High affinity binding site for γ-hydroxybutyric acid in rat brain

Life Sciences, Vol. 30, pp. 953-961 Printed in the U.S.A.

Pergamon Press

HIGH AFFINITY BINDING SITE FOR y-HYDROXYBUTYRIC ACID IN RAT BRAIN J. BENAVIDES*, J.F. RUMIGNY, J.J. BOURGUIGNON**, C. CASH, C.G. WERMU~I**, P. MANDEL, G. VINCENDON and M. MAITRE Centre de Neurochimie du CNRS, INSERM U 44 and **Laboratoire de Pharmacochimie Mol~culaire (ERA 393 du CNRS) 5, rue Blaise Pascal, 67084 Strasbourg Cedex, France. (Received in final form January 13, 1982) SUMMARY The existence of a specific synthesizing enzyme for y-hydroxybutyric acid in rat brain has recently been reported. Here, for the first time, we demonstrate the presence of a high affinity, apparently specific binding site for this compound in the same tissue. This binding does not require Na + and takes place optimally at pH 5.5. The bound y-hydroxybutyric acid is not displacable by GABA or baclofen. We report here on some structurally related compounds of GHB with a similar or better binding capacity than GHB itself. The number of binding sites increases with age up to adulthood and differs depending on the brain region. In primary tissue cultures of pure chicken neurones and glia, y-hydroxybutyric acid binding occurs exclusivelyT in the neuronal preparations. y-Hydroxybutyric acid (GHB) is found in mammalian brain (I) with an uneven distribution at concentrations ranging from I x 10-6 M to i x 10-5 M (2). Administration of GHB to animals and man induce numerous neuropharmacological and neurophysiological effects (for review see 3). These include anesthesia which is accompanied by an EEG pattern similar to that occuring during petit mal epilepsy (4,5). This EEG profile is antagonised by anti petit mal drugs (6) such as valproate (7). GHB induced behavioral depression is accompanied by a large decrease in glucose consumption (8), which is without a correspondingly large depression of oxygen utilization (9,10), an increase in brain acetylcholine specifically in the midbrain and cortex (Ii) and an increase in brain dopamine (12-14) localized in areas rich in dopaminergic nerve terminals (15-17). However, the brain levels of GHB found after administration of sufficient quantities to induce these effects are over five hundred fold higher than the naturally occuring levels (18) and thus it is questionable at present whether endogenous GHB has a neuromodulator role. Some evidence that the presence of GHB in brain tissue may play a functional role is emerging. Firstly, an enzyme apparently specific for its biosynthesis has been isolated from both rat (19) and human brain (20,21). This enzyme which reduces the GABA catabolite, succinic semialdehyde (SSA) to GliB is NADPH-dependent but is not inhibited by barbiturates (19-21) which are inhibitors of most aldehyde reductases previously described (22). In rat brain, the highest levels of this enzyme (23) are found in areas relatively rich in endogenous GHB (24,25).

*in leave of absence from Centro de Biologia Molecular, C.S.I.C. and Universidad Autonoma, Madrid, Spain. 0024-3205/82/110953-09503.00/0 Copyright (c) 1982 Pergamon Press Ltd.

