fJ) Pergamon
`
`Progress in Neurobiology, Vol. 51, pp. 337 to 361 , 1997
`© 1997 Elsevier Science Ltd. All rights reserved
`Printed in Great Britain
`0301-0082/97($32.00
`
`PII: 80301-0082(96)00064-0
`
`THE y-HYDROXYBUTYRATE SIGNALLING SYSTEM IN BRAIN:
`ORGANIZATION AND FUNCTIONAL IMPLICATIONS
`
`MICHEL MAITRE*
`Centre de Neurochimie, Laboratoire de Neurobiologie Moteculaire des Interactions Cellulaires , UPR
`416 CNRS, 5 rue Blaise Pascal, 67084 Strasbourg Cedex , France
`
`(Received 8 August 1996)
`
`Abstract-')'-Hydroxybutyrate is a metabolite of GABA which is synthesized and accumulated by neurons
`in brain. This substance is present in micromolar quantities in all brain regions investigated as well as in
`several peripheral organs. Neuronal depolarization releases y-hydroxybutyrate into the extracellular space
`in a Ca2 +-dependent manner. Gamma-hydroxybutyrate high-affinity receptors are present only in neurons,
`with a restricted specific distribution in the hippocampus, cortex and dopaminergic structures of rat brain
`(the striatum in general, olfactory bulbs and tubercles, frontal cortex, dopaminergic nuclei A,, A 10 and A 12).
`Stimulation of these
`receptors with
`low amounts of y-hydroxybutyrate
`induces
`in general
`hyperpolarizations in dopaminergic structures with a reduction of dopamine release. However, in the
`hippocampus and the frontal cortex, it seems that y-hydroxybutyrate induces depolarization with an
`accumulation of cGMP and an increase in inositol phosphate turnover. Some of the electrophysiological
`effects of GHB are blocked by NCS-382, a y-hydroxybutyrate receptor antagonist while some others are
`strongly attenuated by GABA 8 receptors antagonists.
`Gamma-hydroxybutyrate penetrates freely into the brain when administered intravenously or
`intraperitoneally. This is a unique situation for a molecule with signalling properties in the brain. Thus,
`the y-hydroxybutyrate concentration in brain easily can be increased more than 100 times. Under these
`conditions, y-hydroxybutyrate receptors are saturated and probably desensitized and down-regulated. It
`is unlikely that GABA. receptors could be stimulated directly by GHB. Most probably, GABA is released
`in part under the control of GHB receptors in specific pathways expressing GABA8 receptors.
`Alternatively, GABA8 receptors might be specifically stimulated by the GABA formed via the metabolism
`of y-hydroxybutyrate in brain. In animals and man, these GHBergic and GABAergic potentiations induce
`dopaminergic hyperactivity (which follows the first phase of dopaminergic terminal hyperpolarization), a
`strong sedation with anaesthesia and some EEG changes with epileptic spikes. It is presumed that, under
`pathological conditions (hepatic failure, alcoholic intoxication, succinic semialdehyde dehydrogenase
`defects), the rate ofGHB synthesis or degradation in the peripheral organ is modified and induces increased
`GHB levels which could interfere with the normal brain mechanisms. This pathological status could benefit
`from treatments withy-hydroxybutyric and/or GABA 8 receptors antagonists. Nevertheless, the regulating
`properties of the endogenous y-hydroxybutyrate system on the dopaminergic pathways are a cause for the
`recent interest in synthetic ligands acting specifically at y-hydroxybutyrate receptors and devoid of any role
`as metabolic precursor of GABA in brain. © 1997 Elsevier Science Ltd. All Rights Reserved.
`
`CONTENTS
`
`I. Introduction
`2. Molecular and cellular organization of the y-hydroxybutyrate system in brain
`2.1. y-Hydroxybutyrate is present in small quantities in mammalian brain
`2.2. y-Hydroxybutyrate synthesis in brain
`2.3. y-Hydroxybutyrate degradation
`2.4. y-Hydroxybutyrate is released by depolarization in a Ca' +-dependent manner
`2.5. y-Hydroxybutyrate transport
`2.6. Specific receptor(s) for GHB in brain
`2.7. Neurophysiological events linked to GHB receptor(s) stimulation
`2.8. Modifications of second messenger systems by y-hydroxybutyrate
`2.9. y-Hydroxybutyrate influence upon dopaminergic activity in brain
`2.10. y-Hydroxybutyrate and the serotonergic system
`2.11 . Interactions of y-hydroxybutyrate with opioid mechanisms
`3. Physiopathological implications of the y-hydroxybutyrate system
`3.1. The y-hydroxybutyrate model of generalized absence seizures in rodents
`3.2. The role of y-hydroxybutyrate in the treatment of schizophrenic symptoms
`3.3. y-Hydroxybutyrate and sleep
`3.4. GHB and drug addiction therapy
`3.5. GHB and SSADH deficiency
`4. Conclusion
`Acknowledgements
`References
`
`*Tel.: 03-88-45-66-38; Fax: 03-88-45-66-05; E-mail: maitre@neurochem.u-strasbg.fr.
