Gamma-hydroxybutyric acid as a signaling molecule in brain

doi:10.1016/S0741-8329(99)00092-0

Alcohol

Volume 20, Issue 3, April 2000, Pages 277-283

https://doi.org/10.1016/S0741-8329(99)00092-0 Get rights and content

Abstract

Gamma-hydroxybutyric acid was synthesized 35 years ago to obtain a GABAergic substance that penetrates the brain freely. Since then, gamma-hydroxybutyric acid has been used in human beings for its sedative and anesthetic properties when administered at high doses, and most of the studies on gamma-hydroxybutyric acid have focused on its pharmacological effects. However, gamma-hydroxybutyric acid is also an endogenous substance, which is synthesized and released in the brain by specific neuronal pathways, implicated in the control of the GABAergic, dopaminergic, and opioid systems. This control is mediated by specific gamma-hydroxybutyric acid receptors with a unique distribution in brain and a specific ontogenesis and pharmacology. Stimulation of these receptors induces specific cellular responses. Taken together, these results suggest that gamma-hydroxybutyric acid possesses most of the properties required of a neurotransmitter/neuromodulator in the brain.

Introduction

Until recently, gamma-hydroxybutyric acid (GHB) has been considered a drug to be used in anesthesia and for the regulation of sleep patterns in patients with narcolepsy Hoes et al. 1980, Mamelak et al. 1986. Even after the discovery of its natural occurrence in the brain and in several organs of human beings and various animals species (Roth & Giarman, 1970), its status has remained largely in the pharmacological domain. However, increasing evidence argues for the existence of a GHB system in the brain, implicated in specific signaling between neurons and perhaps between the brain and peripheral organs (Maitre, 1997). Gamma-hydroxybutyric acid administered peripherally penetrates freely into the brain and interacts locally with receptors whose distribution, ontogenesis, kinetics, and pharmacology are specific. These receptors are absent from peripheral organs and influence at least three major neurotransmitter systems in the brain: those of dopamine, opiates, and gamma-aminobutyric acid (GABA). Some brain compartments of this last substance are thought to be directly modulated by GHB acting as a precursor or as a presynaptic signal, leading to the modulation of anxiety, vigilance, and the electroencephalographic profile by means of GABA_A and GABA_B receptors Schmidt-Mutter et al. 1998, Snead 1992. These properties of GHB are used in some therapeutic indications in human beings. However, besides this GABAergic influence, a specific GHBergic entity exists through specific brain synthesis, release, transport, and receptors. At present, the functional specificity of this signal remains largely unknown, but recent study results shed some light on the molecular and cellular organization of the GHB system. This article will focus on new insights concerning GHB synthesis, GHB receptors, and functional links with the GABAergic system.

Section snippets

The reductive route of gamma-aminobutyric acid metabolism leads to GHB

Gamma-aminobutyric acid is first transaminated into an aldehydic product (succinic semialdehyde; SSA) by GABA-T in a manner similar to what has been described for the degradative pathways of catecholamines or serotonin. This transamination is thought to occur largely in the mitochondria. Then SSA either follows the oxidative route inside the mitochondria and is converted into succinic acid by succinic semialdehyde dehydrogenase and enters the Krebs cycle or it could leak out of the mitochondria

Succinic semialdehyde reductase is a neuronal enzyme present in axonal processes and synaptic structures

At the optical level, only neurons in the hypothalamus, cortex, and hippocampus seem to be labeled by SSR antibody. In these last two regions, pyramidal cells are heavily stained, particularly in the CA1 region of the hippocampus. The neuronal cytosol is strongly immunoreactive in general, with labeling of numerous processes and fibers. Glial cells appear not to be labeled.

Triple labeling in the rat substantia nigra and striatum was carried out at the confocal microscopic level with monoclonal

A succinic semialdehyde reductase, able to synthesize gamma-hydroxybutyric acid in the brain, was cloned from rat brain hippocampus

Cloning of brain SSR was undertaken to understand the mechanism of the regulation of GHB synthesis. The SSR was purified from total rat brain by using a series of chromatographic steps followed by a two-dimensional gel electrophoresis on SDS-PAGE. Then the SSR spot was digested in the gel with modified trypsin, and the peptide mixture was separated by high-performance liquid chromatography. Several homogeneous peptides were sequenced, and polymerase chain reaction oligonucleotide primers were

Some regions of the brain express specific receptors sites for gamma-hydroxybutyric acid that are absent from other organs

Radioactive GHB binds to a total membrane preparation of rat brain in a saturable, reversible manner with high affinity. Kinetic analysis supports the existence of two classes of binding sites, one of high affinity (K_d of 30 to 90 nM) and the other of lower affinity (K_d of about 16 μM). The corresponding binding capacities are B_max-1 = 0.5 pmole/mg protein and B_max-2 = 46 pmoles/mg protein, respectively (Benavides et al., 1982). However, if the membranes were washed with CHAPS or TRITON X-100,

