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Interaction of benzodiazepine receptor agonists and inverse agonists with the GABA benzodiazepine receptor complex
Pharmacology Bbwhemisto' & Behavior, Vol. 23. pp. 671-674, 1985. AnkhoInternationalInc. Printed in the U.S.A.
0091-3057/85 $3.00 + .00
Interaction of Benzodiazepine Receptor Agonists and Inverse Agonists With the GABA Benzodiazepine Receptor Complex MANFRED KAROBATH AND PORNTIP SUPAVILAI P r e c l i n i c a l R e s e a r c h , S a n d o z L t d ., 386/216, CH - 4 0 0 2 B a s l e , S w i t z e r l a n d
KAROBATH, M. AND P. SUPAVILAI. Interaction of benzodiazepine receptor agonists and inverse agonists with the GABA benzodiazepine receptor complex. PHARMACOL BIOCHEM BEHAV 23(4) 671-674, 1985.--The effects of the benzodiazepine receptor agonists, antagonists and inverse agonists on the in vitro binding of several ligands which label different recognition sites of the GABA benzodiazepine receptor complex are summarized. Also, results with a novel biochemical in vitro functional model of the GABA benzodiazepine recePtor complex are presented. They are compatible with the concept that drugs which act on benzodiazepine receptors can lead to a bidirectional modulation of the gain of GABAergic neurotransmission. GABA benzodiazepine receptor complex Benzodiazepine receptor agonists Benzodiazepine receptor antagonists Inverse benzodiazepine receptor agonists Functional model
Binding studies
GABA receptors. The investigation of these novel drugs acting on BR has also provided evidence that there appears to be a continuum ranging from drugs with full agonistic efficacy to partial agonists, antagonists, partial inverse agonists to full inverse agonists [6]. This has raised the possibility that partial BR agonists may provide a new opportunity for drug development, since they could retain the desired therapeutic effects like the anxiolytic or antiepileptic actions, but at the same time they could lack some of the undesired side effects of benzodiazepines such as muscle relaxation, ataxia, the potentiation of CNS depressants or sedation. If different ligands acting on BR can allosterically up- or down-regulate the gain of the GABAergic system, then these bidirectional effects should also be detectable in vitro in binding studies or in a functional biochemical in vitro model of the GBRC. In the following these results are briefly summarized.
SOON after the discovery of brain specific benzodiazepine receptors (BR), it became apparent that these receptors are constituents of certain types of GABA-A receptors, which have been termed the GABA benzodiazepine receptor complex (GBRC). As a consequence of the availability of binding assays for the investigation of the affinity of drugs for the BR, several additional chemical classes of drugs have been discovered which interact with benzodiazepine receptors, but have a pharmacological profile which differs from that of the benzodiazepines [1, 2, 5, 11, 12]. Based on their pharmacological properties, these drugs have been subdivided into three different classes of drugs. One class consists of benzodiazepines, but also of other drugs chemically unrelated to benzodiazepines like zopiclone, which have the well described pharmacological actions of benzodiazepines and drugs with this pharmacological profile have been termed benzodiazepine receptor agonists. Other drugs which exert their pharmacological effects as a consequence of their interaction with the BR, induce the opposite actions when compared to benzodiazepines. These drugs can facilitate or induce seizures, and they can provoke anxiety in animals and man. They have been termed inverse BR agonists [6]. In addition, a third class of BR ligands has been proposed, the BR antagonists [11]. These drugs have few intrinsic pharmacological effects of their own in normal animals, but they are capable of antagonizing the effects of BR agonists, as well as those of inverse BR agonists. Benzodiazepine binding sites are constituents of a GBRC, and benzodiazepines have been shown to increase GABAergic neurotransmission [7,10]. The discovery of inverse BR agonists indicates that the alterations of GABAergic neurotransmission by BR ligands can be biphasic and result in an allosteric up- or down-regulation of the gain of
BINDING STUDIES In order to explain the various effects seen in binding studies in vitro a simplified schematic drawing of the GBRC is given (Fig. 1). Ligands exist which permit to directly investigate the binding to the various recognition sites of the GBRC (Fig. 1). The results of these binding studies indicate that changes in the conformation of the GBRC induced by the various classes of drugs which interact with this receptor can be detected by the investigation of the allosteric perturbation of the other ligand recognition sites of the GBRC. For example, the addition of unlabeled GABA will lead to a competitive inhibition of 3H-muscimol binding, as well as to allosteric alterations of the binding of BR ligands and of the binding of 35S-TBPS.
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FIG. 1. Simplified schematic drawing of a GABA benzodiazepine receptor complex. G: GABA recognition site; B: Benzodiazepine receptor: Cago: CNS-depressants binding site (the "'barbiturate site"); Cant: CNS-convulsants binding site (the "picrotoxinin site"); L: Suitable ligand for the respective binding site. There is increasing evidence that the Cago site and the Cant site are different, but closely associated sites ([9,20], and C. Braestrup, personal communication).