`
`337
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`Page 1 of 25
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`JAZZ EXHIBIT 2001
`Par Pharm. Inc. (Petitioner) v. Jazz Pharms. Ireland Ltd. (Patent Owner)
`Case IPR2016-00002
`
`

`
`338
`
`M. Maitre
`
`ABBREVIATIONS
`
`CHAPS
`
`CSF
`EEG
`GABA
`GABA-T
`GABAA
`GABAo
`GBL
`GHB
`
`(3-[3-cholamidopropyl)-dimethylammonio]-1-
`propanesulphonate
`Cerebrospinal fluid
`Electroencephalogram
`y-Aminobutyrate
`GABA-transaminase
`Class A GABA receptors
`Class B GABA receptors
`y-Butyrolactone
`y-Hydroxybutyrate
`
`GHB-DH
`NADP
`NADPH
`
`NCS-382
`
`SSA
`SSADH
`SSR
`T-HCA
`
`GHB-dehydrogenase
`Nicotinamide adenine dinucleotide phosphate
`Nicotinamide adenine dinucleotide
`phosphatereduced form
`Sodium salt of 6,7,8,9-tetrahydro-5-
`[H]benzocycloheptene-5-ol-4-ylidene acetic acid
`Succinic-semialdehyde
`Succinic-semialdehyde dehydrogenase
`Succinic-semialdehyde reductase
`Trans-4-hydroxycrotonatesodium salt
`
`1. INTRODUCTION
`
`Since the early sixties, y-hydroxybutyrate generally
`has been thought to be a drug which enters the brain
`easily and which possesses the general profile of a
`GABAergic ligand (Laborit, 1964). Up to now, the
`majority of the research devoted to this compound
`the neuropharmacological and
`has focused on
`neurophysiological aspects of systemic adminis(cid:173)
`tration. However, GHB is primarily a naturally
`occurring substance in brain which was identified
`about 30 years ago and which is synthesized locally
`(Fishbein and Bessman, 1961; Bessman and Fishbein,
`1963; Roth and Giarman, 1970; Roth, 1970). Like the
`biogenic amines, GABA is converted either by an
`oxidative pathway which produces succinate and
`enters the Krebs cycle or by a reductive pathway
`which gives rise to GHB in the neuronal cytosol. A
`large body of evidence favours a role for GHB as
`neuromodulator released by specific neuronal cir(cid:173)
`cuitry in the mammalian brain. This neuromodu(cid:173)
`lation seems to occur mainly at dopaminergic, but
`also amino-acidergic synapses in the anterior part of
`the central nervous system. The GHB receptors seem
`to possess large functional (and probably structural)
`homologies with the GABA8 receptors. However,
`despite the fact that the GHB system is not as well
`characterized as many other neurotransmitter/neuro(cid:173)
`modulator system, some results are now well
`established. The aim of this review is to focus on these
`results and to suggest some future directions in this
`area of research.
`
`of animals killed by microwave irradiation or in brain
`rapidly frozen after extraction. Probably for this
`technical reason, the concentrations of GHB in the
`brain of small laboratory animals (guinea-pig or rat
`brains) have been found, in general, to be lower than
`in the brain of larger animals (bovine and monkey
`brains). Human brains obtained after autopsy also
`present higher GHB values (Doherty et al., 1978;
`Snead and Morley, 1981).
`The GHB is present in all of the brain regions
`investigated. In the adult rat brain, GHB levels range
`from about 0.4 11M in the frontal cortex, 1.2 11M in
`the hippocampus, 1.8 11M in the striatum to 4.6 11M
`in the substantia nigra. The GHB concentrations are
`highest in human brain and in monkey brain,
`reaching about 11-25 11M in the striatum, but the
`values found for guinea-pig brain are similar to those
`found for rat brain. In developing brain,
`the
`concentrations of GHB have been found to be higher
`than in adult brain (rat, monkey and human). In the
`rat,
`the highest concentration
`is found
`in
`the
`immature hypothalamus and cortex with a decrease
`occurring between postnatal days 12 and 14.
