0022-3565/97/2812-0753$03.00/0
`THE J OURNAL OF PHARMACOLOGY AND EXPEE.1MENTAL THERAPEUTICS
`Copyright© 199.7 by The American Society for Pharmacology a nd Experimental Therapeutics
`JPET 281:753- 760, 1997
`
`Vol. 281, No. 2
`Prinl.<d in U.S.A
`
`1'-Hydroxybutyrate Conversion into GABA Induces
`Displacement of GABA8 Binding that is Blocked by Valproate
`and Ethosuximide 1
`
`VIVIANE HECHLER, CHARLINE RATOMPONIRINA and MICHEL MAITRE
`
`L.N.M./.C, UPR 416 CNRS, Centre de Neurochimie, Strasbourg Cedex, France
`Accepted for publication January 30, 1997
`
`ABSTRACT
`y-Hydroxybutyrate (GHB) has been reported to be a ligand for
`GABA8 receptor(s), although with low or very low affinity (IC50
`= 150-796 fLM). In addition, several reports argue for a role of
`GHB via GABA8 receptors in both in vivo and in vitro electro(cid:173)
`physiological experiments. In the present study, we demon(cid:173)
`strate that the inhibition of GHB's conversion into GABA by rat
`brain membranes blocks the ability of GHB to interfere with
`GABA8 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-Iike effect of GHB at GABA8 , but also at GABAA,
`receptors. This finding could explain the misinterpretation of in
`vifro or in vivo experiments where GHB possesses a GABA-Iike
`effect. But in addition, it is postulated that the normal metab(cid:173)
`olism of GHB in brain induces GABA8 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(cid:173)
`tional role only in some restricted brain areas, a view sup(cid:173)
`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(cid:173)
`cluding the dorsal and ventral striatum, olfactory tracts,~.
`A1 0 and A 12 (Hechler et al., 1992). The major part of the
`hypothalamus, pons-medulla and cerebellum are totally de(cid:173)
`void of high-affinity binding sites for GHB, as are peripheral
`tissues such as liver, muscles and kidneys. Specific high(cid:173)
`affinity GHB binding sites have also been found in cell mem(cid:173)
`branes prepared from human brain (Snead and Liu, 1984).
`This binding does not require N a + and is not displaceable by
`GABA, muscimol, baclofen, isoguvacine, dopamine or picro(cid:173)
`toxin, but only by GHB and structurally rela ted analogs
`(Benavides et al., 1982).
`Electrophysiological studies have shown an effect of GHB
`on about 50% of the cells examined in the nigra-striatal
`pathway (H arris 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 pt1blication Sep tember 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 ~ 100 fLM in vitro and ~3 00 mg/kg in vivo), GHB
`induces a membrane hyperpolarization that is bicuculline(cid:173)
`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(cid:173)
`rons appears to be much lower than the number of GABA(cid:173)
`r esponsive 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 nigra-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 r eceptors 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(cid:173)
`tribution to the pharmacological effects of GHB must be
`considered. This contribution can be explained by a direct
`interaction of GHB with GABAB sites, becau se GHB dis(cid:173)
`placed GABAB binding with an IC50 value of 100-200 fLM
`(Bernasconi et al., 1992), 500 fLM (Ito et al., 1995) or 796 fLM
`(Ishige et al. , 1996). These values largely exceed endogen ous
`GHB levels in brain, which peaked at maxima of 5 to 6 fLM
`(Vayer et al. , 1988).
`
`ABBREVIATIONS: GHB, , -hydroxybutyrate; SSA, succinic semialdehyde; GABA-T, , -aminobutyrate transaminase.
`
`Ranbaxy Ex. 10 15
`IPR Petition - USP 8,772,306
`
`753
`
`

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`754
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`Hechler et al.