GABA and Barbiturate Effects GABA, or other GABA-A receptor agonists like muscimol, enhance the affinity of BR agonists for BR, do not markedly alter that of antagonists and decrease that of inverse BR agonists [5,16] (Fig. 2). Certain CNS depressants, like pentobarbital or etazolate, modify the affinity of ligands for BR in a similar mode as GABA does [4]. The actions of the CNS depressants and GABA are additive. This modulation is fairly consistent with the pharmacological profile of the investigated drugs when the binding experiments are performed at more physiological temperatures (~23 to 37°C) and not at 0°C. It has already previously been observed that more physiological temperatures are required for several allosteric interactions to occur within the GBRC [19].
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FIG. 2. Ratio of the affinity of BR ligands for the BR in the absence of GABA over that in the presence of GABA or muscimol (IC~. [(without GABA)/(with GABA or muscimol)]). AResults from Braestrup et al. [5], using membranes from rat cerebellum and '~Hdiazepam as ligand. IIResults from Mohler and Richards [16]. using membranes from rat cerebral cortex and ~H-Ro 15-1788 as ligand. @Results from Borea et al. [4], using membranes from the cerebral cortex of 5 days old rats and :~H-Ro 15-1788 as ligand.
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FIG. 3. Lol of the ratio of the affinity of BR ligands for the BR in membranes which have been photoaffinity labelled with flunitrazepam over that observed in control membranes. Log PAL ratio is the log (IC~. [(in photolabelled membranes)/(not photolabelled membranes)]). ~Results with membranes from rat cerebellum [13], using 3H-/3CCM as ligand; @Results with membranes from the cerebral cortex of 5-days-old rats using 3H-Ro 15-1788 as ligand [4].
Phtoaffinity Labelling o f BR There is some evidence that a GBRC contains four BR binding sites [15]. When flunitrazepam is irreversibly coupled to one of these four BR binding sites by photoaffinity labelling, the remaining three BR binding sites undergo conformational changes which result in a decreased affinity for BR agonists, but not for BR antagonists or inverse BR agonists [13]. The loss of affinity for BR agonistic benzodiazepines is more pronounced than that of zopiclone, a BR agonist with a different chemical structure. The affinity of BR antagonists and of inverse BR agonists is not markedly altered by photoaffinity labelling (Fig. 3). It has already been pointed out in the initial report that this method permits a distinction of BR agonists from BR antagonists, but it does not allow a distinction between the BR antagonist Ro 15-1788 and the (partial) inverse BR agonists flCCE or/3CCM [13]. There have been attempts to explain the differences of the photoaffinity labelling induced affinity changes alternatively by assuming that the binding of different chemical classes of ligands may occur to different domains of the BR [ 14].
GABA Receptor Binding Diazepam has been reported to increase 3H-GABA binding to crude synaptosomal membranes [8] and these observations have recently been extended [17]. Using 3Hmuscimol as ligand in binding studies performed at 23°C we also have been able to observe small increases of '~Hmuscimol binding by several BR agonists ([3], P. Supavilai and M. Karobath, unpublished results). More potent allosteric effects on aH-muscimol binding by various ligands for the BR can be seen when these investigations are performed in the presence of a CNS depressant like pentobarbital or etazolate (Fig. 4). In these investigations it has been found that BR agonists increase the stimulation of 3H-muscimol binding induced by etazolate, and that inverse BR agonists attenuate the effects of etazolate on 3H-muscimol binding (Fig. 4). The BR antagonist Ro 15-1788 does not markedly alter the actions of etazolate on 3H-muscimol binding but dose dependently antagonizes the effects o f a BR agonist and of an inverse BR agonist [3].
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cerebral cortex was investigated at 23°C using a centrifugation separation method [3]. 3H-muscimol binding was stimulated by 3 /zM etazolate and its perturbation by BR ligands was investigated. Data are expressed as % enhancement of 3H-muscimol binding by etazolate in the absence of other drugs. Three/.tM etazolate alone stimulates binding by 15(V~ (see dashed line). Ligands for the BR were added in concentrations which gave ~>80% BR occupancy in the assay conditions.
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Binding experiments were performed with membranes from rat cerebral cortex at 23°C in the presence of 200 mM NaC1 [20]. Data are expressed as % binding in the absence of drugs (dashed line). BR ligands were added in concentrations which gave ~>8(FA, BR occupancy in the assay conditions.
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FIG. 6. Perturbation of muscimol inhibition of 3sS-TBPS binding to membranes from rat cerebral cortex by ligands for the BR. Displayed is the ICs,, of muscimol [(without BR ligand)/(with BR ligand)]. BR ligands were added in concentrations which gave ~>8ff~ BR occupancy in the assay conditions.
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FIG. 7. Potentiation by BR agonists and attenuation by inverse BR agonists of the effect of muscimol on electrically induced acetylcholine release from rat striatum slices. The BR ligands were added in concentrations which give ~>80%. BR occupancy in the assay conditions. In the absence of muscimol these drugs did not alter electrically induced acetylcholine release [21]. The release of 3Hacetylcholine derived from prelabelling with 3H-choline was investigated and analyzed as described [21 ]. Muscimol at 2/zM gave 49% of the maximal inhibitory effect which is a 75% inhibition of electrically induced acetylcholine release. The results are mean values +S.E.M.; n~>4.
