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[3H]tifluadom binding in guinea-pig brain membranes
European Journal of Pharmacology, 97 (1984) 337-338 Elsevier
337
Rapid communication l aHITIFLUADOM BINDING IN GUINEA-PIG BRAIN MEMBRANES WILLY P. BURKARD
Pharmaceutical Research Department, F. Hoffmann-La Roche & Co., Ltd., CH-4002 Basle, Switzerland Received 12 December 1983, accepted 19 December 1983
Subclasses of opioid receptors can be distinguished e.g. by their membrane binding characteristics for various opioids in vitro. In such competition binding assays the x-agonists dynorphin and ethylketazocine were more potent in displacing [3H]ethylketazocine than in displacing typical/~- or g-opioid ligands like normorphine or [D-AlaE,D-LeuS]enkephalin (DADLE; Chavkin et al., 1981; Goodman and Snyder, 1982). Recently, the 1,4-benzodiazepine derivative tifluadom was shown to have the pharmacodynamic properties of an opiate x-receptor agonist but to be devoid of typical benzodiazepine-like activity (R6mer et al., 1982). We now report that the binding of [3H]tifluadom to guinea-pig brain membranes appears to be associated with great specificity to neuronal x-binding sites. The binding of [3H]tifluadom (spec. activity 14.5 Ci/mmol) was assayed under conditions similar to those utilized for dihydromorphine ([3H]DHM, spec. activity 35 Ci/mmol, 0.5 nM) or [3H]DADLE (spec. activity 30 Ci/mmol, 1.8 nM) binding (Gillian et al., 1980). Whole brain (without cerebellum) or various brain regions were homogenized in 100 vol ice-cold buffer (50 mM Tris-HC1, pH 7.4) by polytron and centrifuged 50 000 × g for 10 min. Pellets were rehomogenized, stirred at 37°C for 1 h and recentrifuged. The membrane preparation (1 ml) was incubated in Tris-HC1 buffer with [3H]tifluadom (0.4 nM) for 40 min at 25°C in a total volume of 2 ml. After incubation, the samples were filtered through Whatmari G F / B glass fibre filters under reduced pressure and washed once with 5 ml and once with 10 ml Tris-buffer. Specific binding was calculated by subtracting the nonspecific binding in the presence 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
of 10/~M bremazocine from the total bound radioactivity. This non-specific binding accounted for about 10% of the total binding. The specific binding of [3H]tifluadom to whole brain homogenate was linear over a range of 0.15-1 mg of protein and was time-dependent, reaching a plateau at about 30 min. Specific binding was saturable as measured in the range of 0.05-20 nM TABLE 1 Regional distribution and inhibition of [3H]tifluadom binding in brain. All experiments were performed in triplicate and replicated at least three times with similar results. Drugs were examined at 4 or more concentrations.
(a) Regional distribution Brain region
Specific binding in fmol/mg protein
Guinea pig Whole brain Striatum Frontal cortex Hypothalamus Cerebellum Midbrain Thalamus Brain stem Hippocampus Spinal cord Rat Whole brain
102 122 99 78 73 64 54 46 28 22 39
(b) Binding inhibitors Drug
Dynorphin-(1-13) DADLE Morphine
IC50 (nM) [3H] Tifluadom
[3H] DHM
[3H] DADLE
0.21 7 500 170
0.49 5.8 1.8
5.2 1.7 128
338
[3 H]tifluadom. Scatchard analysis indicated at least two components of binding with apparent dissociation constant K D = 0.43 nM and 5.6 nM with a maximal number of binding sites of 170 and 330 fmol/mg protein, respectively. Addition of 200 mM NaC1 decreased specific binding by more than 50%. The amount of specific tifluadom binding in guinea-pig brain was highest in striatum and frontal cortex and lowest in brain-stem and hippocampus (table 1). This distribution parallels roughly that described for benzomorphan receptors in the rat (Chang et al., 1981). The high specificity of [3H]tifluadom binding in comparison with [3H]DHM and [3H]DADLE binding was revealed by the inhibitory potencies of dynorphin, DADLE and morphine, respectively. While the latter two compounds showed relatively little difference in the IC50 values with [3H]DHM and [3H]DADLE binding, they yielded IC50 values which differed by more than 10000 fold for [3H]tifluadom binding (table 1). The ability of [3H]tifluadom to label sites associated with K-receptors provides a powerful tool to evaluate ~-receptor properties. Further studies with [3H]tifluadom may clarify the existence of the various classes of opiate receptor subpopulations.
