0022-3565/97/2812-0753$03.00/0
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 281:753–760, 1997
`
`Vol. 281, No. 2
`Printed in U.S.A.
`
`␥-Hydroxybutyrate Conversion into GABA Induces
`Displacement of GABAB Binding that is Blocked by Valproate
`and Ethosuximide1
`
`VIVIANE HECHLER, CHARLINE RATOMPONIRINA and MICHEL MAITRE
`L.N.M.I.C, UPR 416 CNRS, Centre de Neurochimie, Strasbourg Cedex, France
`Accepted for publication January 30, 1997
`
`ABSTRACT
`␥-Hydroxybutyrate (GHB) has been reported to be a ligand for
`GABAB receptor(s), although with low or very low affinity (IC50
`⫽ 150 –796 ␮M). In addition, several reports argue for a role of
`GHB via GABAB receptors in both in vivo and in vitro electro-
`physiological experiments. In the present study, we demon-
`strate that the inhibition of GHB’s conversion into GABA by rat
`brain membranes blocks the ability of GHB to interfere with
`GABAB binding. In particular, the inhibition of GHB dehydroge-
`
`nase by valproate or ethosuximide and the blockade of
`GABA-T by aminooxyacetic acid induce the disappearance of
`the GABA-like effect of GHB at GABAB, but also at GABAA,
`receptors. This finding could explain the misinterpretation of in
`vit´ro or in vivo experiments where GHB possesses a GABA-like
`effect. But in addition, it is postulated that the normal metab-
`olism of GHB in brain induces GABAB mechanisms that could
`be blocked by the administration of valproate or ethosuximide.
`
`GHB is a naturally occurring substance that is located in
`almost all brain regions (Vayer et al., 1988), together with
`succinic semialdehyde reductase, the enzyme responsible for
`its synthesis. However, it is thought to play a direct func-
`tional role only in some restricted brain areas, a view sup-
`ported by the heterogeneous distribution of its receptor sites.
`These are located largely in the cortex, hippocampus and
`thalamus, together with dopaminergic brain structures in-
`cluding the dorsal and ventral striatum, olfactory tracts, A9,
`A10 and A12 (Hechler et al., 1992). The major part of the
`hypothalamus, pons-medulla and cerebellum are totally de-
`void of high-affinity binding sites for GHB, as are peripheral
`tissues such as liver, muscles and kidneys. Specific high-
`affinity GHB binding sites have also been found in cell mem-
`branes prepared from human brain (Snead and Liu, 1984).
`This binding does not require Na⫹ and is not displaceable by
`GABA, muscimol, baclofen, isoguvacine, dopamine or picro-
`toxin, but only by GHB and structurally related analogs
`(Benavides et al., 1982).
`Electrophysiological studies have shown an effect of GHB
`on about 50% of the cells examined in the nigro-striatal
`pathway (Harris et al., 1989), in the neocortical region (Olpe
`and Koella, 1979) and in the parietal cortex (Kozhechkin,
`1980). When used at low doses in vivo (5–10 mg/kg), GHB
`induces a depolarizing effect that is blocked by the GHB
`receptor antagonist NCS-382 (Godbout et al., 1995). How-
`
`Received for publication September 3, 1996.
`1 This work was supported by a grant from DRET 93-172.
`
`ever, when used at higher doses both in vivo and in vitro (in
`general ⱖ100 ␮M in vitro and ⱖ300 mg/kg in vivo), GHB
`induces a membrane hyperpolarization that is bicuculline-
`resistant (Olpe and Koella, 1979) but that has been reported
`to be sometimes inhibited by GABAB antagonists (CGP 35
`348 or CGP 55 845) (Xie and Smart, 1992; Williams et al.,
`1995; Ito et al., 1995). The number of GHB-responsive neu-
`rons appears to be much lower than the number of GABA-
`responsive neurons in the brain regions investigated. The
`neuronal hyperpolarization induced by GHB in vivo or after
`incubation of brain tissue slices with GHB probably explains
`the decrease in dopaminergic neuronal activity resulting in a
`decreased dopamine release in the nigro-striatal pathway
`after administration of GHB (Walters et al., 1973). Baclofen
`has similar effects on dopaminergic neurons (Da Prada and
`Keller, 1976).
