P. Mandel

High affinity binding site for γ-hydroxybutyric acid in rat brain

Abstract The existence of a specific synthesizing enzyme for γ-hydroxybutyric acid in rat brain has recently been reported. Here, for the first time, we demonstrate the presence of a high affinity, apparently specific binding site for this compound in the same tissue. This binding does not require Na + and takes place optimally at pH 5.5. The bound γ-hydroxybutyric acid is not displacable by GABA or baclofen. We report here on some structurally related compounds of GHB with a similar or better binding capacity than GHB itself. The number of binding sites increases with age up to adulthood and differs depending on the brain region. In primary tissue cultures of pure chicken neurones and glia, γ-hydroxybutyric acid binding occurs exclusively-- in the neuronal preparations.

The influence of ionic strength and a polyanion on transcription in vitro: I. Stimulation of the aggregate RNA polymerase from rat liver nuclei

Abstract Studies of the aggregate RNA polymerase from rat liver nuclei with or without a stimulating effect of ammonium sulphate or polyethylene sulphonate suggest that only one type of RNA polymerase is operative in the aggregate enzyme complex. Most of the stimulating effect of high ionic strength is not due to an inhibition of degradative enzymes, as shown by incubating at 17°, a temperature at which the activity of degradative enzymes is drastically decreased. It is likely that the effect of ammonium sulphate or polyethylene sulphonate is mostly to dissociate bound histones from the DNA. Our results indicate that the RNA polymerase is bound to the DNA in the aggregate enzyme as part of an initiation or transcription complex. Kinetic data suggest that several times as many RNA polymerase molecules are engaged in RNA synthesis when polyethylene sulphonate or ammonium sulphate are present. Nearest-neighbor base frequencies indicate that at 37° and at low ionic strength or in the absence of polyethylene sulphonate, the RNA synthesized resembles that of ribosomes, whereas at 17° some RNA rich in A and U is synthesized. In the presence of ammonium sulphate or polyethylene sulphonate much more RNA rich in A and U is synthesized. These results indicate that the small amount of A-U-rich RNA which is synthesized at 37° is immediately destroyed by degradative enzymes, whereas the G-C-rich RNA is much more resistant. It is suggested that at low ionic strength or in the absence of polyethylene sulphonate, the active RNA polymerase is localized mainly in the nucleolus, while in the presence of these agents it is in the extranucleolar portion of the chromatin.

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 (Km= 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.

Gamma hydroxybutyrate distribution and turnover rates in discrete brain regions of the rat

Gamma-hydroxybutyric acid and trans-gamma-hydroxycrotonic acid levels have been determined in 24 regions of the rat brain after sacrifice by microwave irradiation. Concentration ranges are from 4 pmol/mg protein (frontal cortex) to 46 pmol/mg protein (substantia nigra) for gamma-hydroxybutyric acid and from 0.4 pmol/mg protein (striatum) to 11 pmol/mg protein (hypothalamus) for trans-gamma-hydroxycrotonic acid. It appears that gamma-hydroxybutyric acid levels correlate well with GABA distribution in the same region. However this correlation is not evident with regard to the distribution of the gamma-hydroxybutyric acid synthesizing enzyme, specific succinic semialdehyde reductase. Using the antiepileptic drug, valproate which strongly inhibits gamma-hydroxybutyric acid release and degradation, we estimated the turnover rate of this compound in six regions of the rat brain. Turnover numbers ranged from 6.5 h(-1) in hippocampus to 0.76 h(-1) in cerebellum.

Conversion of γ-Hydroxybutyrate to γ-Aminobutyrate In Vitro

Abstract: [3H]γ‐Hydroxybutyric acid ([3H]GHB) at physiological concentration incubated with brain slices in Krebs‐Ringer medium produced [3H]γ‐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 γ‐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.

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.

The effect of partial hepatectomy on the ribonucleic acid polymerase of rat liver.

Abstract Weiss (1960) described, in the liver of normal rats, a nuclear enzyme which polymerizes nucleoside triphosphates into RNA ∗ . We used his techniques, slightly modified, to ascertain if and how partial hepatectomy affected this enzyme.

Depolarization-Evoked Release of γ-Hydroxybutyrate from Rat Brain Slices

Abstract: The release of γ‐hydroxybutyrate from preloaded rat brain striatal slices was investigated. K+‐induced depolarization caused an efflux of γ‐hydroxybutyrate of about 50 fmol min‐1 mg‐1 (wet weight), but in a Ca2+‐free medium containing Mg2+, the evoked release was reduced by 50–60%. The release was higher when 100 μM veratridine was used as a depolarizing agent. The efflux of γ‐hydroxybutyrate is related to veratridine and K+ concentration, and is strongly inhibited by 10 μM tetrodotoxin. The Ca2+ channel blocker verapamil induces a large decrease in the efflux of γ‐hydroxybutyrate after both K+‐ and veratridine‐induced depolarization. These results are in favour of a possible transmitter function for γ‐hydroxybutyrate in rat striatum.

γ-Hydroxybutyrate uptake by rat brain striatal slices

Abstractγ-Hydroxybutyrate uptake by rat brain striatal slices was studied. The uptake was saturable with aKm of 702±107.10−6M. γ-Hydroxybutyrate uptake was sodium dependent and inhibited by the omission of potassium. In addition, the effect of ouabain suggests that the transport is dependent on a cation gradient. Several analogues of γ-hydroxybutyrate inhibit the transport system. GABA has no significant effect. This energy and cation dependent transport system is in favor of a transmitter or modulator role of γ-hydroxybutyrate in the rat brain striatum.

Evidence that a specific succinic semialdehyde reductase is responsible for λ-hydroxybutyrate synthesis in brain tissue slices

7-Hydroxybutyric acid (GHB) is a minor brain catabolite of 7-aminobutyric acid (GABA) which, when administered systematically to animals and man, induces in both multiple neuropharmacological effects (review [ 1 ]). From rat and human brain, two enzymes have been isolated which can reduce succinic semialdehyde (SSA) to GHB [2-4]. In each case, one of these enzymes was non-specific for this reaction and could reduce a wide range of aldehydes, whereas the other enzyme showed a high degree of specificity for SSA reduction. The non-specific SSA reductase from both species was strongly inhibited by barbiturates and various branched-chain fatty acids, whereas the specific enzyme was little affected by most of these compounds. It is of great interest to determine to what extent these two enzymes participate in GHB formation in vivo. To investigate this, we studied the biosynthesis of GHB from labelled GABA in rat brain cerebellar slices, an area rich in SSA reductase enzymes [5]. These experiments were performed in the absence and presence of 3 compounds differentially inhibiting the purified enzymes capable of catalyzing the oxidation of SSA by succinic semialdehyde dehydrogenase and its reduction to GHB.