`Gamma-hydroxybutyrate also has been found in
`rat peripheral organs (Nelson et al., 1981) such as
`heart (12.4 11M), kidney (28.4 11M), liver (1.4 11M),
`muscle (10.2 11M) and brown fat (37 11M). Studies of
`the apparent subcellular distribution of GHB have
`been carried out in the rat brain: GHB appeared to
`be concentrated in cytosolic and synaptosomal
`fractions (Snead, 1987), which most probably implies
`a mechanism for its presynaptic accumulation.
`
`2. MOLECULAR AND CELLULAR
`ORGANIZATION OF THE
`y-HYDROXYBUTYRATE SYSTEM IN BRAIN
`
`2.1. y-Hydroxybutyrate is Present in Small
`Quantities in Mammalian Brain
`
`y-Hydroxybutyrate real concentration in brain has
`been a matter of debate because it needs gas
`chromatography with preferably mass spectrometric
`detection to measure actual levels (Doherty et al.,
`1975a, 1978; Ehrhardt et al., 1988). In addition,
`endogenous GHB concentrations fluctuate rapidly in
`the ischaemic brain, so that brain dissection must be
`carried out rapidly after death (Snead and Morley,
`1981; Eli and Cattabeni, 1983; Vayer et al., 1988).
`The lowest levels of GHB have been found in brain
`
`2.2. y-Hydroxybutyrate Synthesis in Brain
`
`Gamma-aminobutyrate is the major precursor of
`GHB in brain (Roth and Giarman, 1968). Labelled
`GABA (13C or 3H-GABA) administered into the
`lateral ventricles of awake rats rapidly gave rise to
`labelled GHB, with a maximum concentration after
`20 min (Gold and Roth, 1977). Radioactive gluta(cid:173)
`mate, the precursor of GABA in brain, also rapidly
`induced the formation of radioactive GHB (San(cid:173)
`taniello et al., 1978). The GABA-transaminase
`inhibitors (y-vinylGABA or aminooxyacetic acid)
`blocked the metabolism of GABA to GHB, which
`implicates GABA-T in this transformation (Snead
`eta!., 1989; Eli and Cattabeni, 1983). This enzyme,
`classically located in the mitochondria of brain cells
`
`Page 2 of 25
`
`

`
`y-Hydroxybutyrate Signalling System in Brain
`
`339
`
`succinic
`synthesizes
`(Schousboe et al., 1980),
`semia1dehyde (SSA) (Matsuda and Hoshino, 1977),
`which could give rise to GHB after its reduction.
`Thus GABA, like the biogenic amines, possesses two
`the
`types of catabolite: oxidation of SSA by
`mitochondrial enzyme semialdehyde succinic dehy(cid:173)
`drogenase (SSADH) produces succinic acid which
`enters the Krebs cycle of brain cells; reduction of SSA
`gives rise to GHB present in the cytosol of some
`neurons (Fig. 1 ).
`The respective importance of these two pathways
`is very unequal. From about 0.05% (in vitro,
`Rumigny et al., 198la) to 0.16% (in vivo, Gold and
`Roth, 1977) of the metabolic flux coming from
`GABA takes the reductive route in order to form
`GHB. As GHB is formed in the cytosol of a restricted
`population of neurons (see below), the amount of
`
`SSA transported from the mitochondria to the
`is critical for GHB formation and
`cytoplasm
`probably is strictly regulated. Assuming an average
`concentration of about 2 mM for GABA in brain, the
`level of GHB in the whole brain represents a value
`not far from 0.1% of the GABA concentration.
`Other precursors of GHB in brain have been
`postulated. 1,4-butanediol and y-butyrolacetone are
`present in rat brain, at concentrations of about 1/10
`of those of GHB (Barker et al., 1985; Doherty eta!.,
`1975a). The former compound is transformed rapidly
`into GHB when introduced directly into the brain
`in vivo, the transformation being catalysed by a
`pyrazole-insensitive dehydrogenase (Maxwell and
`Roth, 1972; Snead et a!., 1982). The role of
`y-butyrolactone is more obscure, since no lactonase
`activity has been described in the brain cell (Fishbein
`
`: socci ria.ie :
`. . .
`. ..
`···~···
`·: s oi:J:
`. .
`. ..
