Conversion of γ-Hydroxybutyrate to γ-Aminobutyrate In Vitro

[3H]gamma-Hydroxybutyric acid [( 3H]GHB) at physiological concentration incubated with brain slices in Krebs-Ringer medium produced [3H]gamma-aminobutyric acid [( 3H]GABA). This compound was identified by its Rf values on thin-layer chromatograms and by analysis of the dansyl derivatives of the free amino acid fraction. No labelled glutamate could be detected. Brain slices incubated with labelled glutamate and nonradioactive GHB generated labelled 2-oxoglutarate, suggesting that gamma-aminobutyrate-2-oxoglutarate transaminase (GABA-T) is involved in catalyzing this reaction. Furthermore, specific inhibitors of GABA-T blocked the production of labelled GABA from labelled GHB and of labelled 2-oxoglutarate from labelled glutamate. Transformation of [3H]GHB into [3H]GABA was not inhibited by malonate, demonstrating that the succinate-linked pathway is not involved in the generation of GABA. The kinetic characteristics of the multienzyme system involved in GHB degradation studied in vitro are compatible with the production of GABA in vivo.     

Guanosine Triphosphate Binding to β-Subunit of Tubulin in Alzheimer's Disease Brain: Role of Microtubule-Associated Protein τ

Abstract: In Alzheimer's disease, paired helical filaments composed mainly of abnormally phosphorylated τ accumulate in certain selected neurons of the brain, and microtubules are rarely seen in the affected cells. In the present study, the binding of ³²P-labeled 8-azidoguanosine triphosphate ([γ-³²P]8N₃GTP), the photoaffinity analogue of GTP, to the β-subunit of tubulin in brain homogenates was found to be markedly lower in patients with Alzheimer's disease than in aged control human cases. No significant differences were observed in the levels of the α- and β-subunits of tubulin between Alzheimer's disease and control brains obtained 2–7 h postmortem. In nine of 19 Alzheimer's disease and 11 of 12 control autopsied brains (2–7 h postmortem and stored at ?75°C) tubulin was isolated successfully from brain cytosol by in vitro polymerization induced with DEAE-dextran. The GTP binding was observed in the two cycled assembled microtubule preparations from all the normal control, and in eight of nine Alzheimer's disease cases. Alzheimer's disease microtubule preparations contained varying amounts of abnormally phosphorylated τ, whereas no abnormal τ was detected in the control brain preparations. Addition of bovine τ to bovine, normal human, and Alzheimer's disease brain tubulin preparations markedly increased GTP binding to the β-subunit. An alkaline phosphatase-treated paired helical filament-enriched preparation increased by approximately twofold the GTP binding to bovine brain tubulin. GTP binding to tubulin prepared by phosphocellulose chromatography of two cycled microtubules from three Alzheimer's disease and three normal control brains, revealed insignificant differences between the two groups. These findings have suggested that (1) τ protein promotes the GTP binding to the β-subunit of tubulin, and (2) the breakdown of the microtubule system in brains of patients with Alzheimer's disease might in part be due to the abnormal phosphorylation of τ which depresses the GTP binding.     

αγ-Enolase in the Rat: Ontogeny and Tissue Distribution

Abstract: The rat brain enolases are dimers composed of α and γ subunits. At pH 8.6 αγ-enolase seemed to be stable, and no evidence was found for the possible formation of αγ-enolase from αα-enolase and γγ-enolase in the course of rat brain homogenization. During ontogeny of the rat forebrain, αγ-enolase was formed before γγ-enolase. The half-maximal specific concentrations were reached at postnatal days 14 and 23, respectively. The distribution of αγ- and γγ-enolase in various rat brain areas was also investigated. In all areas both forms were present. In neuroendocrine tissues αγ-enolase was present at a much higher concentration than γγ-enolase. The ratio between γγ-enolase and αγ-enolase may be indicative of the degree of neuronal maturation, a conclusion further substantiated by the high ratio observed in cerebellum and the low ratio observed in olfactory bulbs, both compared with the ratio in forebrain.     

