrosine preparation in the presence of CaCl₂ at a concentration that abolished microtubule formation. Release from microtubules was inferred from the observation that the percentages of [¹⁴C]tyrosine released and the decrease of the specific radioactivity of the recovered microtubules were practically identical and did not change after a 10-fold dilution of the incubated microtubules.
[³H]Phenylalanine was released from a preparation of tubulinyl-[³H]phenylalanine also by an enzymatic activity.
The capacity of a tubulin preparation to incorporate tyrosine was increased 43% by pre-treatment with endogenous carboxypeptidase.
Tubulin tyrosinated in vitro was assembled to the same extent as native tubulin. After a mixture of tubulinyl-[¹⁴C]tyrosine and tubulinyl-[³H]phenylalanine was partially assembled, the ratio of ¹⁴C/³H found in the microtubules was the same as in the non-assembled tubulin fraction.     

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.     

SYNAPTOSOMAL TYROSINE HYDROXYLATION: AFFINITY FOR OXYGEN

Abstract— Crude striatal synaptosomes were used to determine the affinity of brain tyrosine 3-mono oxygenase for oxygen. The rate of tyrosine hydroxylation was determined at several different oxygen concentrations using L-[¹⁴C]tyrosine as substrate and measuring the [¹⁴C]DOPA formed in the presence of a decarboxylase inhibitor. The accumulation of [¹⁴C]tyrosine by synaptosomes was unaffected by incubation in nitrogen or 2.3% oxygen. Preincubation in nitrogen for up to 4 h did not impair the ability of synaptosomes to hydroxylate tyrosine when returned to air. Furthermore, pre-incubation in either nitrogen or air produced similar changes in the ultrastructural appearance of the synaptosomes and mitochondria in the crude preparation used in these studies. Thus tyrosine hydroxylation in these synaptosomes appeared to reflect tyrosine 3-mono oxygenase activity over a range of oxygen concentrations. The apparent K_m for oxygen was 2 × 10⁻⁵ at pH 6.7 and 7.4. The apparent K_m was not significantly altered by the addition of Ca²⁺, and was slightly increased in the presence of N ₆-mono-butyryl cyclic AMP or high K⁺. These data are consistent with the hypothesis that the availability of oxygen may limit catecholamine synthesis in the intact rat brain.     

INCORPORATION OF PHENYLALANINE AS A SINGLE UNIT INTO RAT BRAIN PROTEIN: RECIPROCAL INHIBITION BY PHENYLALANINE AND TYROSINE OF THEIR RESPECTIVE INCORPORATIONS

Abstract— Of seven amino acids studied, glutamic acid and phenylalanine were incorporated in highest amounts into the hot-TCA-insoluble material of the 100,000 g supernatant fraction of rat brain homogenate. The system for incorporation of phenylalanine was RNase-insensitive and required ATP (apparent K_m = 0.64 m m ), KC1 (apparent K_m = 14 m m ) and MgCl₂ (optimal concentration range 4-15 m m ). The apparent K_m for phenylalanine was 2.9 m m . [¹⁴C]Phenylalanine did not undergo modification before incorporation. Tyrosine and phenylalanine inhibited the incorporation, respectively, of [¹⁴C]phenylalanine and [¹⁴C]tyrosine when incubated simultaneously or successively. The K_m and K_t (3.3 m m ) values for phenylalanine in the incorporation reaction and as inhibitor of the incorporation of [¹⁴C]tyrosine were similar. We suggest that both the enzyme and the acceptor for the incorporation of these two amino acids are the same. [¹⁴C]Phenylalanine and [¹⁴C]tyrosine entered into COOH-terminal positions in the reactions described. Brain exhibited a 25- to 100-fold higher capacity to incorporate phenylalanine than that of liver, kidney or thyroid. The acceptor capacity in rat brain rapidly decreased from day 5 to day 15 of postnatal age and then slowly until age 150 days.     