`Thus GHB induces specific physiological responses that
`are dependent on its interaction with GHB receptors that are
`distinct from GABAB receptors in kinetics, pharmacology,
`distribution and ontogeny (Benavides et al., 1982; Hechler et
`al., 1992; Snead, 1994). However, a possible GABAergic con-
`tribution to the pharmacological effects of GHB must be
`considered. This contribution can be explained by a direct
`interaction of GHB with GABAB sites, because GHB dis-
`placed GABAB binding with an IC50 value of 100–200 ␮M
`(Bernasconi et al., 1992), 500 ␮M (Ito et al., 1995) or 796 ␮M
`(Ishige et al., 1996). These values largely exceed endogenous
`GHB levels in brain, which peaked at maxima of 5 to 6 ␮M
`(Vayer et al., 1988).
`
`ABBREVIATIONS: GHB, ␥-hydroxybutyrate; SSA, succinic semialdehyde; GABA-T, ␥-aminobutyrate transaminase.
`
`753
`AMN1006
`IPR of Patent No. 8,772,306
`
`

`
`754
`
`Hechler et al.
`
`Several authors have suggested that labeled GABA is
`formed in vivo after the administration of labeled GHB with
`no increase in GABA concentration (see, for exemple, DeFeu-
`dis and Collier, 1970), although one group has suggested that
`brain GABA levels are increased (Della Pietra et al., 1966). In
`our hands, [3H]-GHB is consistently transformed into [3H]-
`GABA by brain extract (Vayer et al., 1985). This conversion is
`due to the coupled effect of GHB dehydrogenase and NADP to
`yield succinic semialdehyde (SSA); then GABA-T activity
`transaminates SSA into GABA. GHB dehydrogenase is a
`cytosolic enzyme that is inhibited by a wide range of antiepi-
`leptic compounds, including barbiturates, valproate, etho-
`suximide and trimethadione (Kaufman and Nelson, 1991).
`Most of these compounds, when administered to rats, induce
`an accumulation of GHB in the brain (Snead et al., 1980).
`The purpose of this study was to demonstrate that, under
`the conditions used for in vitro GABAB binding experiments,
`under in vivo conditions and in experiments carried out with
`brain slices or cell cultures, GHB is partially degraded by
`brain extract into GABA, which then displaces GABAB bind-
`ing. In our experiments, GHB degradation into GABA was
`prevented by GHB dehydrogenase inhibition with either val-
`proate or ethosuximide or by GABA-T inhibition with ami-
`nooxyacetic acid.
`
`Materials and Methods
`Animals. Male Wistar rats weighing 250 to 300 g were killed by
`a blow on the head; their brains were rapidly extracted and used as
`starting material. Procedures involving animals and their care were
`conducted in conformity with national and international regulations
`(decree n° 87848, October 19, 1987, and EEC council directive 86/
`609, QJ L 358, December 12, 1987).
`GABAB binding to rat brain membranes. The methods of Hill
`and Bowery (1981, method 1) and of Bernasconi et al. (1992, method
`2) were used to assess the ability of GHB to displace GABAB binding.
`Method 1 was used in general, but method 2 was adopted in some
`experiments because an IC50 value of 150 ␮M was measured for GHB
`under these conditions. Crude synaptic membranes (P2 fraction)
`were prepared from total brain or from cerebrum or cerebellum. In
`method 2, the vesicular preparation was further purified by centrif-
`ugation on 0.8 M buffered sucrose. After hypoosmotic shock, the
`membranes were centrifuged and frozen at ⫺20°C overnight (method
`1) or for 2 days (method 2). After several incubations and washings
`at ambient temperature, the pellets were used for GABAB binding
`determinations. Incubations were carried out in 600 ␮l of buffer (50
`mM Tris-HCl, 2.5 mM CaCl2, pH 7.4) at ambient temperature with
`25 nM [3H]-GABA (Dupont-NEN, France, 74 Ci/mmol). Isoguvacine
`(100 ␮M, final concentration) and GHB (concentrations from 10 ␮M
`to 5 mM) were added. In some experiments, media were supple-
`mented with valproate or ethosuximide at a final concentration of 1.5
`mM. Nonspecific binding was determined in the presence of 100 ␮M
`baclofen.
`GABAA binding in the presence of GHB. The effect of GHB on
`GABAA binding was tested using [3H]-muscimol (19 Ci/mmol, Du-
`pont-NEN). Membranes were prepared from a crude synaptosomal/
`mitochondrial fraction of rat brain according to the method of Olsen
`et al. (1981). GABAA receptor binding was measured by a rapid
`filtration assay at 0–4°C in Na⫹-free buffer. [3H]-muscimol was
`included at 25 nM (final concentration) with or without 0.1 mM
`nonradioactive GABA. Samples containing 1 mg of protein in an
`assay volume of 600 ␮l were incubated 15 min at 0–4°C with increas-
`ing concentrations of GHB (10 ␮M to 10 mM). The incubation media
`were rapidly filtered at 4°C under suction and then were rinsed twice
`with 2 ml incubation buffer (50 mM Tris-citrate, pH 7.1, at 0°C).