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Chloride Channel Antagonist Binding With 35S-TBPS as Ligand F|G. 8. Antagonism by Ro 15-1788 of the effects of clonazepam or DMCM on muscimol inhibition of electrically induced acetylcholine release from rat striatum slices. The inhibition of electrically induced acetylcholine release by 2 ~xM muscimol was 42%. This is indicated by the dotted horizontal bar. Shown is the potentiation of the action of muscimol by clonazepam and the attenuation of the action of muscimol by DMCM. Also shown is the antagonism of the modulatory action of these two drugs by Ro 15-1788, which alone is ineffective. The results are expressed as % inhibition of electrically induced acetylcholine release. The results are mean values -+S.E.M.: n=4-10. Ro: 0.5 /~M Ro 15-1788 added; C: 0.25 /zM clonazepam added; Ro+C: 0.5 ~M Ro 15-1788 and 0.25 k~M clonazepam added; D: 0.5 p.M DMCM added; Ro+D: 0.5 /~M Ro 15-1788 and 0.5/zM DMCM added.
'~sS-TBPS is a ligand i n t r o d u c e d by Squires et al. [18], w h i c h labels an a n t a g o n i s t binding site o f the chloride c h a n nel o f the G B R C [20]. BR a g o n i s t s i n c r e a s e , and i n v e r s e BR a g o n i s t s d e c r e a s e asS-TBPS binding (Fig. 5). The BR a n t a g o n i s t Ro 15-1788 w h i c h d o e s not alter 35S-TBPS binding by itself, h o w e v e r , b l o c k s the allosteric p e r t u r b a t i o n s o f a BR agonist and o f an i n v e r s e BR agonist [20]. Similar results w e r e also o b t a i n e d w h e n the inhibitory e f f e c t s o f m u s c i m o l on 35S-TBPS binding by BR ligands have b e e n i n v e s t i g a t e d [20] (Fig. 6). T a k e n t o g e t h e r , t h e s e results f r o m binding studies in vitro, with the p o s s i b l e e x c e p t i o n o f the photoaffinity labelling studies, s u p p o r t the initial c o n c e p t f o r m u l a t e d by Braestrup [6] that ligands for b e n z o d i a z e p i n e r e c e p t o r s can induce
674
KAROBATH
b i d i r e c t i o n a l allosteric effects o n the G B R C . W e t h e r e f o r e w e r e i n t e r e s t e d to i n v e s t i g a t e t h e s e drugs in a f u n c t i o n a l b i o c h e m i c a l in vitro m o d e l o f this r e c e p t o r . UP- AND DOWN-REGULATION OF THE GAIN OF A GBRC IN A FUNCTIONAL BIOCHEMICAL IN VITRO MODEL W e h a v e r e c e n t l y d e m o n s t r a t e d t h a t the electrically ind u c e d release o f a c e t y l c h o l i n e f r o m rat s t r i a t u m slices is m o d u l a t e d by a G A B A - A r e c e p t o r w h i c h h a s the c h a r a c teristics o f a G B R C . T h u s m u s c i m o l or G A B A , but not baclofen c a n inhibit electrically i n d u c e d a c e t y l c h o l i n e release f r o m t h e s e slices a n d this a c t i o n is s e n s i t i v e to inhibition by b i c u c u l l i n e , p i c r o t o x i n a n d the cage c o n v u l s a n t s [21]. In the p r e s e n c e o f c l o n a z e p a m , w h i c h has no a c t i o n s o n acetylc h o l i n e release, the c o n c e n t r a t i o n effect c u r v e o f m u s c i m o l is shifted to the left. F o r e x a m p l e , the c o n c e n t r a t i o n o f muscimol w h i c h yields 10% o f the m a x i m a l r e s p o n s e (ED~0) is shifted f r o m 0 . 2 7 / , M in t h e a b s e n c e o f c l o n a z e p a m to 0.03 ~ M in the p r e s e n c e o f 0.1 p,M c l o n a z e p a m . O n the o t h e r h a n d t h e partial i n v e r s e B R a g o n i s t / 3 C C M at 0.025 p,M shifts the ED~0 for m u s c i m o l f r o m 0.27/,~M to 0.8 p,M. f l C C M , in the a b s e n c e o f m u s c i m o l does not alter electrically e v o k e d acetylcholine release [21].
