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.     

Oxidation of γ-Hydroxybutyrate to Succinic Semialdehyde by a Mitochondrial Pyridine Nucleotide-Independent Enzyme

An antibody that inhibits over 95% of the cytosolic NADP+-dependent gamma-hydroxybutyrate (GHB) dehydrogenase activity of either rat brain or kidney was found to inhibit only approximately 50% of the conversion of [1-14C]GHB to 14CO2 by rat kidney homogenate. A similar result was obtained with sodium valproate, a potent inhibitor of GHB dehydrogenase. The mitochondrial fraction from rat brain and kidney was found to catalyze the conversion of [1-14C]GHB to 14CO2. The dialyzed mitochondrial fraction also catalyzed the oxidation of GHB to succinic semialdehyde (SSA) in a reaction that did not require added NAD+ or NADP+ and which was not inhibited by sodium valproate. The enzyme from the mitochondrial fraction which converts GHB to SSA appears to be distinct from the NADP+-dependent cytosolic oxidoreductase which catalyzes this reaction.     

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.     

Effects of Anticonvulsants on the Formation of γ-Hydroxybutyrate from γ-Aminobutyrate in Rat Brain

Abstract: The conversion of γ-aminobutyrate (GABA) via succinic semialdehyde to γ-hydroxybutyrate has been examined in rat brain homogenates. A number of anticonvulsants, including sodium valproate and phenobarbitone, inhibited this metabolic pathway. These results are interpreted in the light of the characteristics of aldehyde reductases known to reduce succinic semialdehyde.     

Endogenous γ-Hydroxybutyrate in Rat Brain Areas: Postmortem Changes and Effects of Drugs Interfering with γ-Aminobutyric Acid Metabolism

Abstract: The possibility that γ-hydroxybutyrate (GHB), a metabolite of γ-aminobutyric acid (GABA), may play a role in the CNS has recently come to attention. We describe here a sensitive and specific mass fragmento-graphic technique that allows the measurement of picomole amounts of GHB in single rat brain areas. Moreover, we show that GHB can accumulate postmortem, an effect that is blocked by the use of microwave irradiation to kill the animals. To understand further the relationship between GABA and GHB formation, we treated rats with drugs known to inferfere with GABA metabolism at different levels and concomitantly measured GABA and GHB in cerebral cortex and cerebellum. Isoniazide, which blocks the formation of GABA, also decreases GHB. Blockers of the catabolism of GABA, such as aminooxyacetic acid and γ-acetylenic GABA, increase GABA levels and decrease those of GHB. Sodium dipropylacetate increases both GABA and GHB, supporting the hypothesis that this effective antiepileptic drug also blocks in vivo the enzyme that converts succinic semialdehyde to succinic acid.     

γ-Hydroxybutyrate Stimulation of the Formation of Cyclic GMP and Inositol Phosphates in Rat Hippocampal Slices

The presence of gamma-hydroxybutyrate (GHB) (300-600 microM) in the incubation medium of rat hippocampal slices led to an increase of intracellular cyclic GMP and inositol phosphates. This phenomenon is dependent on the time and the dose of GHB used and might be the result of the stimulation of GHB receptor sites which are abundant in rat hippocampus. The increase of cyclic GMP and inositol phosphates is blocked by some anticonvulsants and opiate antagonists. These results seems to indicate that, like many substances inducing epileptic phenomena, GHB provokes neuronal depolarization in hippocampus which is accompanied by formation of cyclic GMP and inositol phosphates. The effect of opiate antagonists can be explained by the possible implication of an opiate synapse which mediates GHB effects in rat hippocampus.     

Extracellular Events Induced by γ-Hydroxybutyrate in Striatum: A Microdialysis Study

The modification of dopamine release and accumulation induced by gamma-hydroxybutyrate (GHB) was studied using both striatal slices and in vivo microdialysis of caudate-putamen. GHB inhibited dopamine release for approximately 5-10 min in vitro, and this was associated with an accumulation of dopamine in the tissue. Subsequently, there was an increase in dopamine release. In the microdialysis experiments, low doses of GHB inhibited dopamine release, whereas higher doses strongly increased release; the initial decrease seen in slices could not be detected in vivo. Thus, GHB had a biphasic effect on the release of dopamine: An initial decrease in the release of transmitter was followed by an increase. A time-dependent biphasic effect was observed when GHB was added to brain slices, and a dose-dependent biphasic effect was seen in dialysate after systemic administration of GHB. Naloxone blocked GHB-induced dopamine accumulation and release both in vitro and in vivo. GHB also increased the release of opioid-like substances in the striatum. A specific antagonist of GHB receptors completely blocked both the dopamine response and the release of opioid-like substances. These data suggest that GHB increases dopamine release via specific receptors that may modulate the activity of opioid interneurons.     

