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Thip and isoguvacine are partial agonists of GABA-stimulated benzodiazepine receptor binding
4~5
European Journal of Pharmacology, 58 (1979) 485--488 © Elsevier/North-Holland Biomedical Press
Short communication THIP AND ISOGUVACINE ARE PARTIAL AGONISTS OF GABA-STIMULATED BENZODIAZEPINE RECEPTOR BINDING MANFRED KAROBATH * and MARGIT LIPPITSCH
Department of Biochemical Psychiatry, Psychiatrische Universita'tsklinik, University of Vienna, Austria Received 7 August 1979, accepted 9 August 1979
M. KAROBATH and M. LIPPITSCH, THIP and isoguvacine are partial agonists of GABA-stimulated benzodiazepine receptor binding, European J. Pharmacol. 58 (1979) 485--488. The effects of THIP and isoguvacine on 3H-flunitrazepam binding to washed membranes prepared from the cerebral cortex of adult rats have been examined. THIP, which has only minimal stimulatory effects on benzodiazepine (BZ) receptor binding, has been found to inhibit the stimulation induced by small concentrations (2 pM) of exogenous GABA. While isoguvacine stimulates BZ receptor binding, although to a smaller extent than GABA, it also antagonizes the stimulation of BZ receptor binding induced by GABA. Thus THIP and isoguvacine exhibit the properties of a partial agonist of GABA-stimulated BZ receptor binding. Benzodiazepine receptor Isoguvacine
Flunitrazepam Receptor interaction
GABA receptor THIP
i. Introduction
Benzodiazepine (BZ) receptor binding to synaptic membrane preparations can be modified by GABA agonists and antagonists in vitro and in vivo (Briley and Langer, 1978; Karobath and Sperk, 1979; Martin and Candy, 1979; Tallman et al., 1978, 1979). Thus GABA, muscimol and other structural analogues of GABA enhance the affinity of BZ receptors for their ligands, while bicuculline antagonizes this effect. However THIP (4,5,6,7-tetrahydroisoxazolo (4,5-c)-pyridin3-ol) and isoguvacine, two compounds with strong GABA agonistic effects in vivo, which also interfere with Na÷-independent GABA binding in vitro (Krogsgaard-Larsen et al., 1977) were found to have either no or only small effects on BZ receptor binding (Karobath et al., 1979; Maurer, 1979). We now * Correspondence to: Dr. M. Karobath, Psychiatrische Universit~itsklinik, Lazarettgasse 14, A-1090, Wien, Austria.
report that these two compounds are partial agonists of GABA-stimulated BZ receptor binding.
2. Materials and methods Rat brain cortex was homogenized in 20 vol of ice-cold 0.32 M sucrose. The homogenate was centrifuged for 10 min at 1000 × g and the supernatant was recentrifuged for 10 min at 30 000 X g. The membranes were resuspended in 30 vol 50 mM Tris--citrate buffer, pH 7.1, frozen, thawed, then washed at least five times by rehomogenization in 30 vol of the same buffer and recentrifugation for 10 min at 3 0 0 0 0 × g (Karobath and Sperk, 1979}. For the binding assay 0.1 ml membrane suspensions were incubated for 90 min at 0°C with 0.3 nM 3H-flunitrazepam (N-methyl-3H-flunitrazepam, New England Nuclear, 84.8 Ci/mmol) in 1 ml of an incubation medium which contained 150 mM NaC1, 50 mM Tris--citrate buffer, pH 7.1, and the
486
M. KAROBATH, M. LIPPITSCH
compounds to be tested. The samples were then filtered under vacuum through Whatman GF/B filters and immediately washed twice with 5 ml ice-cold buffer. Radioactivity on the filters was then determined by liquid scintillation counting. Non-specific binding determined in the presence of 3 pM unlabeled diazepam represented less than 5% of the total binding of 3H-flunitrazepam and was subtracted from total binding to give the specific binding.