AND SUPAV|LAI
T h e s e r e s u l t s i n d i c a t e t h a t in this f u n c t i o n a l m o d e l a BR a g o n i s t i n c r e a s e s the efficacy o f m u s c i m o l to e x e r t its functional r e s p o n s e w h e r e a s the i n v e r s e B R agonist h a s the opposite effect. T h e a c t i o n o f a n u m b e r o f l i g a n d s for the B R on the effect o f 2 p,M m u s c i m o l , a c o n c e n t r a t i o n w h i c h leads to a p p r o x i m a t e l y 50% o f its m a x i m a l effect o n electrically ind u c e d a c e t y l c h o l i n e release is s h o w n in Fig. 7. It c a n also be s e e n that Ro 15-1788 w h i c h has w e a k a n d not significant agonistic a c t i o n s (Fig. 7), is able to a n t a g o n i z e the m o d u l a t ory effects o f c l o n a z e p a m a n d o f D M C M on m u s c i m o l inhibition o f electrically i n d u c e d a c e t y l c h o l i n e release f r o m rat s t r i a t u m slices (Fig. 8). T h e results with this m o d e l p r o v i d e direct s u p p o r t for the c o n c e p t p r o p o s e d initially by Haefely et al. [ 10] a n d C o s t a et al. [7] t h a t b e n z o d i a z e p i n e s , in p h a r m a c o l o g i c a l l y r e l e v a n t c o n c e n t r a t i o n , h a v e no intrinsic p h a r m a c o l o g i c a l effects but e n h a n c e G A B A e r g i c n e u r o t r a n s m i s s i o n . F u r t h e r , the o b s e r v a t i o n that B R a g o n i s t s e n h a n c e , a n d i n v e r s e BR a g o n i s t s a t t e n u a t e the e f f e c t i v e n e s s o f m u s c i m o l at the G B R C whilst the B R a n t a g o n i s t Ro 15-1788 is a l m o s t ineffective but b l o c k s the effect o f B R a g o n i s t s a n d i n v e r s e B R a g o n i s t s , a g r e e s with the c o n c e p t t h a t the i n t e r a c t i o n of ligands with the B R c a n lead to b i d i r e c t i o n a l f u n c t i o n a l effects o n G A B A e r g i c neurotransmission.
REFERENCES 1. Bernard, P., K. Berger, R. Sobiski and R. D. Robson. CGS 8216 (2-phenylpyrazolo[4,3-c]quinolin-3(5H-one), an orally effective benzodiazepine antagonist. Pharmaeologist 23: 150, 1981. 2. Blanchard, J. C., A. Boireau, C. Garret and L. Julou. In vitro and in vivo inhibition by zopiclone of benzodiazepine binding to rodent brain receptors. Life Sci 24: 2417-2420, 1978. 3. Borea, P. A., P. Supavilai and M. Karobath. Differential modulation of etazolate or pentobarbital enhanced [aH] muscimol binding by benzodiazepine agonist and inverse agonists. Brain Res 280: 383-386, 1983. 4. Borea, P. A., P. Supavilai and M. Karobath. Effect of GABA and photoaffinity labelling on the affinity of drugs for benzodiazepine receptors in membranes of the cerebral cortex of five-day-old rats. Biochem Pharmaeol 33: 165-168, 1984. 5. Braestrup, C., R. Schmiechen, G. Neef, M. Nielsen and E. N. Peterson. A new type of interaction with benzodiazepine receptors: convulsive ligands. Science 216: 1241-1243, 1982. 6. Braestrup, C., M. Nielsen, T. Honore, L. H. Jensen and E. N. Petersen. Benzodiazepine receptor ligands with positive and negative efficacy. Neuropharmaeology 22: 1451-1457, 1983. 7. Costa, E., A. Guidotti and C. C. Mao. Evidence for involvement of GABA in the action of benzodiazepines: studies on rat cerebellum. Adv Bioehem Psychopharmacol 14:113-130, 1975. 8. Costa, E., A. Guidotti and G. Toffano. Molecular mechanisms mediating the action of diazepam on GABA receptors. Br J Psychiatry 133: 239-248, 1978. 9. Drexler, G. and W. Sieghart. [35S]tert.-Butylbicyclophosphorothionate and avermectin bind to different sites associated with y-aminobutyric acid-benzodiazepine receptor complex. Neurosei Lett 50: 273-277, 1984. 10. Haefely, W., A. Kulcs~ir, H. M6hler, L. Pieri, P. Polc and R. Schaffner. Possible involvement of GABA in the central actions of benzodiazepines. In: Mechanisms ~f' Action o f Benzodiazepines, edited by E. Costa and P. Greengard. New York: Raven Press, 1975, pp. 131-151. I1. Hunkeler, W., H. M6hler, L. Pieri, P. Polc, E. P. Bonetti, R. Cumin, R. Schaffner and W. Haefely. Selective antagonists of benzodiazepines. Nature 290: 514-516, 1981.
