Bicuculline-insensitive GABA receptors: Studies on the binding of (−)-baclofen to rat cerebellar membranes

Neuroscience Letters, 52 (1984) 317-321

317

Elsevier Scientific Publishers Ireland Ltd. NSL 03068

B I C U C U L L I N E - I N S E N S I T I V E G A B A RECEPTORS: STUDIES ON T H E B I N D I N G OF ( - ) - B A C L O F E N TO RAT C E R E B E L L A R M E M B R A N E S

C O L L E E N A. D R E W , G R A H A M A.R. J O H N S T O N * and ROBERT P. W E A T H E R B Y

Department of Pharmacology, University of Sydney, Sydney, NSW 2006 (Australia) (Received and accepted September 27th, 1984)

Key words: 7-aminobutyric acid - baclofen - binding - bicuculline - 7-aminobutyric acid analogues

The binding of [3H](- )-baclofen to synaptic m e m b r a n e s prepared from rat cerebellum was studied. Consistent with pharmacological data that ( - ) - b a c l o f e n is the more active stereoisomer, studies on the binding of [3H](- )-baclofen showed increased specific binding, a higher affinity Ka and a lower Bmax than equivalent studies using [3H](+)-baclofen. Divalent metal ions (mercury, lead, calcium and zinc) inhibited the binding of [3H]( - )-baclofen. The effects of some conformationally restricted analogues of 7-aminobutyric acid (GABA) on [3H](-)-baclofen binding indicated that G A B A interacts with ( - ) baclofen-sensitive binding sites (GABAB) in extended rather than folded conformations, and that folded analogues o f G A B A m a y interact with a class of binding site (GABAc?) insensitive to (-)-baclofen and bicuculline.

Baclofen (lioresal; ~-(p-chlorophenyl)-GABA) may act as a 7-aminobutyric acid (GABA) agonist for a class of receptors that is not antagonised by the 'classic' GABA antagonist bicuculline [3]. The bicuculline-insensitive depressant action of baclofen on the firing of CNS neurons in vivo was first described in 1974 [7, 8] and subsequently a number of conformationally restricted analogues o f GABA were found to have similar actions, including cis-4-aminocrotonic acid [12] and cis-2(aminomethyl)cyclopropane carboxylic acid [1]. The structural similarities between these analogues led to the proposal that they constitute another class of GABA agonist acting on bicuculline-insensitive receptors [10, 11]. On the basis of structure-activity considerations, it appeared that GABA might interact with bicucullineinsensitive receptors in folded conformations, whereas extended conformations interacted with bicuculline-sensitive receptors. Studies on the action of baclofen on monoamine release in vitro led Bowery and his colleagues to propose that baclofen is a selective agonist for a novel class of receptors, designated GABAB, which are insensitive to bicuculline and have a different agonist profile to bicuculline-sensitive receptors, designated GABAA [4, 5]. The receptors activated by baclofen were not linked to chloride channels, their activation involving changes in calcium fluxes. The *Author for correspondence.

0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

318 action of baclofen was stereoselective with the (-)-stereoisomer being some two orders of magnitude more active than the ( + )-stereoisomer [4]. Studies on baclofen action have been complemented by studies on the binding of radioactive baclofen [5, 9]. We have recently resolved the commercially available [3H](+_)-baclofen by high-performance liquid chromatography (HPLC) procedure [15] and now report on binding studies using the pure active stereoisomer [3H](-)-baclofen. Rat cerebellar membranes were prepared from pooled tissue which was homogenised in cold 0.32 M sucrose (10 vols.) in a glass homogenizer with teflon pestle using 8 up and down strokes, using a procedure similar to that described by Bowery et al. [4]. The homogenate was centrifuged (1000 g, 10 min, 4°C) and the nuclear pellet discarded. The supernatant was recentrifuged (20,000 g, 20 min, 4°C) and the resulting P2 pellet resuspended in distilled water (10 vols.). The suspension was centrifuged (8000 g, 20 min, 4°C) and the supernatant and buffy coat collected. The minor membranous tissue remaining fixed to the centrifuge tube was discarded. The supernatant was centrifuged (40,000 g, 20 min, 4°C), the pellet resuspended in distilled water, and divided into aliquots sufficient for individual binding experiments (approximately 1.3 g wet weight tissue). These aliquots were centrifuged (40,000 g, 20 rain, 4°C) and the pellets stored at - 15°C. On the day of assay the frozen membrane pellets were thawed at 20°C (20 min), suspended in Tris-HC1 buffer (10 vols., 50 mM, pH 7.4, containing 2.5 mM calcium chloride) at 20°C for 45 rain in a shaking water bath, then centrifuged (8000 g, 10 min, 4°C). The pellet was resuspended in Tris-HCl buffer and incubated for 15 min prior to recentrifugation. This wash was repeated twice more and the final pellet resuspended in assay buffer (see below) to a final protein concentration of 5 mg/ml. The binding assays were carried out at 20°C for 11 min in 1.5-ml microcentrifuge tubes, the incubation mixture (1 ml final volume) containing assay buffer (50 mM Tris-HCl, pH 7.4, 2.5 mM calcium chloride, 40 /zM isoguvacine; 0.7 ml), membrane suspension (0.1 ml), radioligand solution (0.1 ml) and unlabelled ligand or test substances (0.1 ml). Assays were terminated by centrifugation (10,000 g, 5 min, 20°C, in an Eppendorf 5412 centrifuge) and the pellets superficially rinsed three times with distilled water. Bound radioactivity was determined by liquid scintillation counting. Scatchard analyses were performed using [3H](-)- or [3H](+_)-baclofen (8-10 nM) in the presence of unlabelled ( - ) - or (+)-baclofen (8.8-560 nM). Non-saturable binding was determined in the presence of GABA, ( - )- or ( +_)-baclofen (100/~M). [3H]( - )Baclofen (spec.act. 26 Ci/mmol) was prepared from racemic material (Amersham Int.) by chiral H P L C [15]. [3H]GABA (spec.act. 60 Ci/mmol) was purchased from Amersham Int. Isoguvacine was purchased from Research Biochemical (Wayland). ( - ) - B a c l o f e n was a gift from Ciba-Geigy (Basle). [3H](-)-Baclofen bound to rat cerebellar membranes in Tris-HCl buffer (containing 40/~M isoguvacine and 2.5 mM calcium chloride) in a bicuculline-insensitive, saturable manner. Scatchard analysis (Fig. 1) of the displacement of bound radioactivity by unlabelled ( - ) - b a c l o f e n indicated that binding was to a single population