Distribution of α-(γ-Aminobutyryl)-Hypusine

The distribution of alpha-(gamma-aminobutyryl)-hypusine was examined in several organs of the rabbit and in the brain of the rat, rabbit, dog, ox, and monkey. The peptide occurred only in the brains, but appeared to be absent from dog brain. Concentrations were higher in the cerebral hemispheres than in other portions of the brain. No significant difference between white and gray matter was observed.     

γ-Glutamyl Transpeptidase Activity in Brain Microvessels Exhibits Regional Heterogeneity

Brain microvessels form a tight blood-tissue permeability barrier and express high levels of specific enzymes, including gamma-glutamyl transpeptidase (GGTP). This differentiation is thought to be induced by perivascular astrocytes. By using histochemical methods, we found that the percentage of GGTP-positive vessels varied considerably in different areas of rat brain. Enzyme activity was not found in the pineal gland or the median eminence, where the blood-brain barrier is not expressed. In areas where the blood-brain barrier is expressed, the percentage of GGTP-positive vessels varied from 8% in the optic nerve to 100% in the anterior commissure. The neocortex showed a lower percentage of GGTP-positive vessels (2-15%) than anterior olfactory nucleus (42%), subiculum (70%), hippocampus (48%), and striatum (50-58%). Alkaline phosphatase, another brain microvessel-enriched enzyme, did not show these marked regional differences. The morphometric histochemical results were verified by enzymatic assays in homogenates of different regions from rat and bovine brain and in microvessel preparations of bovine putamen and neocortex. During the postnatal development of rat brain, the difference between neocortex and striatum appeared after day 20. The regional heterogeneity of brain microvessels may be caused by astrocytic heterogeneity and reflect regional heterogeneity in microvascular function.     

γ-Hydroxybutyric Acid in Subcellular Fractions of Rat Brain

The presence of gamma-hydroxybutyric acid (GHB) in synaptosome-enriched fractions of rat brain was ascertained using a GLC technique. The stability of GHB in synaptosomes was evaluated by addition of various gamma-aminobutyric acid (GABA) transaminase (GABA-T) inhibitors, GHB, or ethosuximide to the homogenizing medium. Furthermore, changes in whole brain GHB levels were compared with those in the synaptosomal fraction in animals treated with GABA-T inhibitors, GABA, or ethosuximide. GHB was present in synaptosome-enriched fractions in concentrations ranging from 40 to 70 pmol/mg of protein. There was no evidence for redistribution, leakage, or metabolism of GHB during the preparation of synaptosomes. The elevations of whole brain GHB level associated with GABA-T or ethosuximide treatment were reflected by a parallel increase in synaptosomal GHB content. These data add to the growing evidence that GHB may have neurotransmitter or neuromodulator function.     

Formation of γ-Amino-β-Hydroxybutyric Acid from 2-Hydroxyputrescine in Rat Brain

gamma-Amino-beta-[3H]hydroxybutyric acid ([3H]-GABOB) was formed in rat brain from 2-[3H]-hydroxyputrescine that had been chemically synthesized from 2-oxoputrescine and [3H]sodium borohydride. After the injection of 2-[3H]hydroxyputrescine into the lateral ventricle of a rat brain, the rat was killed and then the brain was removed. [3H]GABOB in the brain was identified by a combination of ion-exchange chromatography, high-voltage paper electrophoresis, and recrystallization of the radioactive compound with authentic GABOB.     