12. Jensen, L. H., E. N. Petersen and C. Braestrup. Audiogenic seizures in DBA/2 mice discriminate sensitively between low efficacy benzodiazepine receptors agonists and inverse agonists. Li[? Sei 33: 393-399, 1983. 13. Karobath, M. and P. Supavilai. Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro. Neurosei Lett 31: 65-69, 1982. 14. Martin, 1. L., C. L. Brown and A. Doble. Multiple benzodiazepine receptors: structures in the brain or structures in the mind? A critical review. Lt:/~, Sci 32: 1925-1933, 1983. 15. Mohler. H., M. K. Battersby and J. G. Richards. Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label. Proc Nail Aead Sci USA 77: 1666-1670, 1980. 16. Mohler, H. and J. G. Richards. Agonist and antagonist benzodiazepine receptor interaction in vitro. Nature 294: 763-765, 1981. 17. Skerritt, J. H., M. Willow and G. A. R. Johnston. Diazepam enhancement of low affinity GABA binding to rat brain membranes. Neurosei Lett 29: 63-66, 1982. 18. Squires, R. F., J. E. Casida, M. Richardson and E. Saederup. [asS]t-Butylbicyclophosphorothionate binds with high affinity to brain specific sites coupled to y-aminobutyric acid-A and ion recognition sites. Mol Pharmacal 13: 326-336, 1983. 19. Supavilai, P. and M. Karobath. The effect of temperature and chloride ions on the stimulation of [:~H]flunitrazepam binding by the muscimol analogues THIP and piperidine-4-sulfonic acid. Neurosei Lett 19: 337-341, 1980. 20. Supavilai, P. and M. Karobath. I:~='S]-t-Butyl-bicyclophos phorothionate binding sites are constituents of the yaminobutyric acid benzodiazepine receptor complex. .I Neurasci 4: 1193-1200, 1984. 21. Supavilai, P. and M. Karobath. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex. L~/k, Sei 36: 417-426, 1985.
0091-3057/85 $3.00 + .00
Interaction of Benzodiazepine Receptor Agonists and Inverse Agonists With the GABA Benzodiazepine Receptor Complex MANFRED KAROBATH AND PORNTIP SUPAVILAI P r e c l i n i c a l R e s e a r c h , S a n d o z L t d ., 386/216, CH - 4 0 0 2 B a s l e , S w i t z e r l a n d
KAROBATH, M. AND P. SUPAVILAI. Interaction of benzodiazepine receptor agonists and inverse agonists with the GABA benzodiazepine receptor complex. PHARMACOL BIOCHEM BEHAV 23(4) 671-674, 1985.--The effects of the benzodiazepine receptor agonists, antagonists and inverse agonists on the in vitro binding of several ligands which label different recognition sites of the GABA benzodiazepine receptor complex are summarized. Also, results with a novel biochemical in vitro functional model of the GABA benzodiazepine recePtor complex are presented. They are compatible with the concept that drugs which act on benzodiazepine receptors can lead to a bidirectional modulation of the gain of GABAergic neurotransmission. GABA benzodiazepine receptor complex Benzodiazepine receptor agonists Benzodiazepine receptor antagonists Inverse benzodiazepine receptor agonists Functional model
Binding studies
GABA receptors. The investigation of these novel drugs acting on BR has also provided evidence that there appears to be a continuum ranging from drugs with full agonistic efficacy to partial agonists, antagonists, partial inverse agonists to full inverse agonists [6]. This has raised the possibility that partial BR agonists may provide a new opportunity for drug development, since they could retain the desired therapeutic effects like the anxiolytic or antiepileptic actions, but at the same time they could lack some of the undesired side effects of benzodiazepines such as muscle relaxation, ataxia, the potentiation of CNS depressants or sedation. If different ligands acting on BR can allosterically up- or down-regulate the gain of the GABAergic system, then these bidirectional effects should also be detectable in vitro in binding studies or in a functional biochemical in vitro model of the GBRC. In the following these results are briefly summarized.
SOON after the discovery of brain specific benzodiazepine receptors (BR), it became apparent that these receptors are constituents of certain types of GABA-A receptors, which have been termed the GABA benzodiazepine receptor complex (GBRC). As a consequence of the availability of binding assays for the investigation of the affinity of drugs for the BR, several additional chemical classes of drugs have been discovered which interact with benzodiazepine receptors, but have a pharmacological profile which differs from that of the benzodiazepines [1, 2, 5, 11, 12]. Based on their pharmacological properties, these drugs have been subdivided into three different classes of drugs. One class consists of benzodiazepines, but also of other drugs chemically unrelated to benzodiazepines like zopiclone, which have the well described pharmacological actions of benzodiazepines and drugs with this pharmacological profile have been termed benzodiazepine receptor agonists. Other drugs which exert their pharmacological effects as a consequence of their interaction with the BR, induce the opposite actions when compared to benzodiazepines. These drugs can facilitate or induce seizures, and they can provoke anxiety in animals and man. They have been termed inverse BR agonists [6]. In addition, a third class of BR ligands has been proposed, the BR antagonists [11]. These drugs have few intrinsic pharmacological effects of their own in normal animals, but they are capable of antagonizing the effects of BR agonists, as well as those of inverse BR agonists. Benzodiazepine binding sites are constituents of a GBRC, and benzodiazepines have been shown to increase GABAergic neurotransmission [7,10]. The discovery of inverse BR agonists indicates that the alterations of GABAergic neurotransmission by BR ligands can be biphasic and result in an allosteric up- or down-regulation of the gain of
BINDING STUDIES In order to explain the various effects seen in binding studies in vitro a simplified schematic drawing of the GBRC is given (Fig. 1). Ligands exist which permit to directly investigate the binding to the various recognition sites of the GBRC (Fig. 1). The results of these binding studies indicate that changes in the conformation of the GBRC induced by the various classes of drugs which interact with this receptor can be detected by the investigation of the allosteric perturbation of the other ligand recognition sites of the GBRC. For example, the addition of unlabeled GABA will lead to a competitive inhibition of 3H-muscimol binding, as well as to allosteric alterations of the binding of BR ligands and of the binding of 35S-TBPS.