`
`a-ketoglutarate ·
`
`Newonaf cvtosol
`
`NADPH
`
`1 ,4 Butanediol
`I
`I
`I
`alcohol dehydrogenase ?
`I
`I
`
`-r~I}{)W@[}jl@)XW~I1!J'i!1l[}jl~ II'~
`
`I •
`(GHIB) • I
`GHB + ~,
`' , '
`
`I
`I
`I -
`
`-
`
`Blood brain barrier
`
`'
`
`I
`peri;theral
`lactrase
`y-butyrolactone
`
`' peripheral administration
`of GHB
`
`Fig. 1. Biosynthesis of GHB in brain. The GABA is metabolized in brain by a mitochondrial GABA-T
`(GABA-TM) which gives rise to a succinic semialdehyde (SSA) pool in the mitochondria. The SSA then
`is oxidized by succinic semialdehyde dehydrogenase (SSADH) to succinate. This oxidative pathway is the
`main pathway of GABA degradation. The SSA is reduced by succinic semialdehyde reductase (SSR),
`present exclusively in the neurons cytoplasm. This enzyme is fairly specific for SSA (Km = 30 JLM) and
`is not inhibited by valproate, ethosuximide or barbiturates, but only by phthalaldehydic acid (competitive
`inhibition) and 4-n-propylheptanoic acid (non-competitive inhibition). An alternative pathway for SSA
`synthesis directly in the cytosol is the possible degradation of GABA via a supposed cytoplasmic GABA-T
`(GABA-T c). A minor route for GHB synthesis is the reduction, possibly by alcohol dehydrogenase, of
`1,4-butanediol which is present in low amount in brain. The concentration of GHB in brain could be
`into the brain.
`increased easily by peripheral administration of GHB which penetrates freely
`Gamma-butyrolactone (GBL) is sometimes used as a GHB precursor because it is transformed by
`peripheral tissue into GHB.
`
`Page 3 of 25
`
`

`
`340
`
`M. Maitre
`
`the equilibrium
`and Bessman, 1966). However,
`between y-butyrolactone and GHB in brain could be
`the result of a chemical rather than an enzymatic
`process.
`The reduction of SSA to GHB is carried out by an
`aldehyde reductase which possesses a low Km for SSA.
`Enzymes purified from several species (human, rat,
`pig, bovine) have been described which are able to
`reduce SSA (Cash et al., 1979; Rumigny et al., 1980,
`198la; Rivett et al., 1981; Rivett and Tipton, 1981;
`Cromlish and Flynn, 1985; Cromlish et al., 1985;
`Hearl and Churchich, 1985; Cho et al., 1993).
`Generally, two SSA reductases have been character(cid:173)
`ized in each species: one with a "high" Km for SSA
`(50-200 JtM), the other with a lower Km (20--30 JtM),
`the co-factor being in each case NADPH. The high
`Km SSA reductase (ALR 1 ) exhibits a broad substrate
`specificity, reducing a wide range of aldehydes
`(including p-nitrobenzaldehyde, 4-carboxybenzalde(cid:173)
`hyde, DL-glyceraldehyde and 3-pyridine carboxalde(cid:173)
`hyde), whereas the low Km SSA reductase shows a
`fairly high degree of specificity for SSA and structural
`analogues of SSA.
`The high Km SSA reductase of human and rat brain
`have been reported to be
`inhibited by several
`compounds including anti-epileptic drugs (valproate,
`ethosuximide, barbiturates) and some branched chain
`fatty acids (Cash et al., 1979; Vayer et al., 1985c).
`When administered in vivo, most of these compounds
`induce an increase in brain GHB levels (Snead et al.,
`1980). It is thus difficult to implicate the high Km SSA
`reductase in the in vivo synthesis of cerebral GHB.
`From a
`theoretical point of view,
`the SSA
`reductase which synthezises GHB in brain must meet
`certain criteria: (1) it must possess a high affinity and
`a high specificity for SSA; (2) the enzyme must be
`preferably localized in the cell compartment which
`has the highest concentration of GHB (cytosolic and
`synaptosomal fractions);
`(3)
`finally,
`the GHB
`synthesizing enzyme must not be
`inhibited by
`valproate and related compounds (short-chain fatty
`acids and anti-epileptics) which lead to the accumu(cid:173)
`lation of GHB in brain. In rat brain, these criteria
`lead to the selection of what has been called SSR2 and
`which is now designated as succinic semi-aldehyde
`reductase (SSR). This enzyme is mainly cytosolic but
`is present also
`in
`the synaptosomal
`fraction
`(Rumigny et al., 198lb, 1982; Weissmann-Nanopou(cid:173)
`los et a!., 1982). It is a monomeric protein of
`molecular weight of about 43 000--45 000 kDa with a
`pH optimum of 5.0. The NADPH is the co-substrate
`of the SSA reduction; the activity is five times less
`with NADH. The role of rat brain SSR has been
`characterized by selective inhibition of the enzyme in
`brain slices incubated under physiological conditions.