in an approximately 10-fold higher concentration (fig. 1). THIP had weak agonistic effects and in several experiments, we observed maximal stimulation of BZ binding which was about 20% of that obtained with GABA or muscimol. However, when membrane preparations were examined where contaminating endogenous GABA was not sufficiently removed, then THIP, due to its antagonistic effects, had either no effect or could even inhibit BZ receptor binding {data not shown). Isoguvacine also exhibited properties of a partial agonist, since it inhibited GABA-stimulated BZ binding with a p o t e n c y similar to that of THIP to approximately those values which were obtained in the presence of high concentrations of isoguvacine alone (fig. 1). Isoguvacine had a stronger agonist potency than THIP and the maximal stimulation we observed was a b o u t 40% of that obtained with GABA or muscimol (fig. 1). Similar effects of THIP and isoguvacine were found in experiments where BZ binding was stimulated
3. Results THIP and isoguvacine had, compared to GABA and muscimol, rather weak effects as stimulators of 3H-flunitrazepam binding. However, THIP caused a concentrationdependent inhibition of BZ binding stimulated by low concentrations of GABA (2 pM) and this stimulatory effect of GABA was reduced by 50% when THIP was present i20 • GABA
iO0
0 Muscimol
" ~ ' ~ ~)
• THIP
• Isoguvacine
0 THIP + 2gM GABA
0 Isoguvacine + 2pM GABA
80 60
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40 20 0
I
I
I
I
I
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-7
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CONCENTRATION
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( log M)
Fig. 1. Effect of THIP, isoguvacine, G A B A and muscimol on H-flunitrazepam binding to washed membranes from rat cerebral cortex. Results are mean values (+S.E.M.) of at least triplicate incubations. The binding of 0.3 n M 3H-flunitrazepam to washed rat brain cortex membranes was determined in incubations (1 ml volume) which contained 50 m M Tris--citrate buffer, p H 7.1,150 m M NaCl and the compounds to be tested. Binding to membranes in the absence of drugs was 9.51 -+ 0.05 (n = 3) fmol/mg tissue. Ordinate: % increase of specifically bound [3H]flunitrazepam.
THIP AND ISOGUVACINE ON BENZODIAZEPINE BINDING
by low concentrations of muscimol (data not shown).
4. Discussion
In contrast to GABA, muscimol and other GABA analogues, THIP and isoguvacine have little effect as stimulators of 3H-diazepam binding (Karobath et al., 1979; Maurer, 1979), although these compounds have potent effects on Na÷-independent GABA binding (Krogsgaard-Larsen et al., 1977). These observations of apparent differences in the pharmacological properties of GABA stimulation of BZ binding and of Na÷-inde pendent GABA binding have led to the suggestion that these two sites might represent different types of GABA receptors (Karobath et al., 1979). However, the present experiments showed that THIP and isoguvacine do interact with the GABA receptor which is responsible for the stimulation of BZ binding. While THIP had barely detectable agonist effects on 3H-flunitrazepam binding, it antagonized the stimulation of BZ binding induced by low concentrations of GABA or muscimol. The agonistic effects of THIP were not found in all experiments, since contamination of the membrane fractions with small amounts of endogenous GABA may have masked this effect. Isoguvacine had stronger stimulatory effects on BZ binding b u t it also exhibited the properties of a partial agonist. The observation of the partial agonist effects of THIP and isoguvacine on GABAstimulated BZ binding was rather unexpected in view of the evidence for the potent direct agonist effects of these drugs on the postsynaptic GABA receptor. Thus THIP and isoguvacine interfere with Na÷-independent GABA binding (Krogsgaard-Larsen et al., 1977). They induce a bicuculline-sensitive depression of cat spinal neurons (KrogsgaardLarsen et al., 1977) and they induce contralateral bicuculline-sensitive turning behaviour after intra-nigral injection (Arnt et a1.,1979).