`Radioactive filters were counted by liquid scintillation.
`
`Vol. 281
`
`Effects of antiabsence drugs on the conversion of [3H]-GHB
`to [3H]-GABA by rat brain membranes. Crude synaptic mem-
`branes were prepared according to Hill and Bowery (1981). These
`membranes were incubated at ambient temperature in 50 mM po-
`tassium phosphate buffer, pH 7.4, containing 200 ␮M [3H]-GHB (10
`␮Ci/mmol) and 1.5 mM of either ethosuximide or valproate. The
`kinetics of the [3H]-GABA formed was monitored after separation
`from [3H]-GHB on a Dowex 50W-X8 column (0.5 ⫻ 3 cm, H⫹ form).
`Controls were carried out in the absence of antiepileptic drugs.
`Radioactive GABA eluted from the columns by 0.1 N NaOH was
`counted by means of a liquid scintillation counter (Vayer et al., 1985).
`In another set of experiments, various concentrations of valproate
`or ethosuximide (0–5 mM) were added to the medium and incubated
`for 20 min at ambient temperature in the presence of 200 ␮M
`[3H]-GHB (10 ␮Ci/mmole). The [3H]-GABA formed at each inhibitor
`concentration was measured using the ion-exchange chromato-
`graphic protocol previously described. The Ki value for each inhibitor
`was determined by plotting 1/v ⫽ f([inhibitor]).
`Measurement of [3H]-aminoacids formed from [3H]-GHB in
`the presence of rat brain crude synaptosomal membranes.
`Crude synaptosomal membranes were prepared from a whole rat
`brain according to the method of Hill and Bowery (1981). These
`membranes were incubated 20 min at ambient temperature with 1
`ml of 50 mM Tris-HCl, pH 7.4, containing CaCl2 (2.5 mM) and 200
`␮M [3H]-GHB (100 ␮Ci/200 nmol). Perchloric acid (0.1 M, final con-
`centration) was added to precipitate the proteins, which were re-
`moved by centrifugation. The amino acid content of the supernatant
`was determined by separation of the amino acids’ o-phthalaldehyde
`derivatives by high-performance chromatography/fluorimetric detec-
`tion, using a modification of the method of Allison et al. (1984).
`Briefly, all chromatographic separations were performed with a
`Nucleosil C 18 column (5 ␮m, 25 ⫻ 0.4 cm) with two Waters pumps
`590 and a Waters Baseline 810 integrator. Detection was carried out
`with a Waters fluorimeter 470 (excitation: 345 nm, emission: 455
`nm). The mobile phase was a binary gradient of solution A (0.1 M
`NaH2PO4, pH 6.0, containing 2% methanol, pH 6.0) and of solution B
`(40% 0.1 M NaH2PO4, pH 6.0, 30% methanol and 30% acetonitrile).
`Precolumn autoderivatization (2 min) and injection were achieved
`with a CMA 200 refrigerated Microsampler (Carnegie Medicine,
`Sweden) by adding to 20 ␮l of tissue extract 20 ␮l of the following
`derivatization mixture: 5 ml of 0.1 M sodium tetraborate, pH 9.5,
`containing 10 ␮l of 3-mercaptopropionic acid (Sigma, Aldrich
`Chimie, France) and 15 mg of o-phthalaldehyde (Sigma) in 500 ␮l of
`methanol. Elution was carried out at a rate of 0.8 ml/min and at a
`temperature of 35°C with the following steps: 0 min, 90% A/10% B;
`15 min, 40% A/60% B (linear gradient); 16 min, 40% A/60% B (iso-
`cratic); 19 min, 100% B (isocratic); 24 min, 90% A/10% B (isocratic)
`until 29 min.
`The different peaks of the amino acids derivatives were collected
`after chromatographic separation, and their radioactivities were de-
`termined by liquid scintillation spectrometry.
`Statistical analysis. Nonlinear regression fitting and IC50 cal-
`culations were performed using the Graphpad-Prism program. Com-
`parison between regression curves was analyzed using the two-way
`ANOVA statistical test.