`into [3H]-GHB
`is
`Incorporation of [3H]-GABA
`reduced only when the activity of SSR is inhibited
`(Rumigny et al., 198la). On the contrary, blockade
`of high Km SSA reductase increases the radioactivity
`in the GHB pool which indicates that this enzyme has
`no role in GHB synthesis but could be implicated in
`GHB degradation.
`The regional and cellular distribution of SSR has
`been studied using a specific polyclonal antibody
`produced against the pure enzyme (Weissmann(cid:173)
`Nanopoulos eta!., 1982). The SSR is present only in
`
`the cytoplasm of numerous neurons of various sizes;
`glial cells appear not to be labelled. At the light
`microscopic
`level,
`the
`large majority of SSR
`immunoreactive neurons are also labelled with an
`antibody directed against glutamate decarboxylase,
`the enzyme that synthesize GABA (Weissmann(cid:173)
`Nanopoulos et al., 1984). Thus, GHB formation
`occurs in GABAergic neurons or in neurons which
`are able to synthesize GABA. At the electron
`microscopic level, SSR staining appears in the somata
`of neurons and in fibres or axonal terminals. In the
`hippocampus (results not published), SSR
`im(cid:173)
`munoreactivity is associated closely with pyramidal
`cells (CAl, CA2 and CA3). Regional distribution
`studies of SSR activity in rat brain shows that the
`enzyme is present in all regions investigated, with a
`maximum
`in cerebellum, colliculi and median
`hypothalamus (Rumigny et al., 198lb, 1982).
`In human and pig brain, the same SSR activity has
`been isolated with about the same characteristics
`(Cash et al., 1979; Cromlish and Flynn, 1985).
`However, the enzyme appears to be a dimer of
`molecular weight of about 80 000 kDa. Beside this
`SSA
`reductase activity, a mitochondrial SSA
`reductase has been described in pig brain which
`possesses high activity for malonic semialdehyde and
`p-nitrobenzaldehyde (Hearl and Churchich, 1985).
`Evidence that this enzyme is
`implicated in the
`synthesis of a GHB pool involved in interneuronal
`signalling is very poor, mainly due to its subcellular
`localization. An SSA reductase from bovine brain has
`been purified more recently but its high Km for SSA
`(67 JtM), its high activity with p-nitrobenzaldehyde
`and the absence of any inhibition profile make its
`(Cho et al., 1993). No
`identification difficult
`immunocytochemical localization of SSA reductase
`isolated from the brain of species other than the rat
`has been performed.
`
`2.3. y-Hydroxybutyrate Degradation
`
`The disappearance of 14C-GHB after intraventricu(cid:173)
`lar administration appears to be very rapid, one-half
`of the isotope being eliminated in less than 5 min
`(Doherty et al., 1975b). As finally most of the
`radioactivity is found in succinic acid and in the
`Krebs cycle,
`the general opinion favours
`the
`transformation ofGHB first into SSA (Mohler et al.,
`1976; Doherty and Roth, 1978). The reaction is most
`probably catalysed by the low Km SSA reductase
`(ALR 1)
`in the presence of NADP (Vayer et al.,
`1985c). As already mentioned, this enzyme is present
`in the brain of most of the species investigated
`(bovine, human, rat and pig brain) and is located in
`the cytosol, but no precise immunocytochemical
`distribution has so far been carried out. The low Km
`SSA
`reductase
`is now
`referred
`to as GHB
`dehydrogenase (GHB-DH) because of the following
`properties (Vayer et al., 1985c; Kaufman and Nelson,
`1979; Kaufman et al., 1983; Kaufman and Nelson,
`1987). Firstly, as already quoted, the enzyme is
`strongly inhibited by various anti-epileptic drugs,
`short-chain fatty acids
`for valproate = 60--
`(K;
`80 ,uM). When tested in vivo,
`these compounds
`induce a significant increase in brain GHB levels,
`most probably by inhibiting GHB catabolism (Snead
`
`Page 4 of 25
`
`

`
`y-Hydroxybutyrate Signalling System in Brain
`
`341
`
`et a!., 1980). In brain slices, inhibitors of GHB
`dehydrogenase lead to the accumulation of radio(cid:173)
`active GHB after incubation of the tissue with
`radioactive GABA (Rumigny eta!., 198la). In brain
`homogenates or when isolated in vitro, the enzyme