Gamma-hydroxybutyric acid receptors possess a specific pharmacological profile

A number of substances have been tested for their ability to displace radioactive GHB from its binding sites. Of these substances, the principal ligands of GABA receptors (muscimol, isoguvacine, baclofen, bicuculline, picrotoxin) and GABA itself are without effect (Benavides et al., 1982). Substances structurally similar to GHB (ethanol) or capable of acting as precursors (butanediol, gamma-butyrolactone) also are inactive, as are the principal antiepileptics capable of interfering with

Gamma-hydroxybutyric acid receptors are coupled to specific cellular responses

In vitro, the binding of radioactive GHB to a crude membrane fraction from the rat brain is sensitive to nonhydrolyzable analogues of GTP (GTPγS) and to pertussis toxin (Ratomponirina et al., 1995). In vivo, intraventricular pertussis toxin followed by autoradiographic study of the [³H]GHB binding on brain slices of the treated rats showed a decrease in GHB-specific binding, which attained statistical significance only in the frontal cortex. These results suggest that GHB receptors belong to a

Gamma-hydroxybutyric acid modulates GABAergic activity in some regions of the brain

No apparent direct interaction of GHB occurs with the GABA_A receptor complex despite the fact that, in some studies, bicuculline partly reverses the inhibitory properties of GHB in electrophysiological tests (Kozhechkin, 1980). However, GHB might sometimes mimic GABA_A receptor stimulation in tissue-slice experiments or in cell cultures or in pharmacological tests Snead & Liu 1993, Snead et al. 1992. These results are thought to be due either to a GHB-induced modification of GABA release in some

