Significance of γ-hydroxybutyric acid in the brain

Gen Pharmac Vol 23, No 6, pp 1027-1034,1992

0306-3623/92$5 00 +000 Copyright © 1992PergamonPress Ltd

Pnnted m Great Britain All rights reserved

MINIREVIEW

SIGNIFICANCE OF ~,-HYDROXYBUTYRIC ACID IN THE BRAIN G TUNNICLIFF Laboratory of Neurochemlstry, Indmna University School of Medicine, 8600 University Boulevard, Evanswlle, IN 47712, U S A [Tel (812) 464 1833, Fax (812) 465 1662] (Recewed 10 February 1992)

Abstract--I Administration of the endogenous compound ~,-hydroxybutyncacid (GHB) can reduce a sleep-hke state m experimental ammals and, indeed, it has been used as a general anaesthetic in cllmcal me&cane 2 Although GHB appears to be a CNS depressant, there is evidence it possesses epfleptlform activity resembling petit real epilepsy In the brain GHB is ewdently derived from GABA, the final step being catalyzed by succmlc semmldehyde reductase, a cytosohc NADP+-dependent enzyme 3 Two differentoxldoreductases, GHB dehydrogenaseand hydroxyacld-ketoacld dehydrogenase,acting mdependently, are responsible for the reverse reaction when GHB is being metabohcally mactwated 4 Brain contains a Na+-dependent GHB uptake system which exhibits two components, one with a K~ of 46 #M and the other w~th a Kmof 325/~M GHB also binds to receptor sites m brain homogenates and exhibits two dlstmct aflinmes One binding site displays a K a of 95 nM whereas the second site has a Ka of 16 laM Bmdlngto both sites is inhibited m the presence of NCS-382, a GHB receptor antagonist 5 GHB might play a role as a neurotransmltter, particularly being revolved m influencing dopamme release m the substantm mgra

INTRODUCTION Butyric acid as well as other short-chain fatty acids are known to induce sleep-hke behaviour after intravenous administration (White and Samson, 1956, Jouany et a l , 1960) As the majority of the butyric acid undergoes fl-oxldatlon, y-hydroxybutyrlc acid (GHB) was synthesized in an attempt to slow down metabolic degradation and thus to enhance the pharmacoiogacal actions In addmon, it was thought that GHB might influence brain y-ammobutyric acid (GABA) actmty because of its structural resemblance to the inhibitory neurotransmmer (Labont, 1964) Results from several studies confirmed that GHB was an effectwe hypnotic agent and, indeed, the drug has been used m general anaesthesia (e g Laborit et a l , 1961) Later experiments, though, found that GHB produced epdeptlform activity (Winters and Spooner, 1965) and it has been suggested that its pharmacological effects, especmlly at lower doses, more closely resemble petit mat epilepsy than sleep (Godschalk et a l , 1977, Snead, 1988) This subject Is controversml, however, since other studies have coneluded just the reverse (e g Nakamura et a l , 1987) Despite the fact that GHB is a close structural analogue of GABA, little ewdence suggests that GHB interferes with GABA neurotransmlssion Instead, It appears to produce its effects by activating specific GHB receptors which may be linked to cGMP and mosltol phosphate mtracellular mechanisms (Vayer and Maitre, 1989, Mmtre et a l , 1990)

Relatively low concentrations of GHB have been detected in the brain, where it exhibits an uneven distribution It appears to be synthesized from GABA via the reduction of succmm semmldehyde It is intriguing to note that considerably higher levels of GHB have been detected in kidney, heart and skeletal muscle (Nelson et a l , 1981) As a result of its pharmacological actions, the ewdence for specific receptor sites, the demonstration of an uptake system, and the existence of metabohzlng enzymes, it is thought that GHB might function as a neurotransmmer m the central nervous system This article reviews the exadence BIOCHEMISTRY