`behaves like a non-specific SSA reductase, strongly
`inhibited by low concentrations of valproate, and
`present in all the rat brain regions investigated
`(Rumigny eta!., 198lb). Its specific activity in these
`brain regions is about 15-fold higher than the specific
`activity of SSR (Rumigny et a!., 1982). However, in
`the presence ofNADP, purified GHB-DH has a very
`high apparent Km for GHB (Km = 2 mM) which is the
`result of the competitive inhibition by both SSA
`(K, = 14 tlM) and NADPH (K, about 7-21 tlM) for
`the random binding of GHB to the enzyme (Vayer
`eta!., 1985c). The SSA and NADPH are the products
`formed by GHB-DH from GHB and NADP,
`therefore GHB-DH activity appear to be strictly
`controlled by the negative feedback activity of the
`reaction products. This phenomenon could play a
`role in the regulation of GHB concentrations in
`brain. In vitro, the problem of SSA and NADPH
`accumulation can be avoided by coupling GHB-DH
`activity to the reduction of D-glucuronate which
`releases NADPH accumulation (Kaufman and
`Nelson, 1981, 1991). This result occurs mainly
`because, as already described, GHB-DH can actively
`catalyse the reduction of glucuronate and make the
`pentose phosphate pathway more active (a property
`attributed to GHB administration; Taberner et al.,
`1972). In addition, in vitro accumulation of SSA can
`be reduced by its being metabolized by GABA-T, e.g.
`(Vayer eta!., 1985b, 1985c). Under these conditions,
`when SSA and NADPH concentrations remain low,
`the apparent Km GHB and Km NADP for GHB-DH
`have been measured at 175 and 1.4 tlM, respectively.
`Hence, physiological concentrations of GHB (2-
`60 tlM) could be rapidly catabolized under these
`conditions in vitro (Vayer et a!., 1985c) (Fig. 2).
`In vivo, NADPH concentrations in GHBergic
`neurons could be maintained low by the reduction of
`glucuronate and the cytosolic pool of SSA, always
`very
`low, could be rapidly
`transported to
`the
`mitochondria and transformed into succinic acid. In
`addition, a direct transport of GHB itself to the
`mitochondria cannot actually be ruled out. A
`GHB-oxoacid-transhydrogenase, capable of reducing
`to SSA
`GHB
`is
`located
`in the mitochondria
`(Kaufman eta!., 1988; Kaufman and Nelson, 1991).
`However, this enzyme does not appear to be involved
`in GHB catabolism since it is not inhibited by
`valproate and is absent from foetal and neonatal
`brain (Nelson and Kaufman, 1994).
`Several authors have suggested that at least a part
`of the cytosolic pool of SSA coming from GHB
`degradation is transformed into GABA. In vivo,
`labelled GABA is formed from labelled GHB with no
`increase in the brain GABA concentration (Mitoma
`and Neubauer, 1968; Margolis, 1969; Doherty eta!.,
`1975b), although one group has reported a GABA
`increase in rat brain 120 min after i.p. administration
`of 500 mgjkg of GHB (Della Pietra eta!., 1966). De
`Feudis and Collier (1970) also reported an increase in
`GABA radioactivity 60 and 120 min after l-[1 4C](cid:173)
`GHB injected i.p. Others studies show very little
`
`incorporation of [14C]-GHB into [' 4C]-GABA, how(cid:173)
`ever these studies measured the brain radioactive
`amino acid pool less than 20 min after [14C]-GHB
`administration (Doherty and Roth, 1978; Mohler
`et a!., 1976).
`In vitro, radioactive GHB is consistently trans(cid:173)
`formed by brain extract into radioactive GABA
`(Vayer et a!., 1985b). Semicarbazide, a GAD
`inhibitor, reduced radioactive GABA production
`when [14C]-glutamate was the precursor but not
`when [14C]-GHB was the precursor, indicating that
`GHB is converted directly to GABA by the brain
`homogenate without passing through glutamic acid
`(Mitoma and Neubauer, 1968). In our hands,
`in vitro experiments carried out on brain homogen(cid:173)
`ates or on brain slices incubated under physiological
`conditions always gave rise to significant amounts of
`radioactive GABA (Vayer et a!., 1985b). In the
`presence of brain slices, 30 min
`incubation of
`labelled glutamate and non-radioactive GHB gener(cid:173)
`ated labelled 2-oxoglutarate, suggesting that GABA(cid:173)
`is
`involved
`in catalysing GABA synthesis.