Amino acids as respiratory substrates in aphids: an analysis of Aphis fabae reared on plants and diets

Abstract. The fate of radioactively labelled amino acids injected into the haemolymph of the aphid Aphis fabae was investigated. Radioactivity from each of L-[U-¹⁴C]-glutamic acid, L-[U-¹⁴C]-serine and L-[U-¹⁴C]-threonine in the aphid tissues declined exponentially, at rates of 32, 9.3 and 1.0 pmol/aphid/min, respectively. For ¹⁴C-glutamic acid, radioactivity lost from the aphids was recovered quantitatively as carbon dioxide, and radioactivity in aphid saliva and honeydew was undetectable. When expressed on a per unit aphid biomass basis, the rate of respiratory loss of glutamic acid from aphids reared on chemically-defined diets was more than double that of aphids reared on the host plant, Vicia faba . It is concluded that respiration is a quantitatively important component to the aphid metabolism of glutamic acid and other amino acids.     

ISOLATION OF HYDROPHOBIC PROTEINS BINDING NEUROTRANSMITTER AMINO ACIDS: γ-AMINOBUTYRIC ACID RECEPTOR OF THE SHRIMP MUSCLE

Abstract— The total lipid extract of shrimp muscle ( Artemisia longinaris ) was precipitated with ether. The supernatant containing 95 per cent of the phospholipids and 50 per cent of the protein showed binding for L-[¹⁴C]glutamatc in the first peak of protein. The sediment, redissolved in chloroform-methanol was chromatographed on a Sephadex LH-20 column. A single peak was eluted in the chloroform (20-40 ml) having no lipid phosphorous and high affinity binding for [¹⁴C]GABA. The saturation was achieved at 1 mole per 80.000 g protein and the curve revealed a single type of binding site. The purification achieved was of about 4000-fold. There was no binding of L-[¹⁴C]glulamate to the ether precipitate. The specificity of the binding of [¹⁴C]GABA was further supported by competition experiments with bicuculline. picro-toxin and muscimol. It is suggested that the hydrophobic protein isolated by us represents the GABA receptor. The findings presented in the two papers of the series suggest that the excitatory and inhibitory receptor from crustacean muscle can be separated as two different proteins.     

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.     

SYNTHESIS AND RELEASE OF [14C]ACETYLCH0LINE IN SYNAPTOSOMES

Abstract— Synaptosomes took up [¹⁴C]choline, about half or more of which was converted to [^I4C]acetylcholine when incubated in an appropriate medium containing 1 to 5 μ M-[¹⁴C] choline and neostigmine. The amount of [¹⁴C]acetylcholine synthesized in synaptosomes increased in parallel with the increase of Na⁺ concentration in the incubation medium. The effect of Na⁺ on the uptake of [^I4C]choline into synaptosomes was dependent on the concentration of choline in the incubation medium.
About 25 per cent of [¹⁴C]acetylcholine synthesized in synaptosomes was released rapidly into the medium by increasing the K⁺ concentration in the medium from 5 m m to 35 m m . The change of Na⁺ concentration hardly affected the release of [¹⁴C]acetylcholine. The effect of K⁺ on the release of [¹⁴C]choline was rather small compared to that on [¹⁴C] acetylcholine. Ouabain promoted the release of [¹⁴C]acetylcholine.     

Effects of Osmotic Pressure on the Oxidative Metabolism of Leishmania major Promastigotes