`
`Several authors have suggested that labeled GABA is
`formed in vivo after the administration oflabeled GHB with
`no increase in Gi\BA concentration (see, for exemple, DeFeu(cid:173)
`dis and Collier, 1970), although one group has suggested that
`brain GABA levels are increased (Della Pietra et al. , 1966). In
`om hands, [3H]-GI-IB is consistently ti·ansformed into [3H](cid:173)
`GABA by brain extract 01 ayer et a.l., 1985). This conversion is
`due to the coupled effect ofGHB dehydrogenase and NADP to
`yield succinic semialdehyde (SSA); then GABA-T activity
`transa.minates SSA into GABA. GHB dehydrogenase is a
`cytosolic enzyme that is inhibited by a wide rangeofantiepi(cid:173)
`leptic compotmds, including barbiturates, valproate, etho(cid:173)
`suximide and trimethadione (Kaufman and Nelson, 1991).
`Most offhese compotmds, 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
`t,he conditions used for iii vitro GABAB binding experiment,s,
`under in vivo condWons and in experime nts carried out with
`brain slices or cell cultmes, GHB is partially degraded by
`brain extract int.o GABA, which then displace.s GABAB birJ,d(cid:173)
`ing. In om experiments, GHB degradation into GABA was
`prevented by GHB dehydrogenase inhibition with either val(cid:173)
`proate or ethosuximide or by GABA-T inhibition with ami(cid:173)
`nooxyacetic acid.
`
`Materials and Methods
`
`Animals. Male Wistar rats weighing 250 to 300 g were killed by
`a blow em the head; their brains were r;,tpidly e)ctr;,tcted and used as
`starting material. Procedures involving animals and their care were
`conducted in conformity with nation;,tl and international regulaticms
`(decree no 87848, October 19, 1987, and EEC council directive 86/
`609, QJ L 358, December 12, 1987):
`GABAB binding to rat lJrain membranes. The methods of Hill
`and Bowery (1981, m ethod 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 p.M was mea:;,ured for GHB
`under these conditions. Crude synaptic membranes (P2 fraction)
`were prepared from. total brain or from cerebrum or cer ebellum. In
`m ethocl 2, the vesicular preparation was further purified by centrif(cid:173)
`ugation on 0.8 M buffered sucrose. After hypoosruotic ;:;h ock; 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 J.d ofbuffer (50
`mM Tris-HCl, 2.5 m.M CaC~, pH 7.4) at ambient temperature with
`25 nM (3 H]-GABA (Dupont-NEN, France, 74 CVrumol). Jsoguvacine
`(100 1-l-M. final concentration) and GHB (concentr ations from 10 f•M
`to 5 mM) were added. 1n some experiments, media vvere supple(cid:173)
`mented with valproat.e or ethosuximide at a final concentration of 1.5
`ruM. Nonspecific binding was determined in the presence of 100 1-l-M
`baclofen.
`GABAA binding in the presence ofGHB. The effect of GHB on
`GABAA biniling was tested using [3H]-muscimol (19 CVrumol, Du(cid:173)
`pont-NEN). Membranes were prepared from a crude synaptosomaV
`mitochondrial fraction ofTat brain according to the method of Olsen
`et at. (1981). GABAA receptor binding was measured by a r apid
`filtration assay at 0-4°C in Na+ -free buffer. fHJ-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 pJ were incubated 15 min at O-re with increas(cid:173)
`ing con centrations ofGBB (10 1-l-M to 10 mM). The incubation media
`were rapidly filtered at 4°C under suction and then were rinsed twice
`with 2 m1 incub;,ttion buffer (50 mM Tris-citrate, pH 7 .1, at 0°C).
`Radioactive filters were counted by liquid scintillation.
`
`Vol. 281
`
`Effects of antiabsence dru g-s on the conversion of [3 H]-GHB
`to (3 H]-GABA by rat brain membranes. Crude syn;,tptic mem(cid:173)
`branes were prepared according to Hill and Bowery (1981). These
`membranes were incubat.ed at ambient temperature in 50 mM po(cid:173)
`tassium phosphate buffer, pH 7.4, conta1ni.ng 200 ~~M fH)-GHB (10
`J.!-CVm.mol) 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 X 3 em, H"'- for.m).
`Controls ~ere 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).