In spite of mmal reports which found that GHB was comparatwely abundant in brain tissue (Bessman and Flshbeln, 1963, Flshbem and Bessman, 1964), it now seems clear that this compound is present in low concentrations (pmol/mg protein), although the exact amount depends on the particular brain region In a detailed regional &stnbution study. Vayer et al (1988) found that substantia mgra, hypothalamus and thalamus contained the highest levels of GHB (30-46pmol/mg protein), amygdala, raphe nuclei and stnatum contained an intermediate level (18-24pmol/mg protein), while the cerebellum and several cortical areas possessed the lowest concentrations of GHB (4-8 pmol/mg protein) Thus a good correlation exists between GHB and GABA levels m the brain

1027

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G TUNNICLIFF

Early lnvest~gaUons found that nervous t~ssue could convert [3H]GABA to [3H]GHB and that sucomc semlaldehyde was an intermediate (Roth and Gmrman, 1969, Anderson et a l , 1977) The first step, controlled by GABA ammotransferase, has already been well characterized (Tunmchff, 1986), but details of the second step are not as clear Furthermore, we know that GHB metabohc degradation revolves conversion to sucomc semmldehyde and succmate before entry into the tncarboxyhc acid cycle (Doherty and Roth, 1978) The most probable explanation for these metabohc events is that GHB xs both derived from and catabohzed to sucomc semmldehyde, although probably by &fferent enzymes

GABA

GABA T

m~tochondna Again, no such mechamsm has been described G H B catabohsm

Several p~eces of ewdence m&cate GHB is eventually converted back to succm~c semlaldehyde before entering the trlcarboxyhc acid cycle as succmate Which enzymes are revolved9 Perhaps a reversal of the NADP+-hnked reductase can account for the first step in GHB catabohsm One disadvantage of this idea ~s that the M~chaells constant of GHB for the enzyme is substantially above the levels of the compound measured m the brain and, mdeed, ~t ~s known

, Succmlc semmldehyde

SSADH

) Succlnate

T*

GHB

where GABA-T and SSADH are GABA ammotransferase and succmlc semlaldehyde dehydrogenase, respectively G H B btosynthests

Several oxldoreductases have been described that are possible can&dates for involvement in GHB metabohsm For instance, a cytosohc NADP ÷dependent oxidoreductase capable of forming GHB from sucomc semmldehyde was described (Tabakoff and Von Wartburg, 1975, Anderson et a l , 1977) and has since been isolated from human, rodent and pig brain (Cash et a l , 1979, Rumlgny et a l , 1980, Hearl and Churchlch, 1985) This enzyme has been named succlnlC semmldehyde reductase There are, however, some physical differences between certain of these enzyme preparations The enzyme from rat bram, for example, ~s a monomer having a Mr between 43,000 and 45,000 (Rumlgny et a l , 1980), whereas the enzyme from pig brain ~s dlmenc w~th a Mr of 110,000 (Hearl and Church~ch, 1985) In the case of the enzyme from human brain, the protein ~s also &merlc with a Mr between 82,000 and 95,000 (Cash et a l , 1979) An lmmunocytochemlcal study of the locahzaUon of this reductase has confirmed that the enzyme occupies the soluble compartment of certain neurons (Welssmann-Nanopoulos et a l , 1982) In hght of th~s evidence, the report that the NADP+-dependent succmlc semmldehyde reductase purified from pig brain ~s also present in substantml amounts in m~tochondna (Head and Churchlch, 1985), seems incongruous Presumably a &fferent enzyme ~s revolved If, as now appears certain, GHB is formed by the reduction of succm~c semmldehyde, does the reaction occur m the m~tochondna or m the cytosol9 If the synthesis of GHB occurs in the m~tochondna there must be a mechamsm to transport ~t out into the cytosol Such a transport system, ff ~t exists, has not been identified If, on the other hand, the reduction of succm~c semmldehyde to GHB takes place m the cytosol, there must be a transport process for succmxc semmldehyde since th~s metabohte ~s made m the