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KAROBATH AND SUPAVILAI
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FIG. 1. Simplified schematic drawing of a GABA benzodiazepine receptor complex. G: GABA recognition site; B: Benzodiazepine receptor: Cago: CNS-depressants binding site (the "'barbiturate site"); Cant: CNS-convulsants binding site (the "picrotoxinin site"); L: Suitable ligand for the respective binding site. There is increasing evidence that the Cago site and the Cant site are different, but closely associated sites ([9,20], and C. Braestrup, personal communication).
GABA and Barbiturate Effects GABA, or other GABA-A receptor agonists like muscimol, enhance the affinity of BR agonists for BR, do not markedly alter that of antagonists and decrease that of inverse BR agonists [5,16] (Fig. 2). Certain CNS depressants, like pentobarbital or etazolate, modify the affinity of ligands for BR in a similar mode as GABA does [4]. The actions of the CNS depressants and GABA are additive. This modulation is fairly consistent with the pharmacological profile of the investigated drugs when the binding experiments are performed at more physiological temperatures (~23 to 37°C) and not at 0°C. It has already previously been observed that more physiological temperatures are required for several allosteric interactions to occur within the GBRC [19].
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AGONISTS
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FIG. 2. Ratio of the affinity of BR ligands for the BR in the absence of GABA over that in the presence of GABA or muscimol (IC~. [(without GABA)/(with GABA or muscimol)]). AResults from Braestrup et al. [5], using membranes from rat cerebellum and '~Hdiazepam as ligand. IIResults from Mohler and Richards [16]. using membranes from rat cerebral cortex and ~H-Ro 15-1788 as ligand. @Results from Borea et al. [4], using membranes from the cerebral cortex of 5 days old rats and :~H-Ro 15-1788 as ligand.
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FIG. 3. Lol of the ratio of the affinity of BR ligands for the BR in membranes which have been photoaffinity labelled with flunitrazepam over that observed in control membranes. Log PAL ratio is the log (IC~. [(in photolabelled membranes)/(not photolabelled membranes)]). ~Results with membranes from rat cerebellum [13], using 3H-/3CCM as ligand; @Results with membranes from the cerebral cortex of 5-days-old rats using 3H-Ro 15-1788 as ligand [4].
Phtoaffinity Labelling o f BR There is some evidence that a GBRC contains four BR binding sites [15]. When flunitrazepam is irreversibly coupled to one of these four BR binding sites by photoaffinity labelling, the remaining three BR binding sites undergo conformational changes which result in a decreased affinity for BR agonists, but not for BR antagonists or inverse BR agonists [13]. The loss of affinity for BR agonistic benzodiazepines is more pronounced than that of zopiclone, a BR agonist with a different chemical structure. The affinity of BR antagonists and of inverse BR agonists is not markedly altered by photoaffinity labelling (Fig. 3). It has already been pointed out in the initial report that this method permits a distinction of BR agonists from BR antagonists, but it does not allow a distinction between the BR antagonist Ro 15-1788 and the (partial) inverse BR agonists flCCE or/3CCM [13]. There have been attempts to explain the differences of the photoaffinity labelling induced affinity changes alternatively by assuming that the binding of different chemical classes of ligands may occur to different domains of the BR [ 14].
GABA Receptor Binding Diazepam has been reported to increase 3H-GABA binding to crude synaptosomal membranes [8] and these observations have recently been extended [17]. Using 3Hmuscimol as ligand in binding studies performed at 23°C we also have been able to observe small increases of '~Hmuscimol binding by several BR agonists ([3], P. Supavilai and M. Karobath, unpublished results). More potent allosteric effects on aH-muscimol binding by various ligands for the BR can be seen when these investigations are performed in the presence of a CNS depressant like pentobarbital or etazolate (Fig. 4). In these investigations it has been found that BR agonists increase the stimulation of 3H-muscimol binding induced by etazolate, and that inverse BR agonists attenuate the effects of etazolate on 3H-muscimol binding (Fig. 4). The BR antagonist Ro 15-1788 does not markedly alter the actions of etazolate on 3H-muscimol binding but dose dependently antagonizes the effects o f a BR agonist and of an inverse BR agonist [3].
EFFECT OF BENZODIAZEPINE RECEPTOR LIGANDS
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cerebral cortex was investigated at 23°C using a centrifugation separation method [3]. 3H-muscimol binding was stimulated by 3 /zM etazolate and its perturbation by BR ligands was investigated. Data are expressed as % enhancement of 3H-muscimol binding by etazolate in the absence of other drugs. Three/.tM etazolate alone stimulates binding by 15(V~ (see dashed line). Ligands for the BR were added in concentrations which gave ~>80% BR occupancy in the assay conditions.