`
`Results
`Effects of GHB on GABAB binding in the presence
`and absence of GHB dehydrogenase inhibitors. In a
`first set of experiments, GABAB binding was carried out on
`rat brain crude synaptosomal membranes prepared accord-
`ing to the method of Bernasconi et al. (1992) or to that of Hill
`and Bowery (1981). The presence of 100 ␮M GHB in the
`incubation medium led to different percentages of displace-
`ment of radioactive GABA (from zero to a maximum of 37%,
`table 1). This heterogeneity was probably due to the variation
`
`AMN1006
`IPR of Patent No. 8,772,306
`
`

`
`1997
`
`in the amount of GABA formed from GHB in the different
`incubation media. However, when valproate (5 mM) was
`present in the medium, GHB was without effect on GABAB
`binding no matter what technique was used for membrane
`preparation (table 1).
`In a second set of experiments, displacement by GHB of
`GABAB binding was studied in the presence and absence of
`concentrations of GHB dehydrogenase inhibitors (1.5 mM
`valproate or 1.5 mM ethosuximide) that blocked the conver-
`sion of GHB into SSA almost completely. Under these condi-
`tions, the IC50 value for GHB (23 ⫾ 0.66 ␮M) was consider-
`ably increased, reaching 0.51 ⫾ 0.012 mM in the presence of
`ethosuximide and 5.1 ⫾ 0.38 mM in the presence of valproate
`(fig. 1A, B and C). To determine that GABAB binding was not
`changed by the presence of the drugs used, we tested the
`displacement of [3H]-GABA by baclofen in the presence of 1.5
`mM valproate (fig. 2). No effect was apparent, and an IC50
`value of 566 nM was calculated for baclofen in the absence of
`valproate, compared with an IC50 value of 964 nM in the
`presence of valproate. Statistical comparison of the two dis-
`placement curves showed no significant difference between
`them (P ⫽ .09, two-way ANOVA, Graphpad-Prism program).
`Effect of GHB on GABAB binding when GHB degra-
`dation was blocked by GABA-T inhibitor. The degrada-
`tion of GHB to GABA implies the presence in the brain
`membrane preparation of GABA-T, which is capable of con-
`verting SSA to GABA. To demonstrate the role of this
`GABA-T activity, GABAB specific binding was measured in
`the presence of GHB alone (300 ␮M) or in the presence of
`GHB (300 ␮M) and aminooxyacetic acid (500 ␮M). The re-
`sults of these experiments are shown in figure 3. GHB alone
`displaced specific GABAB binding by about 35%, whereas the
`presence of aminooxyacetic acid completely blocked this ef-
`fect of GHB. Compared with those in figure 1A, these results
`demonstrate that the ability of GHB to displace GABAB
`binding is not uniform but depends on the batch of mem-
`branes used and their potency to convert GHB into GABA.
`The apparent Ki value for aminooxyacetic acid inhibition of
`GHB conversion into GABA was measured under GABAB
`binding conditions for various concentrations of inhibitor
`
`GHB Effects on GABAB Binding
`
`755
`
`(0–500 ␮M) for a fixed incubation time (20 min) and a fixed
`concentration of GHB (200 ␮M). The graphical representa-
`tion of 1/v ⫽ f ([inhibitor]) gives a Ki value of 339 ␮M, and in
`the absence of inhibitor, 0.35% of GHB was converted into
`GABA (fig. 4).
`Demonstration that GABA is formed from GHB in a
`standard incubation medium used for GABAB binding
`assays. The formation of [3H]-GABA from [3H]-GHB was
`directly quantified in the medium incubated with the crude
`synaptosomal membranes under the conditions required for
`GABAB binding. Membranes prepared from rat brain (meth-
`od 1) were incubated for 20 min at room temperature with
`radioactive GHB. Chromatographic profiles revealed that all
`amino acids were present in significant amounts in the brain
`membrane extract, but only GABA was radioactive. That
`0.36% of [3H]-GHB was converted into [3H]-GABA suggests a
`concentration of about 720 nM GABA in the medium.
`In control experiments, GABAB binding was tested in the
`presence of 200 ␮M GHB or 720 nM GABA. Under these
`conditions, GHB and GABA displaced [3H]-GABA by 58%
`and 63%, respectively (results not shown). These experi-
`ments showed that the concentration of GABA formed from
`GHB under GABAB binding conditions was able to reproduce
`the GHB effect.
`Effects of antiabsence drugs on [3H]-GHB transfor-
`mation into [3H]-GABA by rat brain membranes. On
`incubation with crude brain synaptosomal membranes under
`the same conditions as for the GABAB binding assay, [3H]-
`GHB was rapidly converted to [3H]-GABA. The kinetics of
`this conversion were followed for 30 min (fig. 5). Under con-
`trol conditions, the reaction was linear for about 10 min, and
`the GABA formation was 18.7 pmol/min/mg protein. During
`a 20-min incubation, about 0.37% (0.32%–0.37%) of [3H]-
`GHB was converted. In the presence of 1.5 mM ethosuximide
`or 1.5 mM valproate, GABA synthesis from GHB was linear
`for 30 min, and the activity was reduced to 6.6 pmol/min/mg
`(35% of control activity) or to 1.7 pmol/min/mg (9% of control
`activity), respectively.