that the eqmhbnum of the reaction is much m favor of GHB formaUon A more hkely can&date for catalysis of the oxidation of GHB back to succmlc semlaldehyde ~s GHB dehydrogenase, a cytosohc NADP+-dependent oxldoreductase whose Mr has been shown to be between 31,000 and 38,000 (Kaufman et a l , 1979, 1983, Kaufman and Nelson, 1981) At first sight this enzyme ~s not a good choice for the ox~datlon of GHB m vtvo since the Km values for both GHB and NADP ÷ are comparatively high Moreover, NADPH inhibits the reaction It has been proposed, however, that the kinetic constants for substrate and cofactor can be modified if a second catalytic event occurs with the same enzyme (Kaufman and Nelson, 1981, 1991) The substrate D-glucuronate could be revolved by being reduced by NADPH, thus removing the latter's lnhlb~tory influence At the same Ume the first order rate constant for GHB ox~datlon is increased almost 10-fold m the presence of D-glucuronate These two occurrences are responsible for dnwng the GHB . . . . ~ SSA reacUon GHB + NADP+~---SSA + NADPH 2 D-Glucuronate + NADPH2~L-Gulonate + NADP ÷ The enzyme D-glucuronate reductase (EC 1 1 1 19) is already known, thus GHB dehydrogenase actwlty could represent another property of the same enzyme Rumlgny et al (1980) found two oxtdoreductases capable of mterconvertmg GHB and succm~c semlaldehyde--a specific enzyme as described above responsible for GHB biosynthesis, and a non-specific enzyme now thought to be GHB dehydrogenase (Vayer et a l , 1985) From rat bram th~s enzyme had a Mr of 50,000-54,000 (Rumlgny et a l , 1980), but m human brain the M r was determined to be 40,000-48,000 (Cash et a l , 1979) Compounds which inhibit GHB dehydrogenase m vttro have been administered to rats m order to study the effects on GHB levels ~t-Ketolsocaproate, phenylacetate, valproate and sahcylate were each able to markedly increase brain GHB concentrauons (Kaufman and Nelson, 1987) These results support

GHB in the brain the idea that GHB dehydrogenase is important m the metabohc oxldatmn of GHB in the intact bram If GHB oxidation takes place m the cytosol, then the product succlmc sermaldehyde must be transported into the m~tochondrla since succm~c semialdehyde dehydrogenase is a mltochondnal enzyme On the other hand, ewdence ~s available that another oxldoreductase is present which can convert GHB to SSA m the mltochondrla (Kaufman et al, 1988b) This enzyme is a hydroxyacld-ketoacld transhydrogenase requlnng ~-ketoglutarate as second substrate (Kaufman et al, 1988a) An mborn error of GHB metabolism has been known for about a decade Owing to a lack of succlnlC semlaldehyde dehydrogenase, a substantml accumulatmn of GHB occurs Thls &sorder is accompanied by severe psychomotor retardatmn, ataxla and convulsmns (Jacobs et al, 1981, Dlvry et al, 1983, Rating et al, 1984) In a more recent case, a young patient exhibited mainly severe speech retardatmn (Onkenhout et al, 1989) Vayer et al (1988) have measured GHB turnover rates in several brain regions Highest rates were seen m the hlppocampus, lntermedmte rates m the strlatum, and lowest rates m the cerebellum

PHARMACOLOGY

Hypnotic and anaesthetic aetwns

Admlnlstratmn of GHB at relatwely high doses produces what appears to be a depressmn of the central nervous system since m experimental ammals a hypnoUc state occurs In rats, for example, lntraperitoneal injection of 0 5 g/kg will reduce a type of sleep (Laborlt, 1964) In the eat, the hypnotic state appeared to differ from natural sleep m that the ammal's eyes remained open (Drakontldes et al, 1962) This drug has also been used m humans to reduce general anaesthesia (Blumenfeld et al, 1962, Labont et al, 1962, Solway and Sadove, 1965, Aldrete and Barnes, 1968) Because GHB has poor analgesic properhes, an oplold must be included in the preme&eatmn, or the maintenance of surgical anaesthesia by nitrous oxide must be attained Perhaps partly because of reports that GHB produces seizuretype EEG actwlty (Winters and Spooner, 1965) and that the hypnotic state resembles petit mal epilepsy (Godschalk et al, 1977), the use of GHB m anaesthesia has never gamed wide-spread acceptance y-Butyrolactone is a prodrug of GHB and ~ts admm~stratmn to rats is known to lead to profound EEG changes In one senes of experiments basehne EEG patterns developed into a brief burst of spikes, soon followed by continuous spiking (Snead, 1988) At lugher doses, the hypersynchronous phase changed to sp~ke and slow wave At even greater doses, a burst suppressmn pattern was observed Furthermore, drugs useful m treating petit mal epdepsy were effectwe at suppressing these EEG changes (Snead, 1984) In the chick, GHB produced sedatmn accompamed by myoclomc seizures Electrocort~eograms showed sharp waves and sp~ke actwlty (Osmde, 1972)