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Binding experiments were performed with membranes from rat cerebral cortex at 23°C in the presence of 200 mM NaC1 [20]. Data are expressed as % binding in the absence of drugs (dashed line). BR ligands were added in concentrations which gave ~>8(FA, BR occupancy in the assay conditions.
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FIG. 5. Perturbation o f 3~S-TBPS binding by ligands for the BR.
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FIG. 6. Perturbation of muscimol inhibition of 3sS-TBPS binding to membranes from rat cerebral cortex by ligands for the BR. Displayed is the ICs,, of muscimol [(without BR ligand)/(with BR ligand)]. BR ligands were added in concentrations which gave ~>8ff~ BR occupancy in the assay conditions.
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FIG. 7. Potentiation by BR agonists and attenuation by inverse BR agonists of the effect of muscimol on electrically induced acetylcholine release from rat striatum slices. The BR ligands were added in concentrations which give ~>80%. BR occupancy in the assay conditions. In the absence of muscimol these drugs did not alter electrically induced acetylcholine release [21]. The release of 3Hacetylcholine derived from prelabelling with 3H-choline was investigated and analyzed as described [21 ]. Muscimol at 2/zM gave 49% of the maximal inhibitory effect which is a 75% inhibition of electrically induced acetylcholine release. The results are mean values +S.E.M.; n~>4.
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Chloride Channel Antagonist Binding With 35S-TBPS as Ligand F|G. 8. Antagonism by Ro 15-1788 of the effects of clonazepam or DMCM on muscimol inhibition of electrically induced acetylcholine release from rat striatum slices. The inhibition of electrically induced acetylcholine release by 2 ~xM muscimol was 42%. This is indicated by the dotted horizontal bar. Shown is the potentiation of the action of muscimol by clonazepam and the attenuation of the action of muscimol by DMCM. Also shown is the antagonism of the modulatory action of these two drugs by Ro 15-1788, which alone is ineffective. The results are expressed as % inhibition of electrically induced acetylcholine release. The results are mean values -+S.E.M.: n=4-10. Ro: 0.5 /~M Ro 15-1788 added; C: 0.25 /zM clonazepam added; Ro+C: 0.5 ~M Ro 15-1788 and 0.25 k~M clonazepam added; D: 0.5 p.M DMCM added; Ro+D: 0.5 /~M Ro 15-1788 and 0.5/zM DMCM added.
'~sS-TBPS is a ligand i n t r o d u c e d by Squires et al. [18], w h i c h labels an a n t a g o n i s t binding site o f the chloride c h a n nel o f the G B R C [20]. BR a g o n i s t s i n c r e a s e , and i n v e r s e BR a g o n i s t s d e c r e a s e asS-TBPS binding (Fig. 5). The BR a n t a g o n i s t Ro 15-1788 w h i c h d o e s not alter 35S-TBPS binding by itself, h o w e v e r , b l o c k s the allosteric p e r t u r b a t i o n s o f a BR agonist and o f an i n v e r s e BR agonist [20]. Similar results w e r e also o b t a i n e d w h e n the inhibitory e f f e c t s o f m u s c i m o l on 35S-TBPS binding by BR ligands have b e e n i n v e s t i g a t e d [20] (Fig. 6). T a k e n t o g e t h e r , t h e s e results f r o m binding studies in vitro, with the p o s s i b l e e x c e p t i o n o f the photoaffinity labelling studies, s u p p o r t the initial c o n c e p t f o r m u l a t e d by Braestrup [6] that ligands for b e n z o d i a z e p i n e r e c e p t o r s can induce
674
KAROBATH
b i d i r e c t i o n a l allosteric effects o n the G B R C . W e t h e r e f o r e w e r e i n t e r e s t e d to i n v e s t i g a t e t h e s e drugs in a f u n c t i o n a l b i o c h e m i c a l in vitro m o d e l o f this r e c e p t o r . UP- AND DOWN-REGULATION OF THE GAIN OF A GBRC IN A FUNCTIONAL BIOCHEMICAL IN VITRO MODEL W e h a v e r e c e n t l y d e m o n s t r a t e d t h a t the electrically ind u c e d release o f a c e t y l c h o l i n e f r o m rat s t r i a t u m slices is m o d u l a t e d by a G A B A - A r e c e p t o r w h i c h h a s the c h a r a c teristics o f a G B R C . T h u s m u s c i m o l or G A B A , but not baclofen c a n inhibit electrically i n d u c e d a c e t y l c h o l i n e release f r o m t h e s e slices a n d this a c t i o n is s e n s i t i v e to inhibition by b i c u c u l l i n e , p i c r o t o x i n a n d the cage c o n v u l s a n t s [21]. In the p r e s e n c e o f c l o n a z e p a m , w h i c h has no a c t i o n s o n acetylc h o l i n e release, the c o n c e n t r a t i o n effect c u r v e o f m u s c i m o l is shifted to the left. F o r e x a m p l e , the c o n c e n t r a t i o n o f muscimol w h i c h yields 10% o f the m a x i m a l r e s p o n s e (ED~0) is shifted f r o m 0 . 2 7 / , M in t h e a b s e n c e o f c l o n a z e p a m to 0.03 ~ M in the p r e s e n c e o f 0.1 p,M c l o n a z e p a m . O n the o t h e r h a n d t h e partial i n v e r s e B R a g o n i s t / 3 C C M at 0.025 p,M shifts the ED~0 for m u s c i m o l f r o m 0.27/,~M to 0.8 p,M. f l C C M , in the a b s e n c e o f m u s c i m o l does not alter electrically e v o k e d acetylcholine release [21].