`1n another set ofexperi:illents , various concentrations ofvalproate
`or ethosuximide (0-5 mM) ~ere added to the medium and incubated
`for 20 min at ambient temperature in the presence of 200 J.!-M
`fH]-GHB (10 p.Ci/mmole). The [3H]-GABAformed at eachinhlbi:tor
`concentration was measured using the ion-exchange chromato(cid:173)
`graphic protocol previously described. The K 1 value fiJr each inhlbi:tor
`was determined by plotting llv = fl:linhibitor]).
`Measurement of [3 H]-aminoacids formed from (3 HJ-GHB in
`the presence of rat brain crude synaptosomal membranes.
`Crude synaptosomal membranes were prep;,tred from a whole rat
`brain according to the method of Hill and Bowery (1981). These
`m embranes were incubated 20 min at ambient temperature with 1
`m l of 50 ruM Tris-HCJ, pH 7.4, canta ining CaC~ (2.5 mM) and 200
`1-l-M [3 H]-GHB (100 J.!-Ci/200 nmo1). Perchloric acid (0.1 M, final con(cid:173)
`centration) was added t o precipitate the proteins, which were I e(cid:173)
`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/fluori:illetric detec(cid:173)
`tion, using a modification of the method of Allison et al. (1984).
`Briefly, a ll chromatographic separations were perfurmecl with a
`Nucleosil C 18 column (5 ~em, 25 X 0 .4 em) with two Waters: pumps
`590 and a Waters Bas.eline .810 integrator. Dlet.ection 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
`NaH2P04 , pH 6.0, cont.aining2% methanol, pB 6.0) and of solut ion B
`(40% 0.1 M NaH 2P04 , pH 6.0, 30% methanol and 30% aceton itrile).
`Precolumn autoder:ivatization (2 min) ;,tnd injection vvere achieved
`with a CMA 200 r efrigerated Mkr.osampler (Carnegie Medicine,
`Sweden) by adding to 20 pl of tissue extract 20 pl of the following
`derivatization mixture: 5 ml of 0.1 M sodium tetraborate, pH 9.5,
`containing 10 p.l of 3-mercaptopr opionic acid (Sjgma, Aldrich
`Chimie, France) and 15 mg of o-phthalaldehyde (Sigma) in 500 vl of
`m ethanol. Elution was carried out at a rate of 0.8 ml/min and at a
`tempemture of 35"'C with the following steps: 0 min, 90% NlO% B;
`15 min, 40% N60% B (linear gradient); 16 min, 40% A/60% B (iso(cid:173)
`cratic); 19 ruin, 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(cid:173)
`termined by liquid scintillation ~pectrometry.
`S tatistical analysis. Nonlinear regression fitting and IC5 0 cal(cid:173)
`culations were performed using the Graphpad-Pri.sm program. Com(cid:173)
`parison between regression cmves was analyzed using the two-way
`ANOV A st;,ttistic;,tl test.
`
`Results
`Effects of GHB on GABAs b inding in the p resence
`and absence of GHB dehydrogen ase inhibitors. In a
`first set of experiments, GABAs binding was cru·ried out on
`Tat brain crude synaptosomal membranes prepared accord(cid:173)
`ing to the method of Bernasconi et al. (1992) or to that of Hill
`and Bowery (1981). The presence of 100 r.t-M GHB in the
`incubation medium led to different percentages of displace(cid:173)
`ment of radioactive GABA (from zero to a maximum of 37%,
`table 1). Tb.is he t.erogeneity was probably due to the variation
`
`

`
`1997
`
`in the amount of GABA form ed from GHB in the different
`incubation media. However, when valproate (5 mM) was
`present in the medium, GHB was withollt effect on GABAs
`binding no matter what technique was used for membrane
`preparation (table 1).