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It is well documented that GHB can produce a hypnotic effect But is physiological sleep affected by GHB admmlstratlon9 In normal subjects, GHB &d not alter total sleeping time but did increase slowwave sleep at the expense of stage 1 sleep (Laplerre et al, 1990) In addmon, REM efficiency was increased m the drug-treated subjects An early report had shown that GHB induced drowsiness and unconsoousness at higher doses (Metcalfet al, 1966) EEG analysis revealed the presence of slow-wave sleep In cats, GHB has been reported to Induce REMs (Jouvet et al, 1961, Matsuzakl et al, 1964) but these were not seen m rats or rabbits (Marcus et al, 1967, Godschalk et al, 1977, Godbout and Pwlc, 1982) Several stu&es have concluded that the treatment of narcolepsy with GHB leads to significant improvements m the ehmcal symptoms of catalepsy, sleep paralysis and daytime sleep attacks (Broughton and Mamelak, 1979, Scharf et al, 1985, Mamelak et al, 1986) In a more recent study of the effects of GHB on nocturnal sleep m narcoleptic subjects, GHB was found to increase slow-wave sleep and to reduce the frequency of awakenings (Scrlma et al, 1990) The known improvements m narcoleptlcs induced by GHB may well be related to the beneficial effects on nocturnal sleep Snead and Bearden (1980) descnbed a type of catalepsy induced by GHB It has recently been demonstrated that both catalepsy and sedation can be blocked with NCS-382, a GHB receptor antagonist (Schmldt et al, 1991) Thus sedation and catalepsy appear to be receptor-me&ated Neurophyswlogtcal effects

When added to the perfusate, GHB hyperpolarlzed dorsal root terminals in frog spinal cord In contrast, G A B A also depressed dorsal root potentials but by a depolarlzatmn Only GABA's responses were lnh~blted by GABAA antagomsts (Osono and Davldoff, 1979) Similarly the inhibitory action of GHB on umt cells in neoeortex of rat was unaffected by blcuculhne (Olpe and Koella, 1979) Also, GHB was shown to depress both monosynaptlc and polysynaptxc reflexes (Basil et al, 1964) as well as to mhlbR dorsal root potentmls resulting from segmentary stimulation in the cat spinal cord (Besson et al, 1971) Although most stu&es have concluded that GHB's inhibitory effects are not due to actwatmn of GABA receptors, Kozhechkm (1981) reported that GHB depressed the acUvRy of neurons m the sensorlmotor cortex of the rabbit and that this effect was blocked by blcuculllne Far more neurons were sensmve to G A B A than to GHB, however, suggesting that GHB acted through a subtype of GABA receptor in the rabbit Systemic admlnlstratmn of GHB leads to decreased dopamlnergac actawty (Walters et al, 1973) This is probably a reflectmn of GHB's inhibitory actmn on the cell body of dopamlne-releasmg neurons (Anden et al, 1973, Roth et al, 1973, Walters et al, 1973) In the substantm nlgra this mmally leads to a decrease m dopamme release and an accumulatmn of dopamlne at nerve terminals Finally, a stimulation of dopamme release occurs (Cberamy et al , 1977) Harris et al (1989) earned out in vitro mtracellular recordings m shces of guinea pig substantla mgra They concluded that GHB (1) lowers