AND SUPAV|LAI
T h e s e r e s u l t s i n d i c a t e t h a t in this f u n c t i o n a l m o d e l a BR a g o n i s t i n c r e a s e s the efficacy o f m u s c i m o l to e x e r t its functional r e s p o n s e w h e r e a s the i n v e r s e B R agonist h a s the opposite effect. T h e a c t i o n o f a n u m b e r o f l i g a n d s for the B R on the effect o f 2 p,M m u s c i m o l , a c o n c e n t r a t i o n w h i c h leads to a p p r o x i m a t e l y 50% o f its m a x i m a l effect o n electrically ind u c e d a c e t y l c h o l i n e release is s h o w n in Fig. 7. It c a n also be s e e n that Ro 15-1788 w h i c h has w e a k a n d not significant agonistic a c t i o n s (Fig. 7), is able to a n t a g o n i z e the m o d u l a t ory effects o f c l o n a z e p a m a n d o f D M C M on m u s c i m o l inhibition o f electrically i n d u c e d a c e t y l c h o l i n e release f r o m rat s t r i a t u m slices (Fig. 8). T h e results with this m o d e l p r o v i d e direct s u p p o r t for the c o n c e p t p r o p o s e d initially by Haefely et al. [ 10] a n d C o s t a et al. [7] t h a t b e n z o d i a z e p i n e s , in p h a r m a c o l o g i c a l l y r e l e v a n t c o n c e n t r a t i o n , h a v e no intrinsic p h a r m a c o l o g i c a l effects but e n h a n c e G A B A e r g i c n e u r o t r a n s m i s s i o n . F u r t h e r , the o b s e r v a t i o n that B R a g o n i s t s e n h a n c e , a n d i n v e r s e BR a g o n i s t s a t t e n u a t e the e f f e c t i v e n e s s o f m u s c i m o l at the G B R C whilst the B R a n t a g o n i s t Ro 15-1788 is a l m o s t ineffective but b l o c k s the effect o f B R a g o n i s t s a n d i n v e r s e B R a g o n i s t s , a g r e e s with the c o n c e p t t h a t the i n t e r a c t i o n of ligands with the B R c a n lead to b i d i r e c t i o n a l f u n c t i o n a l effects o n G A B A e r g i c neurotransmission.
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12. Jensen, L. H., E. N. Petersen and C. Braestrup. Audiogenic seizures in DBA/2 mice discriminate sensitively between low efficacy benzodiazepine receptors agonists and inverse agonists. Li[? Sei 33: 393-399, 1983. 13. Karobath, M. and P. Supavilai. Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro. Neurosei Lett 31: 65-69, 1982. 14. Martin, 1. L., C. L. Brown and A. Doble. Multiple benzodiazepine receptors: structures in the brain or structures in the mind? A critical review. Lt:/~, Sci 32: 1925-1933, 1983. 15. Mohler. H., M. K. Battersby and J. G. Richards. Benzodiazepine receptor protein identified and visualized in brain tissue by a photoaffinity label. Proc Nail Aead Sci USA 77: 1666-1670, 1980. 16. Mohler, H. and J. G. Richards. Agonist and antagonist benzodiazepine receptor interaction in vitro. Nature 294: 763-765, 1981. 17. Skerritt, J. H., M. Willow and G. A. R. Johnston. Diazepam enhancement of low affinity GABA binding to rat brain membranes. Neurosei Lett 29: 63-66, 1982. 18. Squires, R. F., J. E. Casida, M. Richardson and E. Saederup. [asS]t-Butylbicyclophosphorothionate binds with high affinity to brain specific sites coupled to y-aminobutyric acid-A and ion recognition sites. Mol Pharmacal 13: 326-336, 1983. 19. Supavilai, P. and M. Karobath. The effect of temperature and chloride ions on the stimulation of [:~H]flunitrazepam binding by the muscimol analogues THIP and piperidine-4-sulfonic acid. Neurosei Lett 19: 337-341, 1980. 20. Supavilai, P. and M. Karobath. I:~='S]-t-Butyl-bicyclophos phorothionate binding sites are constituents of the yaminobutyric acid benzodiazepine receptor complex. .I Neurasci 4: 1193-1200, 1984. 21. Supavilai, P. and M. Karobath. Modulation of acetylcholine release from rat striatal slices by the GABA/benzodiazepine receptor complex. L~/k, Sei 36: 417-426, 1985.