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Secondly, there exists in rat brain a high affinity binding site for GHB which appears to be specific for this ligand. The characteristics of this binding are the subject of this report. MATERIALS AND METHODS [(2,3)3H] GHB potassium salt (specific activity 40 Cimmol -I) was synthesized by the Commissariat ~ l'Energie Atomique (Gif sur Yvette, France). The radiochemical purity was checked by TLC (cellulose plates, Merck) in 3 different solvent systems (Ethanol/water : 50/50 ; n-butanol/diethylamine/water : 85/1/14 ; ethanol/water/ammonia 92/8/I). The plates were scanned with a TLC linear p-counter (Berthold). In the 3 solvent systems, the Rf values were respectively 0.93, 0.25 and 0.39. Unlabelled GHB used as a marker was visualised as the spot obtained after reaction with iodine vapor. In each case the purity was greater than 99%. GHB analogues were prepared as sodium salts by opening their corresponding lactone rings with one equivalent of NaOH and characterized by IH-NMR spectroscopy. Lactonic precursors were obtained from substituted ¥-functionalised butane and butenolides, the syntheses of which are published elsewhere (26). Other reagents were of analytical grade. Membrane preparation. Crude membranes were prepared by homogenization of the cerebral hemispheres from 6 rats in i0 vol. ice cold 0.32 M sucrose. The homogenates were centrifuged at 900 g for i0 min, the supernatants collected and recentrifuged at 26,000 g for 20 min. The resulting pellets were resuspended in 60 ml of 50 mM Tris HCI pH 7.2, and homogenized for 30 sec using a Polytron homogeniser and then centrifuged at 50,000 g for 20 min. The pellets obtained were resuspended in 40 ml Tris HCI medium and stored at -20°C. Incubation procedure. For binding assays, membrane suspensions were thawed, then diluted five fold in iO mM tris HCI, pH 7.2 and centrifuged at 50,000 g for 20 min. The pellets were then resuspended in the incubation medium which consisted unless otherwise s t a ~ d of : Pipes (l,4-piperazine-diethanesulfonic acid) 50 mM, pH 6.5 at O°C, Ca ": 2.5 mM ; Mg ++ : 1.2 mM, K + : 50 mM ; CI- : 12 ~M with a protein concentration of about iO mg per ml. iOO ul of this membrane suspension were added to 500 ~I buffer containing unless otherwise stated, 25 nM [3H] GHB and incubated 30 min. at O°C. Thenthe tubes were centrifuged at 55,000 g for 20 min in a Beckman R25 rotor. The supernatant was aspirated and the pellet rapidly washed twice with 800 ~I ice cold incubation buffer. Then 250 ~I of water was added to each tube which were then frozen, thawed and the pellets resuspended by vortexing before the tritium label was determined by liquid scintillation spectrometry in IO ml of Rotiszint 22 (Roth, West Germany). Dpm were obtained using a quenching curve. As the total amount bound was < 3% of the radiolabel in the incubation medium, the free concentration was taken as equivalent to the total tritium concentration. [3H] GHB displacement by other ligands. For this study, the membranes were added to the incubating buffer containing 25 nM [3H] GHB and iO ~M of the tested substance. Results are expressed as the percentage inhibition of the binding displacable by 5 mM non radioactive GHB. Regional study. For the regional study, 6 rat brains were rapidly dissected on ice according to the method of GLOWINSKI and IVERSEN (27) and the regions pooled. Crude membranes were prepared as described above. Tissue culture. Cultures of isolated neurones and glial cells from chicken embryos were used to test GHB binding. The culture technique of PETTMANN et al.

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(28) for isolated primary neuronal fractions was employed. Cultures of pure glial cells were obtained by the method of BOOHERR and SENSENBRENNER (29). Over 98% of neuronal cells bind tetanus toxin ; toxin binding could not be detected in the glial cultures (28). Ten dishes of neuronal (6 days old) or glial cells (14 days old) were rinsed twice with phosphate saline buffer pH 7.4. The cells were carefully scraped from their plates and centrifuged at 6,000 g for iO min. After freezing (16 h, at -20°C) the resulting pellets were suspended in 20 ml of the incubation medium and homogenized for 60 sec using a Polytron. The homogenates were centrifuged at 50,000 g for 20 min. The pellets were then resuspended in the preceeding buffer and recentrifuged under the same conditions. The final pellets were dispersed in the Pipes medium at a concentration of about 5 mg protein per ml. Protein concentrations were determined by the method of LOWRY et al. (30) using bovine serum albumin as a standard. Statistical analysis of data was carried out using Student's t test. RESULTS Under our incubation conditions (see Methods) the [3H] GHB binding displacable by 5 mM of unlabelled GHB was saturable (Fig. i). The data for this

.,..i ,,~

3

0

~u

~2

O

100

200

Free

300

aH-GHB (nM) FIG. I

Saturation curve of GHB binding to a rat brain membrane fraction : each point is derived from three separate experiments performed in quadruplicate at each concentration. A binding displacable by 5 mM GLIB, Z~non displacable binding, Q total binding;

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specific binding were plotted by the Scatchard method (Fig. 2). Fourteen concentrations of [3H] GHB (specific activity 4 Ci mmol -I) were used. There are

°°vf,~

Kdl 95nM Bml "557 pm°lmg-~

1

.oo] 1

~ ~,

F)