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input resistance of pars compacta neurons, (2) hyperpolanzes the membrane m a concentration-dependent manner and (3) facilitates Ca 2+ conductance In the presence of blcuculhne (200 #M) the effects of GHB were partmlly reduced (by an average of 27%) GHB actions were unaffected by both 4-ammopyndine and tetraethylammonlum, thus K + channels are unlikely to be &rectly revolved It has been reported that C1- channels are mvolved m GHB inhibitory actlvlty (Snead and Nlcholls, 1987), but Harris et al (1989) observed that the GHB effect was blocked by Ba 2÷ sons (known to block calcmm-dependent potassmm and chloride conductances) Consequently, C1- channels are probably not lmphcated directly in the mechanism of action of GHB Neurochemwal actions

Fairly high doses of v-butyrolactone or GHB have been shown to increase acetyichohne levels m the hlppocampus, cerebral cortex and mldbram of the rat (Glarman and Schmldt, 1963, Ladlnsky et a l , 1983) Large doses of GHB also produce an increase m strmtal 5-hydroxytryptamme and 5-hydroxylndole acetic acid (Waldmeler and Fehr, 1978, Hedner and Lundborg, 1983) A plausible explanation for these neurochem~cal changes has not been presented Gessa et al (1966) first noticed that GHB caused a selectwe increase m smatal dopamme levels Tlus has been confirmed by several groups of workers (e g Spano et a l , 1971, Hutchms et a l , 1972, Walters and Roth, 1972) This increase m dopamme might be related to the fact that GHB can inhibit the release of newly synthesized dopamme m strlatal shces (Bustos and Roth, 1972) It now seems likely that after th~s mmal attenuation of dopamme release, an enhancement of tyrosme hydroxylase actwlty occurs followed by a stimulation of dopamme release (Spano et al , 1971, Morgenroth et al , 1976, Cheramy et al , 1977) Thus both mechanisms could contribute to the elevation of dopamlne levels The GHB antagomst NCS-382 has been shown to mh~blt the stimulation of dopamme release by GHB (Maitre et a l , 1990) Th~s increase m dopamme has also been observed m Drosophda melanogaster maintained on a &et rich m y-hydroxybutync acid (Connolly et a l , 1971) It ~s interesting to note that m m~ce, sleeping t~me reduced by GHB was substantmlly prolonged if the ammals were pretreated with L-DOPA No such prolongation m sleeping ume was seen ff the hypnotic agent was pentobarb~tal (R~zzoh et a l , 1969) It m~ght be concluded that the elevation of brain dopamme ~s of ~mportance in the mduct~on of sleep On the other hand, other stu&es have demonstrated that the hypnotic effects of GHB are not mediated wa alterauons m dopammerg~c actw~ty (Waiters and Roth, 1972) Admlmstrat~on of GHB to rats was reported to increase cGMP levels m the h~ppocampus (Vayer et a l , 1987) This observation was confirmed m an m wtro study m which shces of h~ppocampus incubated m 300-600 # M GHB underwent an increase m both mtracellular cGMP and lnositol phosphates (Vayer and Maitre, 1989) These b~ochem~cal changes appear to be receptor medmted since the presence of NCS382 prevented these mcreases m cGMP and m o m o l phosphates (Mmtre et a l , 1990) Presumably the

stimulation of the GHB receptor leads to increases in lntracellular second messengers GHB affects energy metabohsm in the CNS (Fleming and La Court, 1965, Taberner et a l , 1972, Wolfson et a l , 1977, Hailer et a l , 1990) Wolfson et al (1977) administered y-butyrolactone to rats and found a marked decrease in glucose utilization throughout the brain Similarly Hailer et al (1990) reported a substantial reduction in glucose consumptlon in 16 of 37 brain structures of the cat The auditory system was particularly sensitwe to the effects of GHB Interestingly, neither cerebral blood flow nor oxygen consumption underwent concomitant reductxons, indicating that GHB may have acted as an alternative source of fuel Several studies have shown that GHB can protect against the damage of hypoxla Pretreatment with GHB of rats subjected to low oxygen resulted m complete protection against death In contrast in a control group 45% of the animals died (MacMillan, 1978) Addmonally, mtrapentoneal administration of low doses of ybutyrolactone reduced brain damage caused by occlusion of the vertebral and carotid arteries m the gerbd (Lavyne et a l , 1983) Furthermore, energy utilization was reported to be substantmlly spared by y-butyrolactone m an anox~c enwronment (MacMdlan, 1979) Transport and receptor sttes