`The Ki values for inhibition of [3H]-GHB conversion into
`[3H]-GABA were determined for valproate and ethosuximide.
`
`TABLE 1
`Effects of GHB on GABAB binding in the presence and in the absence of valproate
`Crude synaptosomal membranes were prepared according to Bernasconi et al. (1992) or Hill and Bowery (1981). Membranes were incubated in Tris-HCl 50 mM, CaCl2
`2.5 mM, pH 7.4, containing 100 ␮M isoguvacine, [3H]GABA (25 nM, 74 Ci/mmol) and GHB 100 ␮M. In some experiments, valproate (5 mM) was added in order fully
`to inhibit GHB dehydrogenase. After a 15-min incubation at room temperature, bound [3H]GABA was separated from free [3H]GABA by rapid centrifugation at 40,000 ⫻
`g for 30 min.
`
`Crude Synaptosomal Membranes Prepared According to the Method of Bernas-
`coni etal.(1992)
`
`Crude Synaptosomal Membranes Prepared According to the Method of Hill and
`Bowery (1981)
`
`Cerebellum
`Total binding: 5411 ⫾ 217 cpm
`Specific binding: 3390 cpm
`Nonspecific binding: 2021 ⫾ 111 cpm
`GHB 100 ␮M: 1269 cpm displaced 37% of the specific binding
`GHB 100 ␮M ⫹ valproate 5 mM:
`0 cpm displaced
`Cerebrum
`Total binding: 6922 ⫾ 312 cpm
`Specific binding: 3882 cpm
`Nonspecific binding: 3040 ⫾ 52 cpm
`GHB 100 ␮M: 933 cpm displaced 24% of the specific binding
`GHB 100 ␮M ⫹ Valproate 5 mM:
`0 cpm displaced
`
`Cerebellum
`Total binding: 6329 ⫾ 256 cpm
`Specific binding: 1827 cpm
`Nonspecific binding: 4502 ⫾ 318 cpm
`GHB 100 ␮M: 335 cpm displaced 18% of the specific binding
`GHB 100 ␮M ⫹ valproate 5 mM:
`0 cpm displaced
`Cerebrum
`Total binding: 7212 ⫾ 236 cpm
`Specific binding: 2393 cpm
`Nonspecific binding: 4848 ⫾ 613 cpm
`GHB 100 ␮M: 0 cpm displaced
`GHB 100 ␮M ⫹ valproate 5 mM:
`0 cpm displaced
`
`AMN1006
`IPR of Patent No. 8,772,306
`
`

`
`756
`
`Hechler et al.
`
`Vol. 281
`
`Fig. 1. GABAB binding was carried out as de-
`scribed by Hill and Bowery (1981). Crude synaptic
`membranes were prepared from a whole rat brain
`P2 fraction dispersed in distilled water and centri-
`fuged at 8000 ⫻ g for 20 min. The supernatant was
`then centrifuged at 50,000 g, and the resulting
`pellet, after a second wash in distilled water, was
`recentrifuged and stored at ⫺20°C overnight. The
`pellet was then incubated and washed as indicated
`in the original protocol. Binding assays were per-
`formed in 50 mM Tris-HCl buffer, pH 7.4, contain-
`ing 2.5 mM CaCl2 at ambient temperature. Incuba-
`tion media contained [3H]-GABA (25 nM) and 100
`␮M isoguvacine. Total reversible binding was mea-
`sured in the presence of 100 ␮M baclofen. A) Dis-
`placement curve of GHB on GABAB binding from
`rat brain crude synaptosomal membranes.
`In-
`creasing concentrations of GHB displace [3H]-
`GABA in the presence of 100 ␮M isoguvacine with
`an IC50 value of 23 ⫾ 0.66 ␮M (nonlinear regres-
`sion line, Graphpad-Prism program). B) Same ex-
`periment as in panel A, but all the incubation media
`contained 1.5 mM sodium valproate. IC50 is in-
`creased to a value of 5.1 ⫾ 0.38 mM. Under the
`same conditions, the activity of baclofen in dis-
`placing [3H]-GABAB binding was not altered (non-
`linear regression line, Graphpad Prism program).