75

110

Bound (pmol per mg protein) FIG. 2 Analysis by Scatchard plot of [3H] GHB binding to rat brain membranes. The data is derived from two separate experiments performed in quadruplicate at each concentration (variation < 5%) two populations of binding sites and a computer analysis of these data using the mathematical treatment of FELDMAN (31) (two binding sites/one ligand) indicates that the high affinity binding has a Kd! of 95 nM and a Bmax I of 0.56 pmol per mg protein and the lower affinity site, a Kd2 of 16 ~M and Bmax 2 of 46 pmol per mg protein. This experiment was repeated twice and the same results were obtained. Preliminary experiments indicated that equilibrium was reached after 30 min incubation at O°C and TLC analysis (see Methods) of the bound GHB showed that a least 98% of the radioactivity was [3HI GHB. No metabolite could be detected. In order to obtain an estimation of the extent of the binding to the two sites, the binding displacable by I0 ~M (high affinity site) or 5 ~M (sum of the two sites) non-radioactive GHB was utilised in some of the experiments. The displacable binding by i0 uM or 5 mM GHB is strongly pH dependent (Fig. 3). In both cases a sharp optimum at pH 5.5 and a dramatic decrease at neutral pH is observed. However, significant binding occurs at pH 6.5. From pH 7.3 to 8.0, Pipes was substituted by Tris. The following cations : K ÷ (50 mM), Ca ++ (2.5 mM), Mg ++ (1.2 mM) alone or in combination caused a slight but not significant increase in the binding displacable by iO ~M GHB (buffer alone pH 6.5 : 228 ± 43 fmol per mg protein ; buffer pH 6.5 + cations : 303 ± 65 fmol per mg protein, mean ± S.D. 3 experiments p > 0.05) or 5 mM GHB (buffer alone, 370 ! 70 ~mol per mg protein, buffer pH 6.5 and cations : 420 i 86 fmol per mg protein ; mean ± S.D. 3 experiments, p > 0.05). However, to resemble physiological conditions more closely further experiments were performed in Pipes buffer pH 6.5 containing the cations. Na + was omitted, so that the GHB transport system which is strongly Na + dependent ,(results to be published) could not be confused with the binding system.

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5

©

5

S

pH

7

8

FIG. 3 pH dependence of [3H] GHB binding (25 nM) to rat brain membranes. Q binding displaeable by 5 mM GHB ; • binding displacable by IO ~M GHB ; Each point is the mean of 3 determinations performed in quadruplicate (variation < 5%).

A series of GHB ted as inhibitions of some of its analogues analogues are able to

structurally related compounds, including GABA, were tesGHB binding. The results are summarized in Table i. GABA, and y-butyrolactone (GBL) have no effect whereas some GBH displace [~H] GBH.

The distribution of binding in various rat brain regions is shown in Table 2. It is highest in the olfactory bulb and very low in the cerebellum, pons medulla and spinal cord. At 6 days of age, GHB binding represents 41% of the value found in the adult rat brain (see Table 3). In chicken embryo neuronal cultures [3HI GHB binding is present at a level of 50 fmoles per mg protein, whereas ~n glial cultures no binding was apparent (mean of 3 experiments which vary less than 15%).

DISCUSSION The presence of receptor sites for GHB in the CNS is predicted by the multiple effects of this substance. We report here for the first time the presence of a high affinity apparently specific binding site for this natural brain compound. This binding appears to be neuronal in origin and thus these results taken together with the existence of a specific synthesizing system (32) and a specific Na + dependent active transport system (results to be published) strongly suggest that endogenous GHB may play a neuromodulator or perhaps neurotransmitter role. Indeed, both high and low affinity binding sites

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TABLE I [3H] GHB displacement by some analogues and other

Compound

GHB y-butyrolactone 2-methyl GHB 2-phenyl GHB 3-methyl GHB 3-ethyl GHB 3-n-propyl GHB 3-isopropyl GHB 3-hydroxy GliB 3-phenyl GHB 3-benzyl GHB 2,3-dimethyl GHB 4-methyl GHB 3-hydroxypropionic acid 5-hydroxyvaleric acid 2-hydroxyethane sulfonic acid 3-hydroxypropane sulfonic acid 4-hydroxybutane sulfonic acid c~4-hydroxycrotonic acid (cis-HCA) 3-methyl (HCA) 3-isopropyl (HCA) trans 4-hydroxycrotonic acid (trans-HCA) 3-methyl trans-HCA O-hydroxymethylbenzoic acid GABA,(±) baclofen, isoguvacine dopamine picrotoxine ethosuceinimide, trimethadione

compounds

% inhibition of GHB binding at IO uM 52 ± 2 n.s. 24 ± 0.7 n.s. 59 ± 0.7 43 ± 8 5 ± i n.s. 19 ± 4 n.s. n.s. n.s. 45 ± 4 n.s. 43 ± 4 7 ± 0.4 n.s. n.s. n.s. n.s. n.s. 59 ± 0.5 n.s. n.s. n.s. n.s. n.s. n.s.