A High-Affinity, Na+-Dependent Uptake System for γ-Hydroxybutyrate in Membrane Vesicles Prepared from Rat Brain

Abstract: γ-Hydroxybutyrate (GHB) is a compound with numerous neuropharmacological properties. The discovery of its biosynthetic system, together with its endogenous repartition, have prompted its possible implication in neurotransmission. This role is also supported by the existence, reported here, of a high-affinity uptake system for GHB ( K _m= 46.4 μM)in both purified brain plasma membrane vesicles and in the crude mitochondrial fraction. GHB uptake is dependent on a Na⁺ gradient but is independent of the membrane electrical potential. Cl⁻ and K⁺ can also modulate the uptake. As an approach to determine the conformation required for GHB uptake, a series of related compounds, including aryl-or alkyl-derivatives, has been examined for ability to inhibit GHB uptake. The regional distribution of uptake is also indicative of its possible physiological role, since in striatum, an area where GHB has a known pharmacological effect on dopaminergic neurons, this uptake activity is the highest.     

A Histochemical Study of the Distribution of β-Hydroxybutyrate Dehydrogenase in Developing Rat Cerebellum

The distribution of beta-hydroxybutyrate dehydrogenase (3-hydroxybutyrate dehydrogenase, EC 1.1.1.30) in the developing rat cerebellum has been determined using a histochemical method. Staining of Purkinje cells, particularly the soma, was seen at all ages examined. Intense staining of the proximal portions of Purkinje dendrites was noted at 8-11 days postnatally, with less prominent staining of Purkinje dendrites and surrounding structures of the molecular layer seen at later times. Development of glomeruli in the granule cell layer could also be observed due to the intense staining of these structures. (Although noncerebellar structures were not the focus of this study, intense staining of the choroid plexus of the fourth ventricle was also noted.) the transient external germinal layer of the cerebellum did not show appreciable staining. Since beta-hydroxybutyrate dehydrogenase is required for ketone body metabolism, the apparent low level of this enzyme in the external germinal layer suggests that the cells of this layer are not particularly well adapted for utilization of ketone bodies. Thus these results do not provide support for the suggestion that ketone bodies may serve as major substrates for energy metabolism in the external germinal layer of the developing cerebellum. Indeed, the rather restricted distribution of this enzyme in both developing and mature cerebellum (and presumably elsewhere in brain) suggests that ketone body metabolism may be largely confined to relatively few specific cellular compartments.     

Evidence for the Participation of a Cytosolic NADP+-Dependent Oxidoreductase in the Catabolism of γ-Hydroxybutyrate In Vivo

The concentration of gamma-hydroxybutyrate (GHB) in brain, kidney, and muscle as well as the clearance of [1-14C]GHB in plasma have been found to be altered by the administration of a number of metabolic intermediates and drugs that inhibit the NADP+-dependent oxidoreductase, "GHB dehydrogenase," an enzyme that catalyzes the oxidation of GHB to succinic semialdehyde. Administration of valproate, salicylate, and phenylacetate, all inhibitors of GHB dehydrogenase, significantly increased the concentration of GHB in brain; salicylate increased GHB concentration in kidney, and alpha-ketoisocaproate increased GHB levels in kidney and muscle. The half-life of [1-14C]GHB in plasma was decreased by D-glucuronate, a compound that stimulates the oxidation of GHB by this enzyme and was increased by a competitive substrate of the enzyme, L-gulonate. The results of these experiments suggest a role for GHB dehydrogenase in the regulation of tissue levels of endogenous GHB.     

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.     

Regional and Subcellular Localization in Rat Brain of the Enzymes That Can Synthesize γ-Hydroxybutyric Acid

Abstract: Rat brain contains two major NADPH-linked aldehyde reductases that can reduce succinate semialdehyde to 4-hydroxybutyrate. One of these enzymes appears to be fairly specific for succinate semialdehyde and is not significantly inhibited by classic aldehyde reductase inhibitors such as barbiturates. The other enzyme can reduce several aromatic aldehydes and is strongly inhibited by barbiturates and branched-chain fatty acids. Using one such inhibitor, it was possible to distinguish between and measure the two enzyme activities separately in various rat brain regions and in subcellular fractions. Both enzymes are mainly cytoplasmic but there is some activity in the synaptosomal fraction. The activity of the specific succinic semialdehyde reductase is highest in the cerebellum, where it represents 21% of the total activity, and lowest in the cortex, where it represents about 11% of the total activity.     

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.     

Distribution of γ-Tubulin in Higher Plant Cells: Cytosolic γ-Tubulin is Part of High Molecular Weight Complexes

Abstract: γ-Tubulin is a protein found in all eukaryotic cells, where it plays a key role in the nucleation of microtubules. In higher plant cells, γ-tubulin is localized at the nuclear surface, a known microtubule-organizing centre, and is codistributed with all microtubule arrays. Functions of plant γ-tubulin remain to be determined. This study describes some properties of higher plant γ-tubulin. The overall level of γ-tubulin was constant during the cell cycle in synchronized tobacco BY-2 cells. Biochemical analysis of the subcellular distribution of γ-tubulin in maize cells revealed that, in contrast with animal γ-tubulin, plant γ-tubulin is mainly associated with endomembranes. We showed for the first time that the pool of soluble cytosolic γ-tubulin contained two main γ-tubulin complexes. γ-tubulin, Hsp70 and TCP1-related proteins might interact in a small complex of 750 kDa. A second γ-tubulin complex, larger than 1500 kDa was purified. The protein profile of this large complex was very similar to animal γ-tubulin complexes. The putative functions of these two complexes in plant microtubule nucleation are discussed.     