Leishmania major promastigotes were washed and resuspended in an iso-osmotic buffer. The rate of oxidation of ¹⁴C-labeled substrates was then measured as a function of osmolality. An acute decrease in osmolality (achieved by adding H₂O to the cell suspension) caused an increase in the rates of ¹⁴CO₂ production from [6-¹⁴C]glucose and, to a lesser extent, from [1, (3)-¹⁴C]glycerol. An acute increase in osmolality (achieved by adding NaCl, KCl, or mannitol) strongly inhibited the rates of ¹⁴CO₂ production from [1-: ¹⁴C]alanine, [1-¹⁴C]glutamate, and [1, (3)-¹⁴C]glycerol. The rates of ¹⁴CO₂ formation from [1-¹⁴C]laurate, [1-¹⁴C]acetate, and [2-¹⁴C]glucose (all of which form [1-¹⁴C]acetyl CoA prior to oxidation) were also inhibited, but less strongly, by increasing osmolality. These data suggest that with increasing osmolality there is an inhibition of mitochondrial oxidative capacity, which could facilitate the increase in alanine pool size that occurs in response to hyper-osmotic stress. Similarly, an increase in oxidative capacity would help prevent a rebuild up of the alanine pool after its rapid loss to the medium in response to hypo-osmotic stress.     

Lumiflavin and Lumichrome Transport in the Central Nervous System

Abstract: The transport of the lipid-soluble sugarless flavins, [¹⁴C]lumiflavin and [¹⁴C]lumichrome, into and from the isolated choroid plexus and brain slices was studied in vitro. The isolated choroid plexus accumulated both [¹⁴C] flavins by a saturable, energy-requiring process that did not depend on binding or intracellular metabolism of the [¹⁴C] flavins. Both sugar-containing and sugarless flavins, as well as cyclic organic acids, significantly inhibited [¹⁴C]lumiflavin and [¹⁴C]Iumichrome uptake by the isolated choroid plexus. Within 2.5 min, 75% of the [¹⁴C]lumiflavin accumulated by the isolated choroid plexus was released into the medium. Brain slices accumulated [¹⁴C]lumiflavin by a saturable process that did not meet all the criteria for active transport. Ninety-five percent of the [¹⁴C]lumiflavin accumulated by brain slices was released into the medium within 7.5 min. In vivo , 2 h after the intraventricular injection of 6.5 nmol [¹⁴C]lumiflavin, almost all of the [¹⁴C]flavin was cleared from the CNS. Addition of 3.5 μmol FMN to the intraventricular injectate significantly decreased the clearance of [¹⁴C]lumiflavin from the CNS. These studies document that the sugarless flavins are transported by the flavin transport systems in the CNS.     

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).     

A TECHNIQUE FOR MEASURING BRAIN PROTEIN SYNTHESIS

Abstract— Mice were infused intravenously for varying periods of time with L-[U-¹⁴C]- tyrosine. The specific activity of free tyrosine in the blood and the brain, and of protein-bound tyrosine in the brain, was measured and the mean rate of protein synthesis calculated. The half-life of mixed brain proteins was found to be close to 4 days with infusions lasting 0.5–2 h.
The origin of the intracellular tyrosine pool was investigated and it was shown that 60 per cent of this was derived directly from the plasma tyrosine and 40 per cent from protein breakdown within the tissue.     

The Interaction of Dithiothreitol and Acetyl Coenzyme A in a Radiochemical Assay for Rat Brain ATP:Citrate Oxaloacetate Lyase

Abstract: [¹⁴C]Acetyl-CoA was found to react spontaneously with dithiothreitol to give a relatively apolar product which was readily extractable into a butanol-toluene scintillant. This technique was used in a rapid, reproducible assay for rat brain ATP:citrate lyase using [1,5-¹⁴C]citrate as substrate. The tissue extract, a 14,000 g supernatant, exhibited a lyase activity of approximately 7 nmol acetyl-CoA produced/min per mg supernatant protein, and was inhibited ≥79% by α-ketoglutaric acid (10 m m ), Cu²⁺ (1 m m )and Zn²⁺(1 m m ). [¹⁴C]Oxaloacetate, [¹⁴C]malate and endogenous citrate synthase were found not to interfere significantly with lyase estimations, but NADH was required in the reaction mixture to inhibit acetyl-CoA hydrolase activity.     

Isoprenoid biosynthesis in bacteria: Two different pathways?