`In a se.cond set of experiments, displacement by GHB of
`GABAs binding was studied in the presence and absence of
`concentrations of GHB dehych·ogenase inhiliitors (1.5 mM
`valproate or 1.5 mM ethosuximide) that blocked the conver(cid:173)
`sion of GHB into SSA almost completely. Under these condi(cid:173)
`tions, the IC5 0 value foT GHB (23 ::t 0.66 (1-M) was con sider(cid:173)
`ably increased, reaching 0.5 1 ± 0.012 mM in the presence of
`ethosuximide and5 .1 ± 0.38 mM in the presence ofvalproate
`(fig. lA, Band C). To determine that GABAs binding was not
`changed by the presence of the ch·ugs used, we tested the
`displacement of [3 H]-GABA by bac]ofen in the presence of 1.5
`mM valproate (fig. 2). No effect was apparent, and an IC60
`value of 566 nM was calculated for bac1ofen in the absence of
`valproate, compared with an IC50 value of 964 nl\11 in the
`presence of valproate. Statistical compa rison of the two dis(cid:173)
`placement cmves showed no significant differ ence between
`them (P = .09, two-way ANOVA, GTaphpad-Prism program).
`Effect of GHB on GABAa binding when GliB degra(cid:173)
`dation was blocked by GABA-T inhibitor. The degrada(cid:173)
`tion of G HB to GABA implies the presence in the brain
`membrane preparation of GABA-T, which is capable of con(cid:173)
`verting SSA to GABA. To demonstrate the role of this
`GABA-T activity, GABAs specific binding was measured in
`t,he presence of GHB a lone (300 f.I,M) or in t,he presence of
`GHB (300 t-LM) and aminooxyacetic acid (500 (1-M). There(cid:173)
`sults ofthese expel'iments are shown in figure 3. GHB alone
`displaced specific GABAs binding by about 35%, whereas the
`presence of aminooxyacetic acid completely blocked this ef(cid:173)
`fect, ofGHB. Comp:ued with those in figure lA, these r esults
`demonstrate that the ability of GHB to displace GABAs
`binding is not uniform but depends on the batch of m em(cid:173)
`branes used and their pot,ency to convert. GHB into GABA.
`The a ppar en t K; value for a minooxyacetic acid inhibition of
`GHB conversion into GABA was measured under GA.BAB
`binding conditions for various concentrations of inhibitor
`
`GHB Effects on GABAs Binding
`
`755
`
`(0---500 f.l.M) for a fixed incubation t,ime (20 min) and a fixed
`concentration of GHB (200 p..M). Th e graphical r epl·esenta(cid:173)
`t.ion of JJu = f ([inhibitor]) gives a K1 -value of 3:39 ,u.M, and in
`the absence of inhibitor, 0.35% of GHB was converted into
`GABA (fig. 4).
`D emonstration that GABA is formed from GHB in a
`standard incubation medium used for GABAB binding
`assays. The forma.tion of [3H]-GABA from [3H] -GHB was
`directly quantified in the medium incubated with the cr ude
`synaptosomal m embranes w1der the conditions required for
`GABAs binding. Membranes prepared from rat brain (meth(cid:173)
`od 1) were incubated for 20 min at room temperatme with
`radioactive GHB. Ghromatogtaphic 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 [3 H]-GABA s uggests a
`concentration of about 720 nM GABA in the m edium.
`In control experiments, GABAs binding was test,ed in the
`presence of 200 vM GHB or 720 nM GABA. Under these
`conditions, GHB and GABA displaced [3H]-GABA by 58%
`and 63%, respectively (results not shown). These eX!)eri(cid:173)
`ments showed that the concentTation of GABA formed from
`GHB under GABAB binding conditions was able to reproduce
`the GHB effect.
`Effects of antiabsence drugs on [:lff"]-GHB transfor(cid:173)
`mation into [3 H]-GABA by rat brain membranes. On
`incubation with crude brain synaptosomal membranes under
`the same conditions as for the GABAB binding assay, [3H](cid:173)
`GHB was rapidly converted to [3H]-GABA. The kinetics of
`this conversion were followed for 30 min (fig. 5). Under con(cid:173)
`trol conditions, the reaction was linear for about 10 min, and
`the GABA formation was 18.7 pmollminlmg protein. During
`a 20-min incubation, about 0.37% (0.32o/o-0.37%) of [3H](cid:173)
`GHB was converted. In the presence of 1.5 mM et11osuximicle
`or 1.5 mM valproat.e, GABA synthesis from GHB was linear
`for 30 min, and the activity was reduced to 6.6 pmollmin/mg
`(35% of control activity) or to 1.7 pmo1/min/mg (9% of control
`activity), respectively .