Synthesis of γ-Glutamyldopamine and Other Peptidoamines in the Nervous System of Aplysia californica

The synthesis of a series of gamma-glutamyl amines (gamma-Glu-amines), including gamma-Glu-dopamine, gamma-Glu-5-hydroxytryptamine, gamma-Glu-octopamine, gamma-Glu-tryptamine, gamma-Glu-tyramine, and gamma-Glu-phenylethylamine, by nervous tissue of the marine mollusc Aplysia californica is described. After ganglia were incubated in vitro with 14C-amines, the unchanged amine and a new 14C-labeled product, identified as the gamma-Glu conjugate of the amine, were isolated from the tissue extracts. Identification was made by comparing the chromatographic properties (HPLC, TLC, and LC) of the isolated conjugates with chemically synthesized gamma-Glu-amines before and after acid hydrolysis.     

Blood–Brain Barrier Pericytes Are the Main Source of γ-Glutamyltranspeptidase Activity in Brain Capillaries

Cerebral endothelial cells form the selective permeability barrier between brain and blood by virtue of their impermeable tight junctions and the presence of specific carrier systems. These specialized properties of brain capillaries are reflected in the presence of proteins that are not found in other capillaries of the body. gamma-Glutamyltranspeptidase (GGT) has been widely used as a marker for brain capillaries and differentiated properties of brain endothelial cells. By using histochemical and biochemical methods we have investigated the expression of GGT in isolated capillaries, cultured brain endothelial cells and pericytes, and cocultures of astrocytes and brain endothelial cells. It was surprising that the majority of GGT activity was associated with pericytes, but not endothelial cells, suggesting that GGT is a specific marker for brain pericytes. The remaining GGT activity that was associated with endothelial cells rapidly disappeared from cultured cells but was reinduced in cocultures with astrocytes. Our results emphasize the need for pure endothelial cells for the investigation of blood-brain barrier characteristics.     

Quantitative Immunoprecipitation Studies with Anti-γ-Aminobutyric AcidA Receptor γ2 1–15 Cys Antibodies

Antibodies raised against the synthetic peptide NH2-QKSDDDYEDYASNKTC-COOH (gamma 2 1-15 Cys), which corresponds to the N-terminal amino acid sequence with a C-terminal cysteine of the human gamma 2 subunit of the gamma-aminobutyric acidA (GABAA) receptor, were used to study the quantitative immunoprecipitation of agonist benzodiazepine binding sites from bovine brain. Anti-gamma 2 1-15 Cys antibodies were found to immunoprecipitate specifically in parallel [3H]flunitrazepam- and [3H]muscimol-reversible binding sites in a dose-dependent manner. The maximum percentages of [3H]flunitrazepam binding sites immunoprecipitated from detergent extracts of bovine cerebral cortex, cerebellum, and hippocampus were 68, 77, and 83%, respectively. Immunoprecipitation studies with anti-alpha 1 324-341 antibodies carried out in parallel with anti-gamma 2 1-15 Cys antibodies provided evidence for the promiscuity of the gamma 2 subunit within native GABAA receptors. These results substantiate the association of the gamma 2 polypeptide with native GABAA receptors.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Sequence and Regional Distribution of the mRNA Encoding the α2 Polypeptide of Rat γ-Aminobutyric AcidA Receptors

A cDNA from a rat hippocampal cDNA library encodes an isoform of the alpha polypeptide of the gamma-aminobutyric acid (GABA)/benzodiazepine (BZ) receptor. Its deduced amino acid sequence is 96% identical to that of the alpha 2 polypeptide of the bovine GABAA receptor. The polypeptide has features shared by all previously reported GABAA receptor alpha polypeptides and shares 71-76% identity with previously described rat alpha polypeptides. Most of the differences lie in the presumed extracellular and intracellular domains. On Northern blots, the alpha 2 cDNA detects two mRNAs, which are found in cortex, hippocampus, and striatum, brain regions enriched in pharmacologically defined "BZ type II" receptors. Other workers have previously shown that the alpha polypeptides of the GABAA receptor largely determine the BZ binding properties of reconstituted receptors. The distribution of alpha 2 mRNAs in rat brain suggests that the alpha 2 subunit may indeed be involved in the BZ type II receptors.     

