Neuroscience Letters, 12 (1979) 65--68 65 © Elsevier/North-Holland Scientific Publishers Ltd. THE GABA AGONIST THIP, A MUSCIMOL ANALOGUE, DOES NOT INTERFERE WITH THE BENZODIAZEPINE BINDING SITE ON RATS CORTICAL MEMBRANES RICHARD MAURER Biological and Medical Research Division, Sandoz Ltd., CH-4002 Basel (Switzerland) (Received November 3rd, 1978) (Revised version December 22nd, 1978) (Accepted December 22nd, 1978) SUMMARY THIP, a cyclic analogue of muscimol, is a powerful GABA agonist. It is as active as GABA in displacing [3H]muscimol from its binding site to cerebellar membranes (ICs0 = 31.5 + 2.5 raM). However, unlike muscimol or GABA, it is devoid of any modulatory interaction with the benzodiazepine binding site on rat's cortical membranes. Homotaurine, isoguvacine and imidazole acetic acid are less active than muscimol and GABA for increasing the affinity of [3H]dia- zepam to cortical membrane preparations. Biochemical studies failed for a long time to reveal any direct interaction between benzodiazepines and GABA at the receptor level. Recently, however, a modulatory role of GABA agonists and antagonists on the benzodiazepine binding site has been shown [7]. GABA and muscimol increase the affinity of [3H]diazepam for its binding sites on washed membranes of rat cortex, whereas (+)-bicuculline lowers the apparent affinity for [3H]diazepam. A notable exception is imidazole acetic acid, an electrophysiologically and biochemically characterized GABA agonist [1,8], which has no influence on the binding of [3H]diazepam to cortical membranes of the rat. Addition- ally the purification of a heat stable protein from rat brain has been described which seems to act both as a ligand for benzodiazepine receptors and as a modulator of GABA receptors [2]. 4,5,6,7-Tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP), a bicyclic muscimol analogue related to isoguvacine which has been described recently [3] as a member of a new class of GABA agonists, competes effectively for the [3H]GABA binding site (ICs0 = 2.6 + 0.5 uM) and depresses the firing of spinal interneurons, an effect which is antagonized by the GABA antagonist bicuculline. Furthermore, THIP does not affect GABA uptake or inhibit the actions of GAD and GABA-T [4]. 66 In the present paper we compare the potencies of different GABA agonists, and in particular those of THIP, to displace [3H] muscimol with their ability to interact with the benzodiazepine binding site on rat's cortical membranes. The [3H]diazepam-binding assay was performed essentially as described by Mohler et al. [5]. Cerebral cortex of male rats (Ivanovas, 350--400 g) was homogenized in 20 vols. of ice-cold 0.32 M sucrose with a Polytron homo- genizer (position 3 for 15 sec, total setting 10) and centrifuged at 1000 g for 10 min at 4°C. The supernatant was centrifuged for an additional 20 min at 20,000 g and the resulting pellet resuspended with the polytron in Tris--HC1 buffer (0.05 M, pH 7.4) containing 200 mM NaC1. To remove endogenous small molecular weight compound the suspension was dialysed at 0°C against the same buffer for 1--2 days. For [3H]muscimol binding studies rat cerebellar tissue was used. Except for a change in the buffer (Tris-citrate 0.05 M, pH 6.7) the preparation of cere- bellar membranes was identical to the methodology used for cortical mem- branes. Binding assay conditions were the same as described by Snodgrass [6]. [3H] Diazepam (39 Ci-mmo1-1) and [3H]muscimol (12.9 Ci • mol-l) were purchased from New England Nuclear, THIP was obtained through Lundbeck Pharamaceutical Company Denmark, isoguvacine was synthetised at Sandoz, all other compounds being commercially available. The different GABA agonists are listed in Table I in order of their potency in displacing [3H]muscimol from its binding site. Muscimol is the most active compound (ICso = 2.97 • 10 -9 M), followed by a group of compounds including GABA, homotaurine, isoguvacine and THIP, each of which is about 10 times less active than muscimol. Imidazole acetic acid, the weakest GABA agonist tested, is 40 times less active than muscimol. Although muscimol, THIP and isoguvacine are acknowledged to be strong GABA agonists, their effects on [3H]diazepam binding are very different from TABLE I COMPETITION BY GABA AGONISTS FOR SPECIFIC [3H]MUSCIMOL BINDING TO RAT CEREBELLAR MEMBRANE SUSPENSION Each value is the mean of the observed specifically bound ligand +- S.E.M. of 2 to 3 experiments; in each experiment assays were done in triplicate. Compound ICs0 (nM) Muscimol 2.97 ± 0.35 Homotaurine 18.0 -+ 1.0 Isoguvacine 20.5 -+ 1.5 GABA 21.5 ± 0.3 THIP 31.5 -+ 2.5 Imidazole acetic acid 118.0 ± 18.0 67 80- . f' MUSCIMOL -~ 60 / ~D ~- (I) N ' < 40 / / ~-m a / /--ISOGUVACINE ¢J z z ~ 20 i • i Iltl~ O I = ....... 1 ...... 5 ........ I ....... I ....... "1 - -7 -6 -5 -4 -3 CONCENTRATION(log M) Fig. 1. Concentration dependence of specific [ 3H]diazepam binding stimulation with muscimol, isoguvacine and THIP. All symbols represent the mean values of three indepen- dent experiments. TABLE II CHANGES IN [SH]DIAZEPAM BINDING INDUCED BY GABA AGONISTS All experiments were done with triplicate tubes and results of several experiments have been combined. Compound Structure Concentration Stimulation (#M) (% of control ± S.E.M.) Muscimol H2N~ 1 47.3 + 8.7 10 74.7 ± 8.1 GABA H2N ~--I ~H 101 33.7 + 4.3 58.3 + 7.1 Homotaurine H2N~__/--S%H 1 24.0 + 1.0 10 41.0 + 0.2 Isoguvacine H~: 1 0 10 27.0 + 0.2 OH Imidazole .~/--~o 1 0 acetic acid 10 12.0 + 1.6 THIP " 1 10 68 one another (Fig. 1). Whereas THIP is absolutely devoid of any modulating activity on [3H]diazapam binding up to concentrations of 3 • 10 -4 M, isoguva- cine and muscimol increase the affinity of [3H]diazepam binding at concen- trations as low as 10 -6 and 10 -s M respectively. All six GABA agonists were then tested at two different concentrations (10 -5 and 10 -6 M) for their effect on [3H]diazepam binding. Homotaurine increased the affinity of [3H]diaze- pam when used within a concentration range similar to that for GABA and muscimol. Imidazole acetic acid produced only a slight stimulation at 10 -s M and next to THIP was the weakest compound tested (Table II). These results clearly demonstrate that GABA agonists (GABA, homotaurine, isoguvacine, THIP) which are almost equiactive in terms of their ability to displace [3H]muscimol nevertheless display very different potencies for amplifying [3H]diazepam binding. The qualitatively different effects observed with the various substances investigated might be related to the position of the nitrogen atom within the molecule (Table II). GABA agonists like muscimol and homotaurine that have a free amino group modulate the affinity of the benzodiazepine binding site whereas agonists in which the N-atom (correspon- ding to the amino group of GABA) is incorporated into a ring structure show either a marked loss (isoguvacine and imidazole acetic acid} or an inability (THIP) to interact with the benzodiazepine binding site. ACKNOWLEDGEMENTS Thanks go to Drs. B.H. G~dlwiler and E. van Deusen for helpful discussions and assistance in the preparation of the manuscript. 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