487
It has been proposed that this behavioural effect may be a sensitive and quantitative method for evaluation of GABA agonists in vivo (Arnt et al., 1979). The present results are difficult to interpret since the functional role of GABA stimulation of BZ binding is not known at present. At first sight the ,apparent discrepancy between the partial agonist properties of THIP and isoguvacine on GABA-stimulated BZ binding in vitro and their potent agonist effects in vivo seems to support our previous suggestion that GABA-stimulated BZ binding may represent a novel t y p e of GABA receptor (Karobath et al., 1979). On the other hand, THIP and isoguvacine can now be seen to have affinities approximately comparable to that of GABA for the GABA receptor which modifies BZ binding and for Na*-independent GABA binding. The latter binding sites, however do not allow any distinction to be made between agonists or antagonists. Thus, GABA stimulation of BZ binding and Na÷-indepen dent GABA binding exhibit similar affinities for these drugs and could represent the same GABA receptor. However this conclusion is hard to reconcile with the failure to observe partial agonist effects with THIP and isoguvacine in electrophysiological (KrogsgaardLarsen et al., 1977) and behavioural experiments in vivo (Arnt et al., 1979) and would require the assumption that the weak agonist properties of THIP were sufficient to yield the p o t e n t postsynaptic effects that have been observed with this c o m p o u n d in vivo. The hypothesis that GABA stimulation of BZ b!nding and Na+-independent GABA binding reflect the same GABA receptor is also difficult to reconcile with the fact that in cerebellum the ratio of the number of Na÷-inde pendent GABA binding sites over BZ receptors is approx. 3.5 times higher than in various other brain regions (Placheta and Karobath, 1979). Further studies are required to elucidate the functional role of GABA stimulation of BZ binding.
488
Acknowledgements We thank Dr. P. Krogsgaard-Larsen for the donation of THIP and isoguvacine. We also thank P. Placheta and L. Iversen for help in the preparation of this manuscript and J. Reisenhofer and N. Callahan for secretarial help. This work was supported by " F o n d s zur FSrderung der Wissenschaftlichen Forschung in Osterreich".
References Arnt, J., J. Scheel-Kriiger, G. Magelund and P. Krogsgaard-Larsen, 1979, Muscimol and related GABA receptor agonists: the potency of GABA-ergic drugs in vivo determined after intranigral injection, J. Pharm. Pharmacol. 31,306. Briley, M.S. and S.Z. Langer, 1978, Influence of GABA receptor agonists and antagonists on the binding of 3H-diazepam to the benzodiazepine receptors in the brain, European J. Pharmacol. 52, 124. Karobath, M. and G. Sperk, 1979, Stimulation of benzodiazepine receptor binding by 7-aminobutyric acid, Proc. Natl. Acad. Sci. USA 76, 1004.
M. KAROBATH, M. LIPPITSCH Karobath, M., P. Placheta, M. Lippitsch and P. Krogsgaard-Larsen, 1979, Is stimulation of benzodiazepine receptor binding mediated by a novel GABA receptor?, Nature 278, 748. Krogsgaard-Larsen, P., G.A.R. Johnston, D. Lodge and D.R. Curtis, 1977, A new class of GABA agonist, Nature, 268, 53. Martin, I.L. and J.M. Candy, 1978, Facilitation of benzodiazepine binding by sodium chloride and GABA, Neuropharmacology 17,993. Maurer, R., 1979, The GABA agonist THIP, a muscimol analogue, does not interfere with the benzodiazepine binding site on rats cortical membranes, Neurosci. Lett. 12, 65. Placheta, P. and M. Karobath, 1979, Regional distribution of Na÷-independent GABA and benzodiazepine binding sites in rat CNS, Brain Res. (in press). Tallman, J.F., J.W. Thomas and D.W. Gallager, 1978, GABA-ergic modulation of benzodiazepine binding site sensitivity, Nature 274,383. Taliman, J.F., J.W. Thomas and D.W. Gallager, 1979, Identification of diazepam binding in intact animals, Life Sci. 24,873.