Deuterium Isotope Effect in γ-Aminobutyric Acid Transamination: Determination of Rate-Limiting Step

The rate of transamination of gamma-aminobutryic acid (GABA) catalyzed by hog brain gamma-aminobutyrate aminotransferase was substantially reduced when the hydrogen at the gamma-carbon position was replaced by deuterium. The deuterium isotope effect of this reaction has been substantiated by fluorometric, radiometric, and mass spectrometric procedures and assessed kinetically. The ratios of Vmax of the nonlabeled substrate/Vmax of the deuterated substrate obtained under different conditions ranged from 6 to 7. This indicates that the cleavage of the hydrogen from the gamma-carbon is the rate-determining step in GABA transamination. Similar isotope effects have also been shown to occur in the peripheral system in vivo.     

Regulation of Transmitter γ-Aminobutyric Acid (GABA) Synthesis and Metabolism Illustrated by the Effect of γ-Vinyl GABA and Hypoglycemia

The effect of different treatments on amino acid levels in neostriatum was studied to throw some light on the synthesis and metabolism of gamma-aminobutyric acid (GABA). Irreversible inhibition of GABA transaminase by microinjection of gamma-vinyl GABA (GVG) led to a decrease in aspartate, glutamate, and glutamine levels and an increase in the GABA level, such that the nitrogen pool remained constant. The results indicate that a large part of brain glutamine is derived from GABA. Hypoglycemia led to an increase in the aspartate level and a decrease in glutamate, glutamine, and GABA levels. The total amino acid pool was decreased compared with amino acid levels in normoglycemic rats. GVG treatment of hypoglycemic rats led to a decrease in the aspartate level and a further reduction in glutamate and glutamine levels. In this case, GABA accumulation continued, although the glutamine pool was almost depleted. The GABA level increased postmortem, but there were no detectable changes in levels of the other amino acids. Pretreatment of the rats with hypoglycemia reduced both glutamate and glutamine levels with a subsequent decreased postmortem GABA accumulation. The half-maximal GABA synthesis rate was obtained when the glutamate level was reduced by 50% and the glutamine level was reduced by 80%.     

Purification and Properties of Bovine Brain Dopamine β-Hydroxylase

Abstract: Dopamine β-hydroxylase (DBH) was purified from bovine brain by a series of steps including extraction with 0.5% Triton X-100, ammonium sulfate fractionation, and serial chromatographies with Concanavalin A (Con A)-Sepharose, Biogel A-1.5 m, DEAE-Sephadex, and phenyl-Sepharose. The overall purification was approximately 4200-fold and the final specific activity was 147 nmol/min/mg protein. Bovine brain DBH was apparently a glycoprotein and interacted with immobilized Con A. Furthermore, the enLyme bound to phenyl-substituted agarose by hydrophobic interaction. An approximate molecular weight was estimated to be 400,000 by gel filtration; the protein eluted earlier than bovine adrenal DBH with a molecular weight estimated to be 290,000. The K_m values toward tyramine and ascorbate were 1.53 and 1.42 mM, respectively, the optimal pH was 5.0 in the presence of 20 mM tyramine as substrate. Immunological titration studies indicated that bovine brain and adrenal DBH had common antigenic sites. Our data showed a close similarity between the bovine brain and adrenal enzymes.     

OCCURRENCE AND FORMATION OF γ-GLUTAMYLPUTRESCINE IN MAMMALIAN BRAIN

—An unknown radioactive compound was detected in the basic fraction of the trichloroacetic acid extract of rat brain injected with radioactive putrescine. This compound was purified from bovine brain and identified as γ-glutamylputrescine by comparison of its behaviour with that of the synthesized glutamylamides. The amide seemed to be metabolized as rapidly as putrescine in rat brain.     