Abstract The biosynthesis of isopentenylpyrophosphate, a central intermediate of isoprenoid formation, was investigated in six different bacterial organisms. Cell-free extracts of Myxococcus fulvus, Staphylococcus carnosus, Lactobacillus plantarum and Halobacterium cutirubrum converted [¹⁴C]acetyl-CoA or [¹⁴C]hydroxymethylglutaryl-CoA to [¹⁴C]mevalonic acid. Furthermore, [¹⁴C]mevalonic acid, [¹⁴C]mevalonate-5-phosphate and [¹⁴C]mevalonate-5-pyrophosphate were metabolized to [¹⁴C]isopentenylpyrophosphate in bacteria. In contrast, no intermediates of this reaction sequence could be detected using cell-free extracts of Zymomonas mobilis and Escherichia coli . These results indicate that at least two different pathways for the biosynthesis of isopentenylpyrophosphate are present in bacteria.     

Active Transport of Nicotine by the Isolated Choroid Plexus In Vitro

Abstract: In vitro , the transport of [¹⁴C]nicotine into the isolated choroid plexus, the anatomical locus of the blood–CSF barrier, was studied. The isolated rabbit choroid plexus accumulated [¹⁴C]nicotine by two processes: an active saturable transport process and a nonsaturable process. The [¹⁴C]nicotine accumulation process by choroid plexus was not due to binding or intracellular metabolism of the [¹⁴C]nicotine. The [¹⁴C]nicotine accumulation process in isolated choroid plexus was inhibited by weak bases, including tolazoline and lidocaine, but not by the weak acid probenecid. The accumulation process was decreased 60% by iodoacetate and dinitrophenol and by low temperatures. These results are consistent with previous autoradiographic evidence showing the choroid plexus concentrated [¹⁴C]nicotine in vivo , and suggest that the choroid plexus may transfer nicotine between blood and CSF in vivo .     

Axenic incorporation of [U-14C]palmitic acid into the phenolic glycolipid-I of Mycobacterium leprae

Abstract Incorporation of [U-¹⁴C]palmitic acid ([¹⁴C]PA) into the specific phenolic glycolipid-I (PGL-I) of freshly harvested, nude mouse-derived Mycobacterium leprae was investigated in an axenic modified Dubos medium. Incorporation was approximately linear for 10–14 days at pH 7.2, 33°C. No incorporation of radiolabeled phenol, acetate, tyrosine, phenylalanine, bicarbonate, proprionate or UDP-glucose was detected. Procedures known to remove residual host tissue did not diminish the rate of [¹⁴C]PA incorporation, indicating that bacterial metabolism was being measured. The antileprosy compounds, rifampicin and dapsone, significantly reduced incorporation of the label. The ability to quantitate PGL-I synthesis in the extracellular bacillus should facilitate a better understanding of the optimum conditions for metabolism in M. leprae .     

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.     

NEUROTRANSMITTER UPTAKE INTO SLICES OF RAT CEREBRAL CORTEX IN VITRO: EFFECT OF SLICE SIZE

Abstract— The uptake of [¹⁴C]GABA, [¹⁴C]taurine, [³H] β -alanine and [¹⁴C]dopamine was compared in slices of rat cerebral cortex of three different sizes (0.1 × 0.1 × 2 mm, 0.2 × 0.2 × 2 mm and 0.4 × 0.4 × 2 mm prepared with a mechanical tissue chopper). [¹⁴C]Taurine and [³H] β -alanine uptake increased whereas [¹⁴C]GABA uptake decreased with increasing slice size. [¹⁴C]Dopamine uptake was optimal in 0.2 × 0.2 × 2 mm slices. Increasing slice size was shown to decrease inhibition of [³H] β -alanine and [¹⁴C]GABA uptake by l -2,4-diaminobutyric acid. Lactate dehydrogenase activity increased with increasing slice size indicating decreased tissue damage or increased cellular integrity. The possibility that varying slice size can be used to distinguish between neuronal and glial uptake is discussed. It is suggested that taurine uptake in the cerebral cortex is predominantly glial.     

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.