319 6

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BACLOF E N

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0 m

0 2'

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0.2

0.6

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Fig. 1. Scatchard analysis of the binding of [3H](-)-baclofen (solid line and symbols) and [3H](±)baclofen (broken line, open symbols) to membranes prepared from rat cerebellum. Each point is the mean o f quadruplicate determinations and the lines are computer fitted to the data as described by McPherson [14].

of sites with an apparent Ka = 47 _+9 nM and a Bmax = 0.65 + 0.06 pmol/mg protein (means_+S.E.). When (_+)-baclofen was used as the displacing agent of bound [3H]( _+)-baclofen, the apparent kinetic parameters were approximately double (Ka 110 nM, Bmax 1.3 pmo1/mg) those observed with (-)-baclofen displacing [3H]( _+)baclofen. The figures for [3H](_+)-baclofen binding are comparable with those reported (Ka 132 nM, Bmax 1.1 pmol/mg) by Hill and Bowery [9] for [3H](_+)baclofen binding to rat brain synaptosomal membranes. GABA, (_)-baclofen and (-)-baclofen (100 tzM in each case) produced the same displacement of [3H](-)baclofen; this displacement was 30% of the total binding. Thus [3H](- )-baclofen, as expected, showed higher specific binding than that observed (20%, present study and ref. 9) using [3H](+_ )-baclofen. These data are consistent with the non-specific binding of [3H]baclofen being non-stereospecific. Consistent with ( - )-baclofen being the active displacing agent of specifically bound [3H](-)-baclofen binding, essentially the same kinetic parameters were obtained when either ( - ) - or (_+)baclofen was used as displacing agent, expressing the data in terms of pure ( - ) -

320 baclofen. Under these conditions, the binding of baclofen appears to be free of the anomalous binding properties reported for some other racemic radioligands [6] in that the Ka(+ ) is approximately twice the K a ( - ), with binding of the ( + ) isomer not significantly influencing binding of the ( - ) isomer. The increased specific binding observed with the pure ( - ) isomer compared to the ( _+) i.somer would mean that it is preferable to use the pure ( - ) isomer in radioligand binding experiments. There is evidence that the binding of baclofen to rat cerebellar membranes is regulated by divalent cations with manganese and nickel ions enhancing specific binding [13]. In the present study we have found that heavy metal divalent ions inhibit the binding of [3H](-)-baclofen to rat cerebellar membranes. Thus at 100 ~M, the following percentage inhibitions (mean +- S.E.; n =4) were measured: mercurous ions 93+_6, lead ions 67+_ 12, cadmium ions 74_+6, and zinc ions 62_+9. We have considered what properties of the metal ions may contribute to these effects. No relationship can be observed between the effect on baclofen binding and the radii of the ions (crystal ionic, Stokes' hydrated or effective hydrated radii). However, a relationship can be noted with the electronic configuration of the respective metal atoms. The metals (zinc, cadmium, mercury and lead) whose divalent ions inhibit baclofen binding have complete shells (4 f shells) inside the valence shell, while those which enhance (manganese and nickel) have unfilled shells (3 d shells) inside the valence shell. Two pairs of conformationally restricted analogues of GABA were examined as inhibitors of [3H](-)-baclofen binding: cis- and trans-4-aminocrotonic acid [12], and cis- and trans-2-(aminomethyl)cyclopropane carboxylic acid [1]. The trans isomers of these compounds are analogues of extended conformations of GABA and are bicuculline-sensitive inhibitors of neuronal activity, while the cis isomers are analogues of folded conformations of GABA and are bicuculline-insensitive inhibitors of neuronal activity. Unexpectedly, the trans isomers inhibited the binding of [3H](-)-baclofen by 100°70 at 100 ~M, whereas the cis isomers were inactive at this concentration. These findings do not support the proposal [10, 11] that GABA might interact with bicuculline-insensitive receptors in folded conformations, with respect to the bicuculline-insensitive ( - ) - b a c l o f e n binding sites studied under the present conditions. The interaction of analogues of GABA in extended conformations with binding sites for ( - ) - b a c l o f e n is consistent with the inhibition of the binding of (+-)baclofen by muscimol; however, the lack of effect of other extended analogues including T H I P , isoguvacine and piperidine-4-sulphonic acid [4] requires further explanation. The analogues of GABA in extended conformations which interact with baclofen binding sites (muscimol, trans-4-aminocrotonic acid and trans-2-(aminomethyl)cyclopropyl carboxylic acid) have flexible aminomethylene groupings, whereas those that do not interact (THIP, isoguvacine and piperidine-4-sulphonic acid) have the equivalent aminomethylene grouping incorporated into a piperidine ring structure which further restricts conformational mobility.