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The aldehyde dehydrogenase superfamily contains NAD(P)⁺-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Nineteen aldehyde dehydrogenase genes have been identified in the human genome. Mutations in these genes that lead to enzyme inactivation are the molecular basis of several diseases, including microphthalmia/anophthalmia, Sjögren–Larsson syndrome, type II hyperprolinemia, γ-hydroxybutyric aciduria, pyridoxine-dependent seizures, gout, and hyperuricemia, De Barsy syndrome and spastic paraplegia, alcohol-related diseases, cancer, and late-onset Alzheimer’s disease. In addition to these clinical phenotypes, transgenic knockout mouse models have suggested a pivotal role for aldehyde dehydrogenases in physiological processes, such as embryogenesis and development, and in protection against obesity and environmentally induced oxidative damage. Finally, aldehyde dehydrogenases have been shown to be characteristic of cancer stem cells, with high aldehyde dehydrogenase activity relating to cancer prognosis. Collectively, these observations suggest that aldehyde dehydrogenases play crucial roles in the biotransformation of endogenous and exogenous aldehydes that are either toxic or essential for life.
Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death
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Citation Excerpt :
Synthetic or exogenous gamma-hydroxybutyrate (GHB) is used since several years in human clinic for various approved indications, including narcolepsy, cataplexia, anesthesia, sleep induction and alcohol withdrawal treatment. During the 3 last decades, various studies demonstrated that, in fact, the mammalian brain is capable of synthesizing endogenous GHB which acts as a neuromodulator possessing most properties of neurotransmitters (Maitre, 1997; Maitre et al., 2000; Crunelli et al., 2006; Andriamampandry et al., 2007; Carter et al., 2009; Coune et al., 2010; Kemmel et al., 2010). It is well admitted that micromolar concentrations of endogenous GHB may be directly derived from GABA metabolism, particularly from the successive transamination and reduction of GABA in the neuronal compartment by GABA-transaminase and aldo-keto-reductase (Lyon et al., 2007).
Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H₂O₂)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H₂O₂ reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H₂O₂-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H₂O₂-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H₂O₂-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for the development of GHB analog-based strategies against neurodegeneration or nerve cell death.
The role of aldehyde reductase AKR1A1 in the metabolism of gamma-hydroxybutyrate in 1321N1 human astrocytoma cells
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GHB may also act as a neuroprotectant, protecting cells from anoxia and/or oxidative stress [6]. GHB can also act as a neurotransmitter [7,8], and there are specific G-protein coupled receptors with which it has been shown to interact [9,10]. Previously we have shown that the aflatoxin aldehyde reductase AKR7A2 is one of the major enzymes responsible for GHB biosynthesis in human cells, and acts as a succinic semialdehyde reductase (SSAR), converting SSA to GHB [11,12].
The role of the aldehyde reductase AKR1A1 in the biosynthesis of gamma-hydroxybutyrate (GHB) has been investigated in cell lines using a specific double stranded siRNA designed to knock down expression of the enzyme. This enzyme, along with the aldo-keto reductase AKR7A2, has been proposed previously to be one of the major succinic semialdehyde reductases in brain. The AKR1A1 siRNA was introduced into the human astrocytoma cell line (1321N1) and AKR1A1 expression was monitored using quantitative reverse-transcriptase PCR and Western blots. Results show an 88% reduction in mRNA levels and a 94% reduction in AKR1A1 protein expression 72 h after transfection with the siRNA. Aldehyde reductase activity was examined in silenced cells by following the aldehyde-dependent conversion of NADPH to NADP at 340 nm. This revealed a 30% decrease in pNBA reductase activity in cell extracts after AKR1A1 silencing. Succinic semialdehyde reductase activity was significantly lower in silenced cells when measured using high concentrations (1 mM) of succinic semialdehyde, but not with low concentrations (10 μM). The effect of silencing on intracellular and extracellular GHB levels was measured using gas chromatography–mass spectrometry. Results show that AKR1A1 has little effect on the production of GHB, indicating that in this cell line alternative enzymes such as the AKR7A2 are likely to play a more significant role in GHB biosynthesis.
Aldehyde Dehydrogenases
2010, Comprehensive Toxicology, Second Edition
The aldehyde dehydrogenase (ALDH) superfamily contains NAD(P)⁺-dependent enzymes that catalyze the oxidation of aldehydes to their corresponding carboxylic acids. Nineteen ALDH genes have been identified in the human genome. Mutations in these genes leading to enzyme inactivation are the molecular basis of several diseases, including Sjögren–Larsson syndrome (SLS), type II hyperprolinemia, γ-hydroxybutyric aciduria and pyridoxine-dependent seizures, alcohol-related diseases, cancer, and late-onset Alzheimer’s disease (AD). In addition to these clinical phenotypes, transgenic knockout mouse models have suggested a pivotal role for ALDHs in physiological processes, such as embryogenesis and development, and in protection against obesity and environmentally induced oxidative damage. Collectively, these observations suggest that ALDHs play crucial roles in the biotransformation of endogenous and exogenous aldehydes that are either toxic or essential for life.
Enzymes involved in the metabolism of γ-hydroxybutyrate in SH-SY5Y cells: Identification of an iron-dependent alcohol dehydrogenase ADHFe1
2009, Chemico-Biological Interactions
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This revealed that these cells not only synthesize GHB, they are also able to secrete GHB into the media over a 24 h period (Fig. 2). In neurons located in the hypothalamus, cortex and hippocampus, the synthesis of GHB appears to take place in the cytosol, catalyzed by SSAR [21]. In some cases, the enzyme appears to be associated with presynaptic terminals, and release of GHB from neuronal cells is associated with mitochondria-enriched synaptosomes.
The metabolism of the endogenous metabolite γ-hydroxybutyrate (GHB) has been studied in a human neuroblastoma cell line SH-SY5Y as a model for examining neuronal metabolism. We show that GHB can be synthesized and released from these cells, indicating that pathways for GHB synthesis and secretion are present. Activities for the major enzymes that are involved in GHB metabolism are reported, and transcripts for AKR1A1, AKR7A2, ALDH5A1 and GABA-T can be detected by RT-PCR. We also demonstrate the presence of the ADHFe1 transcript, a gene that has been reported to encode a hydroxyacid-oxoacid transhydrogenase (HOT). We show that the ADHFe1 gene is related to bacterial GHB dehydrogenases and has a conserved NAD-binding site. The potential for using the SH-SY5Y cell line for investigating GHB catabolism is discussed.
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To identify the sequence of hydroxyacid-oxoacid transhydrogenase (HOT), responsible for the oxidation of 4-hydroxybutyrate in mammalian tissues, we have purified this enzyme from rat liver and obtained partial sequences of proteins coeluting with the enzymatic activity in the last purification step. One of the identified proteins was ‘iron-dependent alcohol dehydrogenase’, an enzyme encoded by a gene present on human chromosome 8q 13.1 and distantly related to bacterial 4-hydroxybutyrate dehydrogenases. The identification of this protein as HOT was confirmed by showing that overexpression of the mouse homologue in HEK cells resulted in the appearance of an enzyme catalyzing the α-ketoglutarate-dependent oxidation of 4-hydroxybutyrate to succinate semialdehyde.

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ArticlesGamma-hydroxybutyric acid as a signaling molecule in brain

Abstract

Introduction

Section snippets

The reductive route of gamma-aminobutyric acid metabolism leads to GHB

Succinic semialdehyde reductase is a neuronal enzyme present in axonal processes and synaptic structures

A succinic semialdehyde reductase, able to synthesize gamma-hydroxybutyric acid in the brain, was cloned from rat brain hippocampus

Some regions of the brain express specific receptors sites for gamma-hydroxybutyric acid that are absent from other organs

Gamma-hydroxybutyric acid receptors possess a specific pharmacological profile

Gamma-hydroxybutyric acid receptors are coupled to specific cellular responses

Gamma-hydroxybutyric acid modulates GABAergic activity in some regions of the brain

Life Sci.

Brain Res

Eur J Pharmacol

Brain Res

Neuroscience

Neurosci Lett

Brain Res

Neuropharmacology

Neuroscience

Biochem Biophys Res Commun

Eur J Pharmacol

Prog Neurobiol

Eur J Pharmacol

Neurosci Lett

Eur J Pharmacol

Biochem Pharmacol

FEBS Lett

Eur J Pharmacol

Eur J Pharmacol

Eur J Pharmacol

Brain Res

Articles
Gamma-hydroxybutyric acid as a signaling molecule in brain