European Journal of Pharmacology, 58 (1979) 485--488 © Elsevier/North-Holland Biomedical Press
Short communication THIP AND ISOGUVACINE ARE PARTIAL AGONISTS OF GABA-STIMULATED BENZODIAZEPINE RECEPTOR BINDING MANFRED KAROBATH * and MARGIT LIPPITSCH
Department of Biochemical Psychiatry, Psychiatrische Universita'tsklinik, University of Vienna, Austria Received 7 August 1979, accepted 9 August 1979
M. KAROBATH and M. LIPPITSCH, THIP and isoguvacine are partial agonists of GABA-stimulated benzodiazepine receptor binding, European J. Pharmacol. 58 (1979) 485--488. The effects of THIP and isoguvacine on 3H-flunitrazepam binding to washed membranes prepared from the cerebral cortex of adult rats have been examined. THIP, which has only minimal stimulatory effects on benzodiazepine (BZ) receptor binding, has been found to inhibit the stimulation induced by small concentrations (2 pM) of exogenous GABA. While isoguvacine stimulates BZ receptor binding, although to a smaller extent than GABA, it also antagonizes the stimulation of BZ receptor binding induced by GABA. Thus THIP and isoguvacine exhibit the properties of a partial agonist of GABA-stimulated BZ receptor binding. Benzodiazepine receptor Isoguvacine
Flunitrazepam Receptor interaction
GABA receptor THIP
i. Introduction
Benzodiazepine (BZ) receptor binding to synaptic membrane preparations can be modified by GABA agonists and antagonists in vitro and in vivo (Briley and Langer, 1978; Karobath and Sperk, 1979; Martin and Candy, 1979; Tallman et al., 1978, 1979). Thus GABA, muscimol and other structural analogues of GABA enhance the affinity of BZ receptors for their ligands, while bicuculline antagonizes this effect. However THIP (4,5,6,7-tetrahydroisoxazolo (4,5-c)-pyridin3-ol) and isoguvacine, two compounds with strong GABA agonistic effects in vivo, which also interfere with Na÷-independent GABA binding in vitro (Krogsgaard-Larsen et al., 1977) were found to have either no or only small effects on BZ receptor binding (Karobath et al., 1979; Maurer, 1979). We now * Correspondence to: Dr. M. Karobath, Psychiatrische Universit~itsklinik, Lazarettgasse 14, A-1090, Wien, Austria.
report that these two compounds are partial agonists of GABA-stimulated BZ receptor binding.
2. Materials and methods Rat brain cortex was homogenized in 20 vol of ice-cold 0.32 M sucrose. The homogenate was centrifuged for 10 min at 1000 × g and the supernatant was recentrifuged for 10 min at 30 000 X g. The membranes were resuspended in 30 vol 50 mM Tris--citrate buffer, pH 7.1, frozen, thawed, then washed at least five times by rehomogenization in 30 vol of the same buffer and recentrifugation for 10 min at 3 0 0 0 0 × g (Karobath and Sperk, 1979}. For the binding assay 0.1 ml membrane suspensions were incubated for 90 min at 0°C with 0.3 nM 3H-flunitrazepam (N-methyl-3H-flunitrazepam, New England Nuclear, 84.8 Ci/mmol) in 1 ml of an incubation medium which contained 150 mM NaC1, 50 mM Tris--citrate buffer, pH 7.1, and the
486
M. KAROBATH, M. LIPPITSCH
compounds to be tested. The samples were then filtered under vacuum through Whatman GF/B filters and immediately washed twice with 5 ml ice-cold buffer. Radioactivity on the filters was then determined by liquid scintillation counting. Non-specific binding determined in the presence of 3 pM unlabeled diazepam represented less than 5% of the total binding of 3H-flunitrazepam and was subtracted from total binding to give the specific binding.