`C) Same experiment as in panel A, but all the
`incubation media contained 1.5 mM ethosuximide.
`The potency of GHB in displacing GABAB binding
`is greatly decreased (IC50 ⫽ 0.51 ⫾ 0.012 mM)
`(nonlinear regression line, Graphpad-Prism pro-
`gram).
`
`AMN1006
`IPR of Patent No. 8,772,306
`
`

`
`1997
`
`GHB Effects on GABAB Binding
`
`757
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`Fig. 2. Displacement curve of [3H]-GABAB binding according to Hill and
`Bowery (1981) in the absence (䉬) or presence (●) of 1.5 mM valproate.
`Binding was carried out in the presence of 100 ␮M isoguvacine, and
`nonspecific binding was determined with 100 ␮M baclofen. The differ-
`ences between the two curves are not significant (P ⫽ .09, two-way
`ANOVA). Each data point is the mean of three separate determinations.
`
`Under the GABAB binding conditions (membrane prepara-
`tion and incubation medium according to Hill and Bowery,
`1981), valproate and ethosuximide inhibit GABA synthesis
`from GHB with Ki values of 1.0 mM (r ⫽ 0.93) and 2.0 mM
`(r ⫽ 0.98), respectively. GHB concentration was 200 ␮M in
`each case. In the absence of valproate and of ethosuximide,
`0.55% and 0.51% of GHB, respectively, were converted into
`GABA after a 20-min incubation (fig. 6).
`GABAA binding in presence of GHB. Under the condi-
`tions described by Olsen et al. (1981) for GABA binding,
`[3H]-muscimol was displaced by GHB with an IC50 value of
`4.6 ⫾ 0.4 mM (r ⫽ 0.91). However, in the presence of 1.5 mM
`valproate, no significant [3H]-muscimol displacement was
`induced by GHB (fig. 7).
`
`Discussion
`Several authors have described the displacement of [3H]-
`GABA from GABAB sites by GHB, but they have reported
`IC50 values varying from 150 ␮M (Bernasconi et al., 1992), to
`500 ␮M (Ito et al., 1995) and 796 ␮M (Ishige et al., 1996). Our
`own results have ranged from 23 ␮M (the present results) to
`about 520 ␮M (unpublished results) and largely depend on
`the batch of membranes and the protocol used for GABAB
`binding. Using the conditions of Hill and Bowery (1981) or
`Bernasconi et al. (1992), such large variations suggest the
`degradation of GHB by the synaptosomal membranes, which
`can be modified by the methods used for preparing the mem-
`branes and/or the incubation conditions (time, temperature,
`pH and concentrations of GHB). GHB could be converted into
`GABA in vitro by the sequential action of GHB dehydroge-
`nase, which oxidizes GHB to SSA, and then a GABA-T activ-
`ity transaminating SSA to GABA. All the free amino acids
`that could be detected under the present conditions were
`identified in the extract of the synaptosomal/mitochondrial
`membranes, in concentrations of about 0.1 to 0.4 ␮M. This
`result suggests that the cofactors (glutamate, NADP and so
`on) necessary for the enzymatic conversion of GHB to GABA
`are present in significant amounts in the crude synaptosomal
`
`Fig. 3. Displacement of GABAB binding by GHB in the presence or
`absence of a GABA-T inhibitor. Incubation conditions and GABAB
`membranes were identical to those described in the protocol of Hill and
`Bowery (1987). Column A ⫽ control; specific GABAB binding displace-
`able by 100 ␮M baclofen. Column B ⫽ specific GABAB binding dis-
`placeable by 300 ␮M GHB (significantly different from column A, P ⬍
`.01). Column C ⫽ specific GABAB binding in the presence of 300 ␮M
`GHB and 500 ␮M aminooxyacetic acid. The inhibition of GABA-T from
`rat brain crude synaptosomal membranes blocks the synthesis of
`GABA from GHB and inhibits the effect of GHB on GABAB binding. In
`this set of experiments, 300 ␮M GHB displaced [3H]-GABAB binding by
`about 35%. Each data point is the mean of three separate determina-
`tions.
`
`Fig. 4. Determination of the Ki value for aminooxyacetic acid (339 ␮M,
`r ⫽ 0.81). Ordinate ⫽ 1/radioactive GABA produced from 200 ␮M GHB
`after a 20-min incubation, Abscissa ⫽ concentration of aminooxyacetic
`acid. Conditions were those described in the legend for figure 5.
`
`membrane preparation used for GABAb binding experi-
`ments.