γ-Aminobutyric AcidA Receptor α5-Subunit Creates Novel Type II Benzodiazepine Receptor Pharmacology

A cDNA encoding a protein with 70% amino acid identity to the previously characterized gamma-aminobutyric acidA (GABAA) receptor alpha-subunits was isolated from a rat brain cDNA library by homology screening. As observed for alpha 1-, alpha 2-, and alpha 3-subunits, coexpression of this new alpha-subunit (alpha 5) with a beta- and gamma 2-subunit in cultured cells produces receptors displaying high-affinity binding sites for both muscimol, a GABA agonist, and benzodiazepines. Characteristic of GABAA/benzodiazepine type II sites, receptors containing alpha 2-, alpha 3- or alpha 5-subunits have low affinities for several type I-selective compounds. However, alpha 5-subunit-containing receptors have lower affinities for zolpidem (30-fold) and Cl 218 872 (three-fold) than measured previously using recombinantly expressed type II receptors containing either alpha 2- or alpha 3-subunits. Based on these findings, a reclassification of the GABAA/benzodiazepine receptors is warranted.     

Bovine γ-Aminobutyric AcidA Receptor Sequence-Specific Antibodies: Identification of Two Epitopes Which Are Recognised in Both Native and Denatured γ-Aminobutyric AcidA Receptors

Polyclonal antibodies have been raised against synthetic peptides whose sequences correspond to the N-terminal 15 amino acids and the C-terminal 17 amino acids of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1 subunit. These antibodies were shown to react with the denatured GABAA receptor alpha subunit, Mr 53,000, in Western blots with both purified receptor and brain membranes as antigens. Also, both antibodies recognised both the purified and detergent-solubilised GABAA receptor as demonstrated by dose-dependent specific immunoprecipitation of the GABA and benzodiazepine binding sites from solution. Evidence is also presented to show brain-regional distribution of the expression of the alpha 1 subunit.     

Catecholamine Concentration in the Developing Rat Brain after γ-Hydroxybutyric Acid

The effect of the GABA receptor agonist γ-hydroxybutyric acid (GHBA) on brain catecholamine concentration was investigated in 1 to 28 day old rats. The infant rats were given GHBA in various doses (375–1500 mg/kg) and the effects on whole brain or regional brain concentration of dopamine (DA) and noradrenaline (NA) were measured. Brain DA concentration increased in a dose-dependent way already from two days of postnatal age. In the regional brain study of the 14- and 28-day-old animals the increase in DA concentration was found to be almost exclusively located in the striatal region. Generally, no changes in NA concentration were found in the whole brain or various brain regions at any of the ages after GHBA. It is concluded that the inhibitory striatal-nigral neurons, utilizing GABA as a transmitter, are functionally developed during early postnatal age.     

The γ-Aminobutyrate Content of Nerve Endings (Synaptosomes) in Mice After the Intramuscular Injection of γ-Aminobutyrate-Elevating Agents: A Possible Role in Anticonvulsant Activity

Abstract: The intramuscular administration of L-cycloserine, gabaculine, and aminooxyacetic acid caused significant, time-dependent increases in the γ-aminobutyric acid (GABA) content of both whole brain and synaptosomalenriched preparations obtained from the tissue, a linear relationship being observed between the two parameters. In contrast, the administration of hydrazine resulted in a large increase in whole brain GABA level, with little change in the synaptosomal GABA content. The key factor in these different responses appeared to be the degree of inhibition of glutamic acid decarboxylase by the drugs. Pretreatment of mice with the GABA-elevating agents resulted in a delay in the onset of seizures, which was related directly to the increase in synaptosomal GABA content. Although the seizures were delayed, they occurred while the GABA content of nerve endings (synaptosomes) was above that in preparations from untreated animals. The decrease in GABA content at the onset of seizures, expressed as a percentage of the level at the time of injection of the convulsant agent, was, however, reasonably constant. A hypothesis to explain these results is proposed.