in an approximately 10-fold higher concentration (fig. 1). THIP had weak agonistic effects and in several experiments, we observed maximal stimulation of BZ binding which was about 20% of that obtained with GABA or muscimol. However, when membrane preparations were examined where contaminating endogenous GABA was not sufficiently removed, then THIP, due to its antagonistic effects, had either no effect or could even inhibit BZ receptor binding {data not shown). Isoguvacine also exhibited properties of a partial agonist, since it inhibited GABA-stimulated BZ binding with a p o t e n c y similar to that of THIP to approximately those values which were obtained in the presence of high concentrations of isoguvacine alone (fig. 1). Isoguvacine had a stronger agonist potency than THIP and the maximal stimulation we observed was a b o u t 40% of that obtained with GABA or muscimol (fig. 1). Similar effects of THIP and isoguvacine were found in experiments where BZ binding was stimulated
3. Results THIP and isoguvacine had, compared to GABA and muscimol, rather weak effects as stimulators of 3H-flunitrazepam binding. However, THIP caused a concentrationdependent inhibition of BZ binding stimulated by low concentrations of GABA (2 pM) and this stimulatory effect of GABA was reduced by 50% when THIP was present i20 • GABA
iO0
0 Muscimol
" ~ ' ~ ~)
• THIP
• Isoguvacine
0 THIP + 2gM GABA
0 Isoguvacine + 2pM GABA
80 60
//
40 20 0
I
I
I
I
I
-8
-7
-6
-5
-4
i
I
"I
CONCENTRATION
i
I
I
I
I
I
i
( log M)
Fig. 1. Effect of THIP, isoguvacine, G A B A and muscimol on H-flunitrazepam binding to washed membranes from rat cerebral cortex. Results are mean values (+S.E.M.) of at least triplicate incubations. The binding of 0.3 n M 3H-flunitrazepam to washed rat brain cortex membranes was determined in incubations (1 ml volume) which contained 50 m M Tris--citrate buffer, p H 7.1,150 m M NaCl and the compounds to be tested. Binding to membranes in the absence of drugs was 9.51 -+ 0.05 (n = 3) fmol/mg tissue. Ordinate: % increase of specifically bound [3H]flunitrazepam.
THIP AND ISOGUVACINE ON BENZODIAZEPINE BINDING
by low concentrations of muscimol (data not shown).
4. Discussion
In contrast to GABA, muscimol and other GABA analogues, THIP and isoguvacine have little effect as stimulators of 3H-diazepam binding (Karobath et al., 1979; Maurer, 1979), although these compounds have potent effects on Na÷-independent GABA binding (Krogsgaard-Larsen et al., 1977). These observations of apparent differences in the pharmacological properties of GABA stimulation of BZ binding and of Na÷-inde pendent GABA binding have led to the suggestion that these two sites might represent different types of GABA receptors (Karobath et al., 1979). However, the present experiments showed that THIP and isoguvacine do interact with the GABA receptor which is responsible for the stimulation of BZ binding. While THIP had barely detectable agonist effects on 3H-flunitrazepam binding, it antagonized the stimulation of BZ binding induced by low concentrations of GABA or muscimol. The agonistic effects of THIP were not found in all experiments, since contamination of the membrane fractions with small amounts of endogenous GABA may have masked this effect. Isoguvacine had stronger stimulatory effects on BZ binding b u t it also exhibited the properties of a partial agonist. The observation of the partial agonist effects of THIP and isoguvacine on GABAstimulated BZ binding was rather unexpected in view of the evidence for the potent direct agonist effects of these drugs on the postsynaptic GABA receptor. Thus THIP and isoguvacine interfere with Na÷-independent GABA binding (Krogsgaard-Larsen et al., 1977). They induce a bicuculline-sensitive depression of cat spinal neurons (KrogsgaardLarsen et al., 1977) and they induce contralateral bicuculline-sensitive turning behaviour after intra-nigral injection (Arnt et a1.,1979).