`Two types of enzymes in brain are able to catalyze the
`oxidation of GHB to SSA (Kaufman and Nelson, 1991). One of
`these enzymes is a cytosolic NADP⫹-dependent oxidoreduc-
`tase, whereas the other is present in the mitochondrial frac-
`tion and does not require NAD⫹ or NADP⫹. The former
`enzyme, which has been named GHB dehydrogenase, is more
`likely to be the main route for GHB degradation in brain
`because its inhibition by valproate and other antiepileptic
`drugs (trimethadione, ethosuximide) leads to an accumula-
`tion of GHB in brain (Snead et al., 1980). The mitochondrial
`enzyme is not sensitive to valproate. In the in vitro experi-
`ments, the presence of valproate and ethosuximide with syn-
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`Fig. 5. Kinetics of [3H]-GABA formation
`from 200 ␮M [3H]-GHB in the presence
`of rat brain crude synaptosomal mem-
`branes prepared according to Hill and
`Bowery (1981). Incubations were carried
`out at ambient temperature in 50 mM
`potassium phosphate buffer, pH 7.4.
`The [3H]-GABA formed was separated
`from [3H]-GHB by ion exchange chroma-
`tography on a Dowex 50 W-X8 column
`and elution with 0.1 N NaOH.
`å Control; ç In the presence of 1.5 mM
`ethosuximide (65% inhibition compared
`with control, the activity being calculated
`during the linear phase of the kinetics; 䉬
`In the presence of 1.5 mM valproate
`(94% inhibition compared with the linear
`phase of the control).
`
`Fig. 6. Determination of apparent Ki values for valproate (å) and etho-
`suximide (f). Ordinate ⫽ 1/amount of radioactive GABA produced in a
`20-min incubation under the conditions described in the legend for
`figure 5. Abscissa ⫽ concentration of inhibitors, the concentration of
`GHB being fixed at 200 ␮M. During this period of time, the conversion
`of GHB into GABA could be considered linear in the presence or
`absence of inhibitors. The Ki value measured for valproate is 1 mM (r ⫽
`0.93) and for ethosuximide is 2 mM (r ⫽ 0.97). Because the mechanism
`of inhibition is noncompetitive, these Ki values are the real ones mea-
`sured in GABAB binding conditions.
`
`aptosomal/mitochondrial membranes renders GHB ineffec-
`tive for displacing GABA from GABAB binding sites. The
`same result is obtained when GABA-T activity of the mem-
`brane preparation is blocked by incubation with aminooxy-
`acetic acid. Thus inhibition of the conversion of GHB to
`GABA results in a lack of interference with GABAB binding.
`The synthesis of [3H]-GABA from [3H]-GHB has been dem-
`onstrated in vitro under the conditions required for GABAB
`binding. The concentration of GABA in the medium at the
`end of the 20-min incubation period in the presence of 200
`␮M GHB was about 720 nM, a concentration high enough to
`
`Fig. 7. Displacement curve of [3H]-muscimol binding in the presence of
`increasing concentrations of GHB. The methodology of Olsen et al.
`(1981) has been used, because under the conditions, the Kd values for
`GABA are of high affinities. An IC50 value of 4.6 ⫾ 0.4 mM (r ⫽ 0.91) was
`calculated for GHB ({). In the presence of valproate (1.5 mM), the
`displacement of radioactive muscimol disappears (Q). Each data point
`is the mean of three separate determinations.
`
`interfere with GABAB binding. This result explains the ap-
`parent interaction of GHB with GABAB sites described in
`vitro. Interference with the GABAA receptor(s) is probably
`less evident because the Kd value for GABAA binding is
`higher (micromolar range; see Edgar and Schwartz, 1992).
`Even with the membrane preparation and the binding pro-
`tocol of Olsen et al. (1981; Kd values for GABA of 13 and 300
`nM), GHB displaced [3H]-muscimol with a weak affinity.
`This result is in agreement with the studies of Serra et al.
`(1990) and Snead and Liu (1993), which demonstrated no
`modification of [3H]-muscimol or [3H]-flunitrazepam binding
`in the presence of 1 mM GHB. The muscimol-stimulated
`36Cl⫺ uptake by synaptoneurosomes was not altered in these
`studies, probably because of the low EC50 value (8–11 ␮M)
`calculated for muscimol (Edgar and Schwartz, 1992), which
`should be compared with the low concentration of GABA
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`found in the membrane medium after a 20-min incubation. In
`addition, it is possible that conditions of GABAB binding
`(ambient temperature instead of 0–4°C, the nature of the
`membranes and the nature of the incubation medium) favor
`the synthesis of GABA from GHB in vitro.