Human γγ-Enolase: Two-Site Immunoradiometric Assay with a Single Monoclonal Antibody

Abstract: A monoclonal antibody (mAb), termed BBS/NC/VI-H14 (H14), that reacts with the human enzyme γγ-enolase was prepared. It was directed against the γ-subunit and did not cross-react with the α- or β-subunit. The mAb H14 can be used for quantitative determination of γγ-enolase in a two-site immunoradiometric assay (two-site IRMA). It is also suitable for immunostaining formalin-fixed tissues. The specific identification of γγ-enolase provided by the two-site IRMA with H14 is discussed in relation to the cellular distribution of this protein.     

β-Hydroxybutyrate as a Precursor to the Acetyl Moiety of Acetylcholine

Abstract— Rat brain cortex slices were incubated with 10 mm -glucose and trace amounts of [6-³H]glucose and [3-¹⁴C]β-hydroxybutyrate. The effects of (-)-hydroxycitrate, an inhibitor of ATP-citrate lyase; methylmalonate, an inhibitor of β-hydroxybutyrate dehydrogenase; and increasing concentrations of unlabeled acetoacetate were examined. The incorporation of label into lactate, citrate, malate, and acetylcholine (ACh) was measured and ³H:¹⁴C ratios calculated. Incorporation of [¹⁴C]β-hydroxybutyrate into lactate was limited because of the low activity of gluconeogenic enzymes in brain, whereas incorporation of ¹⁴C label into Krebs cycle intermediates and ACh was higher than in previous experiments with [³H-,¹⁴C]-glucose. (–)-Hydroxycitrate (5.0 m^M) reduced incorporation of [³H]glucose and [¹⁴C]β-hydroxybutyrate into ACh. In contrast, slices incubated with methylmalonate (1 mm ) showed a decrease in ¹⁴C incorporation without appreciably affecting glucose metabolism. The effects of high concentrations of methylmalonate were nonselective and yielded a generalized decrease in metabolism. Acetoacetate (1 mm ) also produced a decreased ¹⁴C incorporation into ACh and its precursors. At 10 mm , acetoacetate reduced ³H and ¹⁴C incorporation into ACh without substantially affecting total ACh content. From the results, it is suggested that in adult rats β-hydroxybutyrate can contribute to the acetyl moiety of ACh, possibly via the citrate cleavage pathway, though it is quantitatively less important than glucose and pyruvate. This contribution of ketone bodies could become significant should their concentration become abnormally high or glucose metabolism be reduced.     

Isolation and Identification of α-(γ-Aminobutyryl)-Hypusine

A new dipeptide, alpha-(gamma-aminobutyryl)-hypusine, was identified in bovine brain. This compound was isolated from trichloroacetic acid-soluble fraction of bovine brain with five steps of ion-exchange chromatography. Its structure was postulated by routine chemical analyses and determined by synthesis. The amount of the compound isolated from 1.2 kg of bovine brain was 870 nmol.     

Regional Selectivity of a γ-Aminobutyric Acid-Induced [3H]Acetylcholine Release Sensitive to Inhibitors of γ-Aminobutyric Acid Uptake

The effects of gamma-aminobutyric acid (GABA) on the release of [3H]acetylcholine ([3H]ACh) were studied in synaptosomes prepared from rat hippocampus, cerebral cortex, hypothalamus, and striatum and prelabelled with [3H]choline. When synaptosomes were exposed in superfusion to exogenous GABA (0.01-0.3 mM) the basal release of newly synthesized [3H]ACh was increased in a concentration-dependent way in hippocampus, cortex, and hypothalamus nerve endings. In contrast, the release of [3H]ACh was not significantly affected by GABA in striatal synaptosomes. The effect of GABA was not antagonized significantly by bicuculline or picrotoxin. Muscimol caused only a slight not significant increase of [3H]ACh release when tested at 0.3 mM whereas, at this concentration, (-)-baclofen was totally inactive. The GABA-induced release of [3H]ACh was counteracted by SKF 89976A, SKF 100561, and SKF 100330A, three strong and selective GABA uptake inhibitors. The data suggest that, in selective areas of the rat brain, GABA causes release of [3H]ACh following penetration into cholinergic nerve terminals through a GABA transport system.     

Influence of Sodium-Independent γ-Aminobutyric Acid Binding on the Assay of Sodium-Dependent γ-Aminobutyric Acid Binding in a Membrane Preparation of Rat Brain

A large amount of [³H]GABA was bound to crude synaptic membrane fractions of rat. by sodium-independent process in a medium that contained 100 μM [³H]GABA used for assaying GABA uptake site. This [³H]-GABA binding was different from receptor binding of GABA. It was confirmed that this sodium-independent [²H]GABA binding scarcely occurred in the presence of a physiological concentration of sodium chloride, and that sodium-independent GABA binding had a negligible influence on sodium-dependent GABA binding.