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The lack of interaction between the two analogues of GABA in folded conformations, which act as bicuculline-insensitive neuronal inhibitors, and the ( - )-baclofen binding sites in the present study may indicate the existence of a class of bicucullineinsensitive binding sites (GABAc?) for GABA that is insensitive to ( - ) - b a c l o f e n . Ault and Nadler [2] have provided electrophysiological evidence for two pharmacologically distinct, bicuculline-insensitive actions of GABA in the facia dentata in rat hippocampal slices, only one of which is shared by baclofen. The authors are grateful to Ciba-Geigy (Basle) for a gift of the stereoisomers of baclofen, and the award of a Commonwealth Postgraduate Scholarship to C.A.D., and to the National Health and Medical Research Council of Australia for financial support. 1 Allan, R.D., Curtis, D.R., Headley, P.M., Johnston, G.A.R., Lodge, D. and Twitchin, B., The synthesis and activity of cis- and trans-2-(aminomethyl)cyclopropanecarboxylic acid as conformationally restricted analogues of GABA, J. Neurochem., 34 (1980) 651-655. 2 Ault, B. and Nadler, J.V., Physiological evidence for two pharmacologically distinct, bicucullineinsensitive actions of GABA in the rat hippocampal slice, Soc. Neurosci. Abstr., 9 (1983) 411. 3 Bowery, N.G., Baclofen: 10 years on, TIPS, 3 (1983) 400-403. 4 Bowery, N.G., Hill, D.R. and Hudson, A.L., Characteristics of GABAn receptor binding sites on rat whole brain sympathetic membranes, Brit. J. Pharmacol., 78 (1983) 191-206. 5 Bowery, N.G., Hill, D.R., Hudson, A.L., Doble, A., Midllemiss, D.N., Shaw, J. and Turnbull, M.J., ( - )-Baclofen decreases neurotransmitter release in the mammalian CNS by an action at a novel GABA-receptor, Nature (Lond.), 283 (1980) 92-94. 6 Burgisser, E., Hancock, A.A., Lefkowizt, R.J. and De Lean, A., Anomalous equilibrium binding properties of high-affinity racemic radioligands, Molec. Pharmacol., 19 (1981) 205-216. 7 Curtis, D.~R., Game, C.J.A., Johnston, G.A.R. and McCulloch, R.M., Central effects of beta-(pclorophenyl)-gamma-aminobutyric acid, Brain Res., 70 (1974) 493-499. 8 Davies, J. and Watkins, J.C., The action of beta-phenyl-GABA derivatives on neurones in the cat cerebral cortex, Brain Res., 70 (1974) 501-505. 9 Hill, D.R. and Bowery, N.G., 3H-Baclofen and 3H-GABA bind to bicuculline-insensitive GABAB sites in rat brain, Nature (Lond.), 290 (1981) 149-152. 10 Johnston, G.A.R., Physiologic pharmacology of GABA and its antagonists in the vertebrate nervous system. In E. Roberts, T.N. Chase and D.B. Tower (Eds.), GABA in Nervous System Function, Raven Press, New York, 1976, pp. 394-411. 11 Johnston, G.A.R., Amino acid receptors. In J.R. Smythies and R.J. Bradley (Eds.), Receptors in Pharmacology, Dekker, New York, 1978, pp. 295-333. 12 Johnston, G.A.R., Curtis, D.R., Beart, P.M., Game, C.J.A. McCulloch, R.M. and Twitchin, B., Cis and trans-4-aminocrotonic acid as GABA agonists of restricted conformation, J. Neurochem., 24 (1975) 157-160. 13 Kato, K., Goto, M. and Fukuna, H., Regulation by divalent cations of 3H-baclofen binding to GABA~ sites in rat cerebellar membranes, Life Sci., 32 (1983) 879-887. 14 McPherson, G.A., Analysis of radioligand binding experiments on a microcomputer system, TIPS, 4 (1983) 369-370. 15 Weatherby, R.P., Allan, R.D. and Johnston, G.A.R., Resolution of the stereoisomers of baclofen by high performance liquid chromatography, J. Neurosci. Meth., 10 (1984) 23-28.