`The K1 values for inhibition of [3H]-GHB conversion into
`[3H]-GABA were d eter mined for valproate and ethosuximide.
`
`TABLE 1
`Effects of GHB on GABAs binding In the presence and in the absence of valproate
`Crude synaptosomal m embranes were prepared according to Bernasconi eta/. (1992) or Hill and Bowery (1981 ). Membranes were incubated in Tris-HCI SO mM, CaCI2
`2.5 mM, pH 7 .4, containing 100 ,~M isogwacine, ["H)GABA (25 nM, 74 Ci/mmoQ 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 ["'H]GABA was separ~ed from free ["'H]GABA by rapid centrifugation at 40,000 X
`g for 30 min.
`
`Crude Synaptosomal Membranes Prepar~d According to tha M~thod of Barnas·
`coni et a/. (1992)
`
`Crude Synaptosomal Membranes Prepared Acr.ording to lhe MeU1od of Hill and
`Bowery (1 981)
`
`Cerebellum
`Total binding: 54 11 :t 217 cpm
`Specific binding: 3390 cprn
`Nonspecific binding: 2021 ± 111 cpm
`GHB 100 p.M: 1269 cpm displaced 37% of the specific binding
`GHB 100 J.LM + valproate 5 mM:
`0 cpm displaced
`Cerebrum
`Total binding: 6922 ::+:: 312 cprn
`Specific binding : 3882 cpm
`Nonspecific binding: 3040 ± 52 cpm
`GHB 100 p.M: 933 cpm displaced 24% of the specific binding
`GHB 100 J.LM + Valproate 5 mM:
`0 cpm displaced
`
`Cerebellum
`Total binding: 6329 ± 256 cpm
`Specific binding: 1827 cpm
`Nonspecific binding: 4502 ± 318 cpm
`GHB 100 f!M: 335 cpm displaced 18% of the specific binding
`GHB 100 p.,M + valproate 5 mM:
`0 cpm displaced
`Cerebrum
`Total binding: 7212 :t 236 cpm
`Specific binding: 2393 cpm
`Nonspecific binding: 4848 ± 613 cpm
`GHB 100 f!M: 0 cpm displaced
`GHB 100 p.,M + valproate 5 mM:
`0 cpm displaced
`
`

`
`756
`
`Hechler et al.
`
`Vol. 281
`
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`Fig. 1. GABA8 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 x 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-HCI buffer, pH 7.4, contain-
`ing 2.5 mM CaCI2 at ambient temperature. lncuba-
`tion media contained [3 H]-GABA (25 nM) and 100
`0M isoguvacine. Total reversible binding was mea-
`sured in the presence of 1 00 0M baclofen. A) Dis-
`placement curve of GHB on GABA8 binding from
`rat brain crude synaptosomal membranes. In-
`creasing concentrations of GHB displace [3 H]-
`GABA in the presence of 1 00 0M isoguvacine with
`an IC50 value of 23 ± 0.66 0M (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 [3 H]-GABA8 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 GABA8 binding
`is greatly decreased (IC50 = 0.51 ± 0.012 mM)
`(nonlinear regression line, Graphpad-Prism pro-
`gram).
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`1997
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`GHB Effects on GABA8 Binding
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`~
`
`25
`
`0
`-10
`
`-9
`
`-8
`
`-6
`
`-5
`
`-7
`Log [Baclofen]
`Fig. 2. Displacement curve of [3 H]-GABA8 binding according to Hill and
`Bowery (1981) in the absence(+) or presence (e) of 1.5 mM valproate.
`Binding was carried out in the presence of 100 0M isoguvacine, and
`nonspecific binding was determined with 100 0M baclofen. The differ(cid:173)
`ences between the two curves are not significant (P = .09, two-way
`AN OVA). Each data point is the mean of three separate determinations.