Benawdes et al (1982a) found that [3H]GHB was taken up by an actwe transport system into plasma membrane vesicles derived from rat brain The uptake process consisted of two components, one of which exhlb~ted a Km of 46 4 # M and the other a K,. of 325/~M Both uptake components were Na ÷dependent and required C1- for maximum actlwty Uptake showed a regional varmbdlty w~th stnatum possessing the greatest uptake actw~ty Several compounds were tested as mh~b~tors of uptake GABA, 3-methylGHB, 3-hydroxypropane sulphomc acid, 5hydroxyvalenc acid, and trans-4-hydroxycrotomc acid were all good mh~bltors Rat smatal shces also took up [3H]GHB in a sodmm dependent manner, but only the low affimty uptake system could be detected (Hechler et a l , 1985) Shces of rat strlatum, preloaded with [3H]GHB, released GHB when depolarized by K ÷ or veratndme (Maitre et a l , 1983) In each case the [3H]GHB efflux was related to the concentration of the depolarizing agent In a CaZ+-free medmm, release was markedly reduced Slmdarly, the Ca2+-channel blocker verapared, also slgmficantly attenuated the reduced efflux of GHB [3H]GHB has been shown to brad to a membrane fraction from rat brain (Benawdes et a l , 1982b) Binding was saturable and two populations of binding sites were ldentlfied--a high aftimty site ( K d ~95 nM) and a low affimty site (Kd = 16 #M) (Fig 1) GABA was meffectwe at displacing [3H]GHB binding Olfactory bulbs and hlppocampus showed the greatest amount of binding, with cerebellum and spinal cord showing the least No binding was detectable m the pons/medulla Since NCS-382 potently inhibits both h~gh- and low-affimty [3H]GHB binding (Maitre et a l , 1990), these binding sites probably represent GHB receptors

GHB m the brain oooT,

eo

0 005

0 0025

I 1

I 25

I 5

) 75

I 10

Bound

F~g 1 Scatchard plot of binding of [3H]GHB to rat brain membranes Bound hgand defined as pmol/mg protein Free hgand concentration ~s nmol/1 Redrawn from Benav~des et al (1982b) Also using rat brain synaptlc membranes, Snead and Llu (1984) confirmed the existence of saturable [3H]GHB binding that was unaffected by GABA Again, bmdmg was resolvable xnto two components a hlgh-affimty binding exhibiting a Kd of 580 nM and a lower affimty component whose Kd was 2 3 # M Hlppocampus contained the greatest number of bmdmg s~tes whereas much less binding was detectable m the brain-stem In human brain, hlppocampus was again the region of greatest binding Almost as much binding, however, was reported m the pons, a region m rat brain displaying no apparent bmdlng In an autoradlographlcal study, Hechler et al (1987) surveyed the &stnbut~on of [3H]GHB binding m rat bram H~ppocampus and cortex showed the greatest binding whereas caudal brain areas exhibited minimal binding In both hlppocampus and strlatum [3H]GHB binds to intrinsic neurons and not to dopamlnerg~c presynaptlc terminals (Hechler et a l , 1989) These mtnnslc neurons could be oplo~d in nature since naloxone blocks GHB actions in the hlppocampus and strlatum (Snead and Bearden. 1980) Moreover, naloxone prevents the GHB-mduced increase m cGMP (Vayer et a l , 1987) There is no evidence naloxone has an atfimty for GHB receptors CONCLUSIONS