`Nevertheless, [3H]-GHB binding has been described as
`possessing some of the properties of the GABAA receptor
`complex. It has been claimed that picrotoxin, diazepam and
`pentobarbital enhance [3H]-GHB binding (Snead et al.,
`1992), and an effect of GHB on chloride conductance has been
`proposed (Snead and Nichols, 1987). Under the conditions
`described for the above studies, an effect of GABA synthe-
`sized from GHB cannot be ruled out.
`Moreover, in some studies, an antagonistic effect of bicu-
`culline to GHB-mediated effects has been noted. Ho¨sli et al.
`(1983) described a hyperpolarizing effect of GHB that is
`blocked by bicuculline and is associated with an increase in
`chloride conductance in cultured spinal, brainstem and cer-
`ebellar neurons. No apparent GHB binding sites in these
`structures have been reported in rat brain (Hechler et al.,
`1992). Thus it seems that GABA, formed from GHB in these
`cell cultures, is responsible for the GABAA receptor(s) stim-
`ulation. In other experiments in vivo, GHB possesses prop-
`erties of its own that were bicuculline-resistant, whereas
`under the same conditions, the effects of GABA were antag-
`onized by bicuculline (Olpe and Koella, 1979).
`In several electrophysiological studies carried out by in
`vivo administration of GHB or by application of GHB to
`cerebral tissue slices, GHB behaves like a GABAB ligand, its
`effects being blocked by antagonists at GABAB receptors (see,
`for example, Xie and Smart, 1992). In vivo, conversion of
`radioactive GHB into GABA has been described, and further-
`more, a down-regulation of GABA receptors in the rat brain
`was induced by chronic GHB administration (Gianutsos and
`Suzdak, 1984).
`Thus it appears that besides exerting a specific GHBergic
`effect at GHB receptors, GHB possesses GABAergic proper-
`ties both in vitro and in vivo. When observed in vitro, this
`GABA-like effect is due to GHB conversion by the tissue or
`the tissue extract into GABA, which displaces radioactive
`GABA from its binding sites (GABAA and GABAB). Also in
`vivo, it seems likely that the GABAB response induced by
`GHB is due to local conversion of GHB into GABA. Evidence
`does point to a regional segregation of GABAA and GABAB
`synapses (Misgeld et al., 1995); perhaps GHB selectively
`potentiates the GABAB neurons. This could be realized either
`by regulating GABA release by a GHB-dependent mecha-
`nism at GABAB synapses or by potentiating GABAB syn-
`apses with GHB acting as precursor of a specific GABA pool.
`This phenomenon could explain an in vivo effect of GHB at
`both GABAB and GHB receptors.
`Such a mechanism could also explain the role of GHB in
`inducing general absence epilepsy in rodents. In this model,
`the GABAB agonist baclofen, and GABAergic compounds in
`general, aggravate the symptomatology and EEG distur-
`bances (Snead, 1992). GHB must be given at doses not less
`than 375 mg/kg (about 300–400 ␮M in brain), and the ab-
`sence seizures appear with a latency of 10 to 15 min (Snead,
`1991). Valproate, ethosuximide and trimethadione inhibit
`GHB conversion into GABA in vitro and induce GHB accu-
`mulation in brain after administration in vivo (Snead et al.,
`1980). Despite this GHB accumulation, these compounds
`
`GHB Effects on GABAB Binding
`
`759
`
`normalize the EEG. Thus it seems likely that the GABA
`arising from GHB participates largely in the induction and
`severity of the epileptic syndrome. All the synthetic ligands
`(agonists or antagonist, Maitre et al., 1990; Hechler et al.,
`1993) for the GHB receptor that have been tested so far are
`devoid of epileptic activity, and furthermore, these ligands
`cannot be converted to GABA in vivo.
`
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`BENAVIDES, J., RUMIGNY, J. F., BOURGUIGNON, J. J., CASH, C. D., WERMUTH, C. G.,
`MANDEL, P., VINCENDON, G. AND MAITRE, M.: High affinity binding site for
`gamma-hydroxybutyrate in rat brain. Life Sci. 30: 953–961, 1982.
`BERNASCONI, R., LAUBER, J., MARESCAUX, C., VERGNES, M., MARTIN, P., RUBIO, V.,
`LEONHARDT, T., REYMANN, N. AND BITTIGER, H.: Experimental absence sei-
`zures: Potential role of gamma-hydroxybutyric acid and GABAB receptors.
`J. Neuronal Transm. 35: suppl. 155–177, 1992.
`DA PRADA