`
`Under the GABAB binding conditions (membrane prepara(cid:173)
`tion and incubation medium according to Hill and Bowery,
`1981), valproate and ethosuximide inhibit GABA synthesis
`from GHB with K; values of 1.0 mM (r = 0.93) and 2.0 mM
`(r = 0.98), respectively. GHB concentration was 200 JLM 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(cid:173)
`tions described by Olsen et al. (1981) for GABA binding,
`3 H]-muscimol was displaced by GHB with an IC 50 value of
`[
`4.6 ± 0.4 mM (r = 0.91). However, in the presence of 1.5 mM
`valproate, no significant [3 H]-muscimol displacement was
`induced by GHB (fig. 7).
`
`Discussion
`Several authors have described the displacement of [3 H](cid:173)
`GABA from GABAB sites by GHB, but they have reported
`IC 50 values varying from 150 JLM (Bernasconi et al., 1992), to
`500 JLM (Ito et al., 1995) and 796 JLM (lshige et al., 1996). Our
`own results have ranged from 23 JLM (the present results) to
`about 520 JLM (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 ofGHB by the synaptosomal membranes, which
`can be modified by the methods used for preparing the mem(cid:173)
`branes and/or the incubation conditions (time, temperature,
`pH and concentrations ofGHB). GHB could be converted into
`GABA in vitro by the sequential action of GHB dehydroge(cid:173)
`nase, which oxidizes GHB to SSA, and then a GABA-T activ(cid:173)
`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 JLM. 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
`
`c
`A
`B
`Fig. 3. Displacement of GABA8 binding by GHB in the presence or
`absence of a GABA-T inhibitor. Incubation conditions and GABA8
`membranes were identical to those described in the protocol of Hill and
`Bowery (1987). Column A= control; specific GABA8 binding displace(cid:173)
`able by 100 0M baclofen. Column B = specific GABA8 binding dis(cid:173)
`placeable by 300 0M GHB (significantly different from column A, P <
`.01). Column C = specific GABA8 binding in the presence of 300 0M
`GHB and 500 0M 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 GABA8 binding. In
`this set of experiments, 300 0M GHB displaced [3 H]-GABA8 binding by
`about 35%. Each data point is the mean of three separate determina(cid:173)
`tions.
`
`1.1x1Q10
`
`9.0x10°9
`
`...~
`0
`.§.
`III
`J:
`(!)
`
`7.0x1Q09
`
`5.0x1009
`
`1.0x1 009
`
`-300
`
`-100
`
`100
`
`300
`
`500
`
`Cone (JlM)
`
`Fig. 4. Determination of the K; value for aminooxyacetic acid (339 JLM,
`r = 0.81). Ordinate = 1/radioactive GABA produced from 200 0M 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(cid:173)
`ments.
`Two types of enzymes in brain are able to catalyze the
`oxidation ofGHB to SSA (Kaufman and Nelson, 1991). One of
`these enzymes is a cytosolic NADP+ -dependent oxidoreduc(cid:173)
`tase, whereas the other is present in the mitochondrial frac(cid:173)
`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 G HB degradation in brain
`because its inhibition by valproate and other antiepileptic
`drugs (trimethadione, ethosuximide) leads to an accumula(cid:173)
`tion ofGHB in brain (Snead et al., 1980). The mitochondrial
`enzyme is not sensitive to valproate. In the in vitro experi(cid:173)
`ments, the presence ofvalproate and ethosuximide with syn-
`
`

`
`758
`
`Hechler et al.
`
`Vol. 281
`
`2000
`
`iii
`
`- 1500
`~ c.
`~
`~ 1000
`m
`~
`(!)
`~
`"L
`
`500
`
`l
`
`Fig. 5. Kinetics of [3 H]-GABA formation
`from 200 0 M [3 H]-GHB in the presence
`of rat brain crude synaptosomal mem(cid:173)
`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 [3 H]-GABA formed was separated
`from [3 H]-GHB by ion exchange chroma(cid:173)
`tography on a Dowex 50 W-X8 column
`and elution with 0.1 N NaOH.