There is httle doubt that GHB ~s not merely a by-product of GABA metabohsm Clearly it has &street neurophyslologlcal and pharmacological actions, many of which are undoubtedly the result of the actwatlon of spectfic GHB receptors For instance, catalepsy, sedation and the mcrease m brain cGMP levels are all blocked by NCS-382, a GHB receptor antagomst It is not known ff GHB's effects on energy metabohsm are receptor mediated, but now that NCS-382 is avadable this doubt should soon be removed At the membrane level, increases m Ca :+ conductances may well account for GHB's inhibitory acuons The ewdence ~s fairly substanUal that GHB plays a role m the funcuomng of the central nervous system, perhaps as an inhibitory transmitter acting on dopammergxc neurons Certain of GHB's behavioral effects are receptor medmted, but it is unclear to what extent dopammerglc cells are involved It seems hkely

1031

that the action of GHB at its receptor sites Is terminated by ~ts rapid removal by a h~gh atfimty uptake process In this regard ~t Is typical of most neurotransnutters The cytosollc succmlc semmldehyde reductase seems Important m GHB blosynthes~s, but because succmlc semlaldehyde is produced in the mltochono drla, ~t appears certain a transport system must exist to bring the sybstrate in contact w~th the enzyme There are two can&dates for the catalytic degradation of v-hydroxybutyrlc acl d - - G H B dehydrogenase whose actw~ty ~s enhanced by the concomm~tant reducUon of o-glucuronate, and GHB transhydrogenase which is located m the mltochondrm With the first enzyme the product has to be transported to the mltochondrm before further oxidation to succmate can occur With the second enzyme, the substrate would have to be transported into the mltochondna The pharmacological and physiological actions of GHB make it a wable candidate as a neurotransmltter or neuromodulator m the CNS The presence of metabohzmg enzymes, uptake systems and receptor binding sites provide supporting evidence REFERENCES

Aldrete J A and Barnes D P (1968) 4-Hydroxybutyrate anesthesm for cardmvascular surgery Anesthesta 23, 558-565 Anden N -E, Magnusson T and Stock G (1973) Effect of drugs influencing monoamme mechanisms on the mcrease m brain dopamme produced by axotomy or treatment with gamma-hydroxybutync acid Naunyn-Schmwdebergs Arch Pharmac 278, 363-372 Anderson R A , Rltzmann R F and Tabakoff B (1977) Formation of gamma-hydroxybutyrate m brain J Neurochem 28, 633-639 BasdB, BlalrA M J N and HolmesS W (1964) The action of so&urn 4-hydroxybutyrate on spinal reflexes Br Pharmac 22, 318-328 Benavldes J, Rumlgny J F, Bourgmgnon J J, Wermuth C G, Mandel P and Maitre M (1982a) A hlgh-afllmty, Na+-dependent uptake system for 7-hydroxybutyrate m membrane vesicles prepared from rat brain J Neurochem 38, 1570-1575 Benawdes J , Rumlgny J F , Bourgmgnon J J, Cash C, Wermuth C G , Mandel P, Vmcendon G and Maitre M (1982b) High affimty binding site for y-hydroxybutync acid m rat brain Ltfe Scl 30, 953-961 Bessman S P and Flshbeln W M (1963) Gammahydroxybutyrate, a normal brain metabohte Nature 200, 1207-1208 Besson J M, Ravot J P, Abdelmoumene M and Aleonard P (1971) Effects of gamma-hydroxybutyrate on segmental and cortical control of transmission of the afferent volley at spinal level Neuropharmacology 10, 145-151 Blumenfeld M, Suntay R G and Harmel M H (1962) Sodmm gamma-hydroxybutync acid a new anaesthetic adjuvant Anesth Analg Curr Res 41, 721-726 Broughton R and Mamelak M (1979) The treatment of narcolepsy-catalepsy vath nocturnal gamma-hydroxybutyrate Can J Neurol Scz 6, 1-6 Bustos G and Roth R H (1972) Effect of ~,-hydroxybutyrate on the release of monoarmnes from the rat strmtum Br J Pharmacol 44, 817-820 Cash C D , Maitre M and Mandel P (1979) Punficatmn from human brain and some properues of two NADPHhnked aldehyde reductases which reduce succmlc semmldehyde to 4-hydroxybutyrate J Neurochem 33, 1169-1175

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