`T
`Furthermore, specific inhibitors of GABA-T (etha(cid:173)
`nolamine-0-sulphate, gabaculine or aminooxyacetic
`acid) strongly reduced the production of labelled
`GABA from labelled GHB and of labelled 2-oxoglu(cid:173)
`tarate from labelled glutamate. Under these con(cid:173)
`ditions, transformation of GHB into GABA was not
`inhibited by malonate, demonstrating
`that
`the
`succinate-linked pathway is not involved in the
`generation of GABA. With 2-70 tlM GHB in the
`medium, the apparent Km for the transformation of
`GHB into GABA by the multienzymatic system
`(GHB-DH + GABA-T) was found to vary from 55
`to 145 tlM, which is compatible with the brain GHB
`concentrations that exist in vivo (Vayer eta!., 1985b,
`1985c).
`When administered in vivo, the effects of GABA-T
`inhibitors on GHB levels were found to be apparently
`contradictory. Eli and Cattabeni (1983) report a
`decrease of brain GHB levels after i.p. administration
`of y-acetylenicGABA or aminooxyacetic acid to rats
`120 or 60 min respectively before being sacrificed. In
`apparent contradiction to these results is the report
`of Snead (1987) that indicates an increase in GHB
`concentrations, in vitro and in vivo, in presence of
`GABA-T
`inhibitors (y-vinylGABA, y-acetylenic(cid:173)
`GABA or aminooxyacetic acid). Even if another
`source of GHB exists in brain (other than GABA),
`the increase in brain GHB levels (seen particularly in
`the synaptosomal fraction) seems to indicate that a
`GABA-T activity is involved in the degradation of
`GHB. The discrepancy observed with the results of
`Eli and Cattabeni (1983) is in favour of two different
`GABA-T activities (two different protein species
`andjor with different cellular locations) participating
`in both the synthesis and degradation of GHB. The
`different subcellular
`localization of
`these
`two
`GABA-T activities and perhaps their respective
`sensitivities to the inhibitors make the kinetics of
`inhibition of these two GABA-T pools somewhat
`different. In a particulate fraction of rat brain, the
`cytosolic GABA-T pool is more rapidly exposed to
`the inhibitors than the mitochondrial pool. In the
`in vivo experiments, differences between the times of
`sacrifice after administration of the various inhibitors
`
`Page 5 of 25
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`

`
`342
`
`M. Maitre
`
`Extracellylar space
`
`T-HCA ._ - GIH!Bl
`
`NADP
`
`/
`
`GHB deh drogenase
`
`···················
`...................
`...................
`...................
`
`valproate
`ethosuximide
`barbiturates
`
`SSA
`
`~t·Tc
`
`Fig. 2. Degradation of GHB in brain. The GHB present in the extracellular space (after release by
`GHBergic terminals or peripheral administration of exogenous GHB) is actively transported by neuronal
`cells with a Km of 46 JlM. The main route for GHB degradation is its conversion into succinic
`semialdehyde (SSA) via an NADP-dependent GHB dehydrogenase (GHB-DH) which is inhibited by some
`anti-epileptics (valproate, ethosuximide, barbiturates). The SSA could be then oxidized in the Krebs cycle
`or converted into GABA by the mitochondrial GABA-T (GABA-TM) or perhaps by a cytosolic isoform
`of this enzyme (GABA-Tc). The GABA pool formed from GHB is thought to possess a specific functional
`role, being released in neuronal circuits expressing mainly GABA. receptors. However, GHB also could
`be converted into trans-hydroxycrotonate (T-HCA), possibly through P-oxidation.
`
`and the doses of inhibitors used could explain the
`apparently contradictory results.
`The existence of several GABA-T pools is a matter
`of debate. Classically, GABA-T is a mitochondrial
`enzyme, present both in neuronal and glial cells.
