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Influence of morphine dependence on GABA-stimulated benzodiazepine binding to mouse brain synaptic membranes
European Journal of Pharmacology, 79 (1982) 335-336
335
Elsevier Biomedical Press
Rapid communication INFLUENCE OF MORPHINE DEPENDENCE ON G A B A - S T I M U L A T E D B E N Z O D I A Z E P I N E BINDING T O M O U S E BRAIN SYNAPTIC M E M B R A N E S S U B B I A H P. SIVAM and I N G K. HO *
Department of Pharmacology and Toxicology, University of Mississsppi Medical Center, Jackson, MS 39216, U.S.A. Received 23 March 1982, accepted 24 March 1982
Several reports have indicated that 3,-aminobutyric acid (GABA) may be involved in opiate analgesia and tolerance/dependence (Ho et al., 1976; Sivam et al., 1981). Electrophysiological and biochemical evidence show that benzodiazepines (BDZ) exert their primary actions in the central nervous system by facilitation of GABAergic transmission; at the receptor level, BDZ binding sites are intimately associated with the GABA receptor-ionophore complex (Olsen, 1981). Thus, far, it has generally been believed that BDZ do not influence opiate mechanism. In the present report, we provide evidence that opiate mechanism may be linked to BDZ receptors via GABA receptors. Adult male ICR mice (TIMCO, Houston, TX) were used. Morphine tolerance was induced by a pellet implantation method (75 mg base s.c., for 72 h). Abrupt withdrawal was carried out by extricating the pellets after 72 h of implantation; the animals were allowed to remain for 24 h after the pellet removal. For acute treatment, a dose of morphine sulphate (20 mg/kg) was administered s.c. The animals were sacrificed at the appropriate time schedule and the whole brains were used for the study. Synaptic membranes were prepared, by differential centrifugation and discontinuous sucrose density gradient procedures, as described earlier (Sivam et al., 1981). [3H]Flunitrazepam ([3H]FNA: specific activity 83.6 Ci/mmol, New England Nuclear) bindings to the synaptic membranes were carried out by a filtration assay. Two-tenth ml of the membrane (0.2-0.3 mg protein) preparation was added to a mixture containing the required final concentration of [3H]FNA * To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
with or without other drugs, in a total incubation volume of 1.0 ml and incubated at 4°C for 20 min. The reaction was terminated by rapidly filtering through Whatman G F / B filters. They were then washed twice with 5 ml of 50 mM Tris-HC1 buffer (pH 7.4). The radioactivity was measured by a liquid scintillation system. Non-specific binding was determined in the presence of 1 0 - 6 M unlabelled diazepam. For saturation studies, a concentration range of 0.05-10 nM of [3H]FNA was used. For GABA stimulated [3H]FNA binding, a fixed concentration (0.3 nM) of the ligand was used. The results are the mean -+S.E. of at least three independent determinations each in triplicate. Student's t-test was used for statistical analysis. [3H]FNA binding to synaptic membranes exhibited saturation kinetics. Scatchard analysis of the data revealed a single population of receptors. Neither acute treatment of morphine, nor tolerance development, nor withdrawal affected [3H]FNA binding, as evidenced by the dissociation constant ( K o ) and maximum binding capacity (Bmax). The K o (riM) and Bma x (fmol/mg protein) for the various treatments were: acute control: 1.91-+0.18 and 787-+63, treated: 1.98-+0.2 and 709-+ 78; tolerant control: 1.83-+ 0.23 and 729-+ 81, treated: 1.93 -+ 0.41 and 847 -+ 43; withdrawal control: 2.09-+ 0.33 and 969-+ 92, treated: 1.83-+ 0.36 and 904-+ 53. Morphine or naloxone added in vitro upto 1 0 - a M failed to affect [3H]FNA binding as well as the GABAstimulated [3H]FNA binding. These data indicate that morphine treatments do not affect directly the BDZ binding. The addition of GABA or a potent GABA
336 TABLE 1 Effect of GABAergic compounds on [3H]flunitrazepam ([3H]FNA) to whole brain synaptic membranes obtained from control and morphine tolerant mouse. The binding was determined by a filtration assay as described in the text. Values represent mean -+S.E. The + and - signs denote increase and decrease from the basal binding respectively. Compounds
GABA (4X 10 6M) Muscimol (8 X 10-7 M) Bicuculline(8X 10 -6 M) Picrotoxin (2.4X 10-5 M) GABA + bicuculline Muscimol+ bicuculline GABA + picrotoxin Muscimol+ picrotoxin
% Change of basal [3H]FNA binding Control
Tolerant
+36+2.6 +38--+3.1 +4-+ 1.5 + 1±2.4 +2-+ 1.7 +4 + 1.3 + 35 -+2.4 +37--+3.2
+22+1.2 * +21-+1.3" --3--2.4 --2-+2.6 +5-+2.1 +6--2.3 + 20 +--2.1 * +23 + 1.8 *
* P
cuculline n o r picrotoxin in the c o n c e n t r a t i o n s used, affected the basal [ 3 H ] F N A binding. Bicuculline blocked the G A B A a n d muscimol stimulated [ 3 H ] F N A b i n d i n g , whereas picrotoxin failed to do so. These results indicate that the G A B A receptors a p p e a r to be bicuculline sensitive a n d picrotoxin insensitive. This further suggests that the G A B A receptors involved are postsynaptic a n d p r o b a b l y u n r e l a t e d to the chloride i o n o p h o r e linked to G A B A receptors. G A B A has been shown to stimulate B D Z b i n d ing a n d there is evidence that this process is mediated by specific recognition site, which involves a novel type of a G A B A receptor ( K a r o b a t h et al., 1979; U n n e r s t a l l et al., 1981). If this is indeed so, then one is t e m p t e d to suggest that this particular type of G A B A receptor might be affected b y tolerance development. The results taken together indicate that m o r p h i n e t o l e r a n c e / d e p e n d e n c e appears to affect B D Z b i n d i n g indirectly by a m e c h a n i s m involving G A B A receptors.