487
It has been proposed that this behavioural effect may be a sensitive and quantitative method for evaluation of GABA agonists in vivo (Arnt et al., 1979). The present results are difficult to interpret since the functional role of GABA stimulation of BZ binding is not known at present. At first sight the ,apparent discrepancy between the partial agonist properties of THIP and isoguvacine on GABA-stimulated BZ binding in vitro and their potent agonist effects in vivo seems to support our previous suggestion that GABA-stimulated BZ binding may represent a novel t y p e of GABA receptor (Karobath et al., 1979). On the other hand, THIP and isoguvacine can now be seen to have affinities approximately comparable to that of GABA for the GABA receptor which modifies BZ binding and for Na*-independent GABA binding. The latter binding sites, however do not allow any distinction to be made between agonists or antagonists. Thus, GABA stimulation of BZ binding and Na÷-indepen dent GABA binding exhibit similar affinities for these drugs and could represent the same GABA receptor. However this conclusion is hard to reconcile with the failure to observe partial agonist effects with THIP and isoguvacine in electrophysiological (KrogsgaardLarsen et al., 1977) and behavioural experiments in vivo (Arnt et al., 1979) and would require the assumption that the weak agonist properties of THIP were sufficient to yield the p o t e n t postsynaptic effects that have been observed with this c o m p o u n d in vivo. The hypothesis that GABA stimulation of BZ b!nding and Na+-independent GABA binding reflect the same GABA receptor is also difficult to reconcile with the fact that in cerebellum the ratio of the number of Na÷-inde pendent GABA binding sites over BZ receptors is approx. 3.5 times higher than in various other brain regions (Placheta and Karobath, 1979). Further studies are required to elucidate the functional role of GABA stimulation of BZ binding.
488
Acknowledgements We thank Dr. P. Krogsgaard-Larsen for the donation of THIP and isoguvacine. We also thank P. Placheta and L. Iversen for help in the preparation of this manuscript and J. Reisenhofer and N. Callahan for secretarial help. This work was supported by " F o n d s zur FSrderung der Wissenschaftlichen Forschung in Osterreich".
References Arnt, J., J. Scheel-Kriiger, G. Magelund and P. Krogsgaard-Larsen, 1979, Muscimol and related GABA receptor agonists: the potency of GABA-ergic drugs in vivo determined after intranigral injection, J. Pharm. Pharmacol. 31,306. Briley, M.S. and S.Z. Langer, 1978, Influence of GABA receptor agonists and antagonists on the binding of 3H-diazepam to the benzodiazepine receptors in the brain, European J. Pharmacol. 52, 124. Karobath, M. and G. Sperk, 1979, Stimulation of benzodiazepine receptor binding by 7-aminobutyric acid, Proc. Natl. Acad. Sci. USA 76, 1004.
M. KAROBATH, M. LIPPITSCH Karobath, M., P. Placheta, M. Lippitsch and P. Krogsgaard-Larsen, 1979, Is stimulation of benzodiazepine receptor binding mediated by a novel GABA receptor?, Nature 278, 748. Krogsgaard-Larsen, P., G.A.R. Johnston, D. Lodge and D.R. Curtis, 1977, A new class of GABA agonist, Nature, 268, 53. Martin, I.L. and J.M. Candy, 1978, Facilitation of benzodiazepine binding by sodium chloride and GABA, Neuropharmacology 17,993. Maurer, R., 1979, The GABA agonist THIP, a muscimol analogue, does not interfere with the benzodiazepine binding site on rats cortical membranes, Neurosci. Lett. 12, 65. Placheta, P. and M. Karobath, 1979, Regional distribution of Na÷-independent GABA and benzodiazepine binding sites in rat CNS, Brain Res. (in press). Tallman, J.F., J.W. Thomas and D.W. Gallager, 1978, GABA-ergic modulation of benzodiazepine binding site sensitivity, Nature 274,383. Taliman, J.F., J.W. Thomas and D.W. Gallager, 1979, Identification of diazepam binding in intact animals, Life Sci. 24,873.