`However, Chan-Palay et al. (1979), using immuno(cid:173)
`cytochemical techniques, demonstrated that GABA(cid:173)
`T is also present on post-synaptic membranes and in
`the cytosol. These findings also have been docu(cid:173)
`mented by Walsh and Clark (1976), Bloch-Tardy
`et al. (1980), Vincent et al. (1981) using other
`
`techniques, like histochemistry. It will probably be
`possible to describe the characteristics of GABA-T
`isoforms only after their cloning and expression in
`eucaryotic cells. The existence of this neuronal
`cytosolic GABA-T could explain the local transform(cid:173)
`ation of the cytosolic SSA into GABA. This GABA,
`the metabolism of GHB, could
`resulting from
`constitute a specific pool distinct from the GABA
`pool directly synthesized from glutamate, and could
`possess specific functional properties.
`Another possible route for GHB degradation has
`
`Page 6 of 25
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`

`
`y-Hydroxybutyrate Signalling System in Brain
`
`343
`
`been suggested by Walkenstein et al. (1964) through
`P-oxidation. The existence of this pathway would
`implicate the presence of hydroxycrotonate in brain.
`In
`fact,
`the coexistence of both GHB and
`trans-hydroxycrotonate (T-HCA) has been demon(cid:173)
`strated in renal tissue (Niwa et al., 1982). In brain,
`small amounts of T-HCA have been detected which
`are about 1/10 of those of GHB in the same brain
`regions (Vayer et a!., 1985a, 1988). The T-HCA
`possesses some of the functional role of GHB, in
`particular it is a high-affinity ligand for the GHB
`receptor with agonistic-like properties (Hechler eta!.,
`1990b; Hechler et al., 1993). In addition, T-HCA is
`a substrate in vitro for GHB-DH and thus possibly
`could be transformed into aminocrotonate after
`transamination by GABA-T. The existence of this
`pathway would represent a supplementary link to the
`GABAergic system.
`As the major route for GHB degradation is
`inhibited by valproate, an accumulation of GHB is
`induced following acute treatment with this com(cid:173)
`pound. A dose-dependent increase in brain GHB
`content was demonstrated between 200 and 600 mg/
`kg valproate, mainly due to GHB-DH inhibition
`(Vayer et al., 1988). The distribution of valproate in
`brain 15 min after i.p. administration of200 mg/kg is
`homogeneous at about 700 J.lM, which represent 10
`times the K; for GHB-DH inhibition. Under these
`conditions, it is generally accepted that the biosyn(cid:173)
`thetic pathway for GHB is not affected. Then, after
`i.p. administration of 400 mg/kg valproate, the
`time-dependent accumulation of GHB in different
`regions of the rat brain was used in order to
`determine
`the
`rate of GHB synthesis. Linear
`regression analysis of initial accumulation of GHB
`gives a turnover time of 9 min in the hippocampus,
`35 min in the pineal gland, 47 min in the temporo(cid:173)
`parietal cortex, 30 min in the striatum and 78 min in
`the cerebellum. These turnover times appears to be
`rapid, especially in the forebrain, and are similar to
`what has been reported for well-known neurotrans(cid:173)
`mitters, like serotonin in the hippocampus or GABA
`in the cortex or striatum (Neckers and Meeck, 1976;
`Bernasconi et al., 1982). In total brain, a turnover
`time of 26 min for GHB has been measured by Gold
`and Roth (1977) by the use of [lH]-GABA as the
`GHB precursor.
`
`incubated in a physiological oxygenated medium,
`caused an efflux of radioactive GHB of about 50 fmol
`min- 1 mg- 1
`• This release was reduced by 50-60% in
`a Ca2 +-free medium containing Mg2 + and EGT A,
`and almost completely abolished if the medium was
`supplemented by verapamil (100 J.lM) or tetrodotoxin
`(10 J.lM). Veratridine (10-100 J.lM) induced an even
`stronger release (about 80 fmol min- 1 mg- 1 wet
`weight), also largely reduced by verapamil. The
`amount of GHB released appeared to be dependent
`on the intensity of cell depolarization induced by
`veratridine.
`The same methodology has been used to examine
`the extent of GHB release according to the brain
`region studied (Vayer and Maitre, 1988). The 50 J.lM
`veratridine
`induced a strong release of GHB
`(70-80 fmol min- 1 mg- 1 wet weight) from rat brain
`slices taken from the hippocampus, striatum or
`fronto-parietal cortex. However, this release was low
`from
`slices
`taken
`from
`the pons-medulla or
`cerebellum (about 80-90% reduction). In these last
`two regions, the Ca2 +-component of the release was
`exceptionally low or absent, although in the other
`regions