For this study the membranes were added to the incubating buffer containing 25 nM [3HI GHB and i0 uM of the tested substance. Results are expressed as the percentage inhibition of the binding displacable by 5 mM GHB (mean ± SD of 3 separate experiments performed in quadruplicate which varied less than 5%). n.s. : no significant inhibition.

were detected, however, the Kd of the high affinity site is similar in magnitude to those for established neurotransmitters such as GABA (33) and serotonin (3~) and this may be the real GHB receptor. It is clear that this binding site is not the GABA receptor, since t h e p H o p t i m u m , K d a n d B m a x a r e different (35) and [3HI GHB cannot be displaced by GABA and some of its analogues. This result is in agreement with the data of OLPE and KOELLA (36) who demonstrate that GHB does not elicit its effects on nigral and neocortical cells

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via bicuculline sensitive GABA receptors. On the other hand,the GHB binding site seems to be different to the GABA site described by HILL and BOWERY (37) since GABA and (±) baclofen do not displace [3H] GHB in the presence of Ca ++ and Mg ++ ions. GHB does not displace significantly (-) baclofen from its re-

TABLE 2 Regional distribution of [3HI GHB binding

Region

Whole brain Olfactory bulbs Frontal cortex Striatum Hippocampus Thalamus Hypothalamus Cerebellum Pons Medulla Spinal cord

[3H] GHB binding 10 -15 mole mg -I 220 ± 52 567 ±85 248 ±25 296 ± 14 465 ±27 89 ± 16 107 ± 32 15 ± 3 n.d. 24 ± 5

different from whole brain

p p p p p p p

< > < < < < <

.005 .05 .05 .001 .O1 .02 .O01

p < .001

GHB binding was measured as described under Materials and Methods using 25 nM [3H] GHB (+ 5 mM GHB for estimating background). Results are mean ± SD (3 separate experiments performed in quadruplicate which varied less than 5%). n.d. : no binding detected under our assay conditions.

TABLE 3 [3H] GHB binding in 6 day old and adult rat brain

Adult 6 days after birth

Displacable by 5 mM GHB

Displacable by i0 ~M GHB

353 ± 70 145 ± 26 *

183 + 37 51 ± 9 *

GHB binding([3H] GHB binding, 10 -15 mole per mg protein) was measured as described under Materials and Methods using 25 nM [3HI GHB. Results are mean ± SD (3 separate experiments performed in quadruplicate which varied less then 5%). * : significantly different from adult (p < .005)

ceptor site (37). This fact, and the results reported here, seem to indicate that GHB cannot be considered a GABA agonist as suggested by MELDRUM (38). However, it might be involved in the regulation of GABAergic synapse activity but only via a specific GHB receptor (39-40). The pH optimum of 5.5 cannot be due to lactonization of GHB as the binding is not displacable by GBL (see TABLE i). In addition, IH-NMR spectroscopy performed on GHB incubated in the binding buffer at pH 5.5 and O°C shows that the lactonic form of GHB

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cannot be detected in these conditions. Also the pH optimum of 5.5 for GHB binding suggests that protonation of the ligand (pKa = 4.72 (41)) and/or of the binding site, is a condition necessary for the most active conformation. Some alkyl substituted derivatives of GHB are able to displace [3H] GHB from its binding sites (TABLE i). Among these, 3-methyl-GHB is as good as GHB itself. It is notable that the trans isomer of 4-hydroxycrotonic acid has similar or greater affinity for the binding sites than GHB itself. This result and the inability of GBL to displace GHB indicate that the binding site requires GHB or its analogues in an open structure and semi-extended conformation. The GHB receptor is clearly distinct from the transport process, as it takes place optimally at pH 5.5 and is not cation dependent whereas the GHB transport process is Na + dependent and takes place at pH 7.4 (unpublished results). There are large differences in GHB binding in different brain regions. It is surprising that low binding could be detected in the cerebellum which is an area relatively rich in endogenous GHB (24,25) andinitsbiosynthetic enzyme (23). However, high levels of GHB binding sites are found in the striatum and olfactory bulbs, areas rich in dopaminergic nerve terminals where GHB administration causes an increase in dopamine levels (15-17). The fact that GHB binding increases from infancy to adulthood is of interest as it has recently been demonstrated that the endogenous GHB level in rats decreases by about 300% during this same period (25). It is yet too early to hypothesize on the role of endogenous GHB, although there is some evidence that it may be of significance in some pathological states such as Huntington Chorea (42-43) and epilepsy (3,44). Evidently, excess GHB in brain gives rise to undesirable symptomes as witnessed after administration per os, and thus abnormalities in its biosynthesis, transport, receptors or in the as yet uncertain mechanism of its degradation might also be involved in the aetiology of some forms of neurological disease. The results presented above which show the presence of a GHB receptor with an affinity for GHB similar to that found with neurotransmitter receptors for their ligands suggest a neurotransmitter or neuromodulator function for endogenous GHB and thus further work is needed to elucidate this possible role. ACKNOWLEDGEMENTS This work was supported by CNRS, ATP n ° 1680, "Pharmacologic des r~cepteurs des neurom~diateurs". We thank Mr. Serge GOBAILLE for his expert dissection of rat brain region. REFERENCES I. 2.

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