Acknowledgement This study was supported by a grant from NIDA, DA-01310.
References Ho, I.K., H.H. Loh and E.L. Way, 1976, Pharmacological manipulation of gamma-aminobutyric acid (GABA) in morphine analgesia, tolerance and physical dependence, Life Sci. 18, 1111. Karobath, M., P. Placheta, M. Lippitsch and P. KrogsgaardLarsen, 1979, Is stimulation of benzodiazepine receptor binding mediated by a novel GABA receptor, Nature 278, 748. Olsen, R.W., 1981, GABA-benzodiazepine-barbituratereceptor interactions, J. Neurochem. 37, I. Sivam, S.P., T. Nabeshima and I.K. Ho, 1981, Alterations of regional GABA receptors in morphine tolerant mice, Biochem. Pharmacol. 30, 2187. Unnerstall, J.R., M.J. Kuhar, D.L. Niehoff and J.N. Palacios, 1981, Benzodiazepinereceptors are coupled to a subpopulation of y-aminobutyric acid (GABA) receptors: Evidence from a quantitative autoradiographic study, J. Pharmacol. Exp. Ther. 218, 797.
335
Elsevier Biomedical Press
Rapid communication INFLUENCE OF MORPHINE DEPENDENCE ON G A B A - S T I M U L A T E D B E N Z O D I A Z E P I N E BINDING T O M O U S E BRAIN SYNAPTIC M E M B R A N E S S U B B I A H P. SIVAM and I N G K. HO *
Department of Pharmacology and Toxicology, University of Mississsppi Medical Center, Jackson, MS 39216, U.S.A. Received 23 March 1982, accepted 24 March 1982
Several reports have indicated that 3,-aminobutyric acid (GABA) may be involved in opiate analgesia and tolerance/dependence (Ho et al., 1976; Sivam et al., 1981). Electrophysiological and biochemical evidence show that benzodiazepines (BDZ) exert their primary actions in the central nervous system by facilitation of GABAergic transmission; at the receptor level, BDZ binding sites are intimately associated with the GABA receptor-ionophore complex (Olsen, 1981). Thus, far, it has generally been believed that BDZ do not influence opiate mechanism. In the present report, we provide evidence that opiate mechanism may be linked to BDZ receptors via GABA receptors. Adult male ICR mice (TIMCO, Houston, TX) were used. Morphine tolerance was induced by a pellet implantation method (75 mg base s.c., for 72 h). Abrupt withdrawal was carried out by extricating the pellets after 72 h of implantation; the animals were allowed to remain for 24 h after the pellet removal. For acute treatment, a dose of morphine sulphate (20 mg/kg) was administered s.c. The animals were sacrificed at the appropriate time schedule and the whole brains were used for the study. Synaptic membranes were prepared, by differential centrifugation and discontinuous sucrose density gradient procedures, as described earlier (Sivam et al., 1981). [3H]Flunitrazepam ([3H]FNA: specific activity 83.6 Ci/mmol, New England Nuclear) bindings to the synaptic membranes were carried out by a filtration assay. Two-tenth ml of the membrane (0.2-0.3 mg protein) preparation was added to a mixture containing the required final concentration of [3H]FNA * To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press
with or without other drugs, in a total incubation volume of 1.0 ml and incubated at 4°C for 20 min. The reaction was terminated by rapidly filtering through Whatman G F / B filters. They were then washed twice with 5 ml of 50 mM Tris-HC1 buffer (pH 7.4). The radioactivity was measured by a liquid scintillation system. Non-specific binding was determined in the presence of 1 0 - 6 M unlabelled diazepam. For saturation studies, a concentration range of 0.05-10 nM of [3H]FNA was used. For GABA stimulated [3H]FNA binding, a fixed concentration (0.3 nM) of the ligand was used. The results are the mean -+S.E. of at least three independent determinations each in triplicate. Student's t-test was used for