`_.Control; TIn the presence of 1.5 mM
`ethosuximide (65% inhibition compared
`with control, the activity being calculated
`during the linear phase of the kinetics; +
`In the presence of 1.5 mM valproate
`(94% inhibition compared with the linear
`phase of the control).
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`Time (min)
`
`1.5x1Q10
`
`1.0x1Q10
`
`... ~
`0
`.§.
`m
`:I:
`(!)
`
`5.0x1Q09
`
`<f. 110
`
`OJ c
`'g 100
`:0
`
`0 "" l 90
`"' 0
`E
`'iil
`::J
`E
`I'
`"2.....
`
`80
`
`70
`
`-3
`
`-2
`
`-1
`
`2
`
`3
`
`4
`
`5
`
`Cone (mM)
`
`Fig. 6. Determination of apparent K; values for valproate (_.) and etho(cid:173)
`suximide (II). 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 JLM. During this period of time, the conversion
`of GHB into GABA could be considered linear in the presence or
`absence of inhibitors. The K; 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 K; values are the real ones mea(cid:173)
`sured in GABA8 binding conditions.
`
`aptosomal/mitochondrial membranes renders GHB ineffec(cid:173)
`tive for displacing GABA from GABAB binding sites. The
`same result is obtained when GABA-T activity of the mem(cid:173)
`brane preparation is blocked by incubation with aminooxy(cid:173)
`acetic acid. Thus inhibition of the conversion of GHB to
`GABA results in a lack of interference with GABAB binding.
`The synthesis of [3 H]-GABA from [3 H]-GHB has been dem(cid:173)
`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
`JLM GHB was about 720 nM, a concentration high enough to
`
`-5.0
`
`-4.5
`
`-4.0
`
`-3.5
`
`-3.0
`
`log [GHB,M]
`
`Fig. 7. Displacement curve of [3 H]-muscimol binding in the presence of
`increasing concentrations of GHB. The methodology of Olsen et a/.
`(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 (EB). Each data point
`is the mean of three separate determinations.
`
`interfere with GABAB binding. This result explains the ap(cid:173)
`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(cid:173)
`tocol of Olsen et al. (1981; Kd values for GABA of 13 and 300
`nM), GHB displaced [3 H]-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 [3 H]-muscimol or [3 H]-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 EC 50 value (8-11 JLM)
`calculated for muscimol (Edgar and Schwartz, 1992), which
`should be compared with the low concentration of GABA
`
`

`
`1997
`
`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.
`Neverthe1ess, [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
`pentobarpital enhance [3R J-GHB binding (Snead et al. ,
`1992), and an effect ofGHB on chloride conductance has been
`proposed (Snead and N ichols, 1987). Under the conditions
`described for the above studies, an effect of GABA synthe(cid:173)
`si zed from G EB cannot be ruled out.
`Moreover, in some studies, an antagonistic effect of bicu(cid:173)
`culline to GHB-mediated effects has been noted. Hosli et al.
`(1983) described a hyperpolarizing effect of GHB that is
`blocked by bicuculline and is associated with an increase in
`chloride conductance in cultmed spinal, brainstem and cer(cid:173)
`ebellar nemons. No apparent GHB binding sites in these
`structures have been reported in rat brain (Hechler et al. ,
`1992). Thus it seems tha t GABA, formed from GHB in these
`cell cultur es, is responsible for the GABAA receptor(s) stim(cid:173)
`ulation. In other experiments in vivo, GHB possesses prop(cid:173)
`erties of its own that were bicuculline-reslstant, wher eas
`under the same conditions, the effects of GABA were antag(cid:173)
`onized by bicuculline (Olpe and Koella, 1979).
`In several electrophysiological studies canlecl out by in
`viuo administration of GHB or by application of Gl-IB 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). I n vivo, conversion of
`radioactive GHB into GABA has been described, and fmther(cid:173)
`more, a down-regulation of GABA receptors in the rat brain
`was induced by c1u·onic GHB administration (Gianutsos and
`Suzdak, 1984).
`Thus