statistical analysis. [3H]FNA binding to synaptic membranes exhibited saturation kinetics. Scatchard analysis of the data revealed a single population of receptors. Neither acute treatment of morphine, nor tolerance development, nor withdrawal affected [3H]FNA binding, as evidenced by the dissociation constant ( K o ) and maximum binding capacity (Bmax). The K o (riM) and Bma x (fmol/mg protein) for the various treatments were: acute control: 1.91-+0.18 and 787-+63, treated: 1.98-+0.2 and 709-+ 78; tolerant control: 1.83-+ 0.23 and 729-+ 81, treated: 1.93 -+ 0.41 and 847 -+ 43; withdrawal control: 2.09-+ 0.33 and 969-+ 92, treated: 1.83-+ 0.36 and 904-+ 53. Morphine or naloxone added in vitro upto 1 0 - a M failed to affect [3H]FNA binding as well as the GABAstimulated [3H]FNA binding. These data indicate that morphine treatments do not affect directly the BDZ binding. The addition of GABA or a potent GABA
336 TABLE 1 Effect of GABAergic compounds on [3H]flunitrazepam ([3H]FNA) to whole brain synaptic membranes obtained from control and morphine tolerant mouse. The binding was determined by a filtration assay as described in the text. Values represent mean -+S.E. The + and - signs denote increase and decrease from the basal binding respectively. Compounds
GABA (4X 10 6M) Muscimol (8 X 10-7 M) Bicuculline(8X 10 -6 M) Picrotoxin (2.4X 10-5 M) GABA + bicuculline Muscimol+ bicuculline GABA + picrotoxin Muscimol+ picrotoxin
% Change of basal [3H]FNA binding Control
Tolerant
+36+2.6 +38--+3.1 +4-+ 1.5 + 1±2.4 +2-+ 1.7 +4 + 1.3 + 35 -+2.4 +37--+3.2
+22+1.2 * +21-+1.3" --3--2.4 --2-+2.6 +5-+2.1 +6--2.3 + 20 +--2.1 * +23 + 1.8 *
* P
cuculline n o r picrotoxin in the c o n c e n t r a t i o n s used, affected the basal [ 3 H ] F N A binding. Bicuculline blocked the G A B A a n d muscimol stimulated [ 3 H ] F N A b i n d i n g , whereas picrotoxin failed to do so. These results indicate that the G A B A receptors a p p e a r to be bicuculline sensitive a n d picrotoxin insensitive. This further suggests that the G A B A receptors involved are postsynaptic a n d p r o b a b l y u n r e l a t e d to the chloride i o n o p h o r e linked to G A B A receptors. G A B A has been shown to stimulate B D Z b i n d ing a n d there is evidence that this process is mediated by specific recognition site, which involves a novel type of a G A B A receptor ( K a r o b a t h et al., 1979; U n n e r s t a l l et al., 1981). If this is indeed so, then one is t e m p t e d to suggest that this particular type of G A B A receptor might be affected b y tolerance development. The results taken together indicate that m o r p h i n e t o l e r a n c e / d e p e n d e n c e appears to affect B D Z b i n d i n g indirectly by a m e c h a n i s m involving G A B A receptors.
Acknowledgement This study was supported by a grant from NIDA, DA-01310.
References Ho, I.K., H.H. Loh and E.L. Way, 1976, Pharmacological manipulation of gamma-aminobutyric acid (GABA) in morphine analgesia, tolerance and physical dependence, Life Sci. 18, 1111. Karobath, M., P. Placheta, M. Lippitsch and P. KrogsgaardLarsen, 1979, Is stimulation of benzodiazepine receptor binding mediated by a novel GABA receptor, Nature 278, 748. Olsen, R.W., 1981, GABA-benzodiazepine-barbituratereceptor interactions, J. Neurochem. 37, I. Sivam, S.P., T. Nabeshima and I.K. Ho, 1981, Alterations of regional GABA receptors in morphine tolerant mice, Biochem. Pharmacol. 30, 2187. Unnerstall, J.R., M.J. Kuhar, D.L. Niehoff and J.N. Palacios, 1981, Benzodiazepinereceptors are coupled to a subpopulation of y-aminobutyric acid (GABA) receptors: Evidence from a quantitative autoradiographic study, J. Pharmacol. Exp. Ther. 218, 797.