Acknowledgements The author wishes to thank Dr. P.M. MOiler and Dr. N. FlOck for synthesizing [3H]tifluadom, as well as Mr. S. Henriot for his excellent technical assistance.
References Chang, K.-J., E. Hazum and P. Catrecasas, 1981, Novel opiate binding sites selective for benzomorphan drugs, Proc. Natl. Acad. Sci. U.S.A. 78, 4141. Chavkin, C., I.F. James and A. Goldstein, 1981, Dynorphin is a specific endogenous ligand of the K opioid receptor, Science 215, 413. Gillian, M.G.C., H.W. Kosterlitz and S.J. Patterson, 1980, Comparison of the binding characteristics of tritiated opiates and opioid peptides, Br. J. Pharmacol. 70, 481. Goodman, R.R. and S.H. Snyder, 1982, K Opiate receptors localized by autoradiography to deep layers of cerebral cortex: Relation to sedative effects, Proc. Natl. Acad. Sci. U.S.A. 79, 5703. ROmer, D., H.H. B0scher, R.C. Hill, R. Maurer, T.J. Petcher, H. Zeugner, W. Benson, E. Firmer, W. Milowski and P.W. Thies, 1982, Unexpected opioid activity in a known class of drug, Life Sci. 31, 1217.
337
Rapid communication l aHITIFLUADOM BINDING IN GUINEA-PIG BRAIN MEMBRANES WILLY P. BURKARD
Pharmaceutical Research Department, F. Hoffmann-La Roche & Co., Ltd., CH-4002 Basle, Switzerland Received 12 December 1983, accepted 19 December 1983
Subclasses of opioid receptors can be distinguished e.g. by their membrane binding characteristics for various opioids in vitro. In such competition binding assays the x-agonists dynorphin and ethylketazocine were more potent in displacing [3H]ethylketazocine than in displacing typical/~- or g-opioid ligands like normorphine or [D-AlaE,D-LeuS]enkephalin (DADLE; Chavkin et al., 1981; Goodman and Snyder, 1982). Recently, the 1,4-benzodiazepine derivative tifluadom was shown to have the pharmacodynamic properties of an opiate x-receptor agonist but to be devoid of typical benzodiazepine-like activity (R6mer et al., 1982). We now report that the binding of [3H]tifluadom to guinea-pig brain membranes appears to be associated with great specificity to neuronal x-binding sites. The binding of [3H]tifluadom (spec. activity 14.5 Ci/mmol) was assayed under conditions similar to those utilized for dihydromorphine ([3H]DHM, spec. activity 35 Ci/mmol, 0.5 nM) or [3H]DADLE (spec. activity 30 Ci/mmol, 1.8 nM) binding (Gillian et al., 1980). Whole brain (without cerebellum) or various brain regions were homogenized in 100 vol ice-cold buffer (50 mM Tris-HC1, pH 7.4) by polytron and centrifuged 50 000 × g for 10 min. Pellets were rehomogenized, stirred at 37°C for 1 h and recentrifuged. The membrane preparation (1 ml) was incubated in Tris-HC1 buffer with [3H]tifluadom (0.4 nM) for 40 min at 25°C in a total volume of 2 ml. After incubation, the samples were filtered through Whatmari G F / B glass fibre filters under reduced pressure and washed once with 5 ml and once with 10 ml Tris-buffer. Specific binding was calculated by subtracting the nonspecific binding in the presence 0014-2999/84/$03.00 © 1984 Elsevier Science Publishers B.V.
of 10/~M bremazocine from the total bound radioactivity. This non-specific binding accounted for about 10% of the total binding. The specific binding of [3H]tifluadom to whole brain homogenate was linear over a range of 0.15-1 mg of protein and was time-dependent, reaching a plateau at about 30 min. Specific binding was saturable as measured in the range of 0.05-20 nM TABLE 1 Regional distribution and inhibition of [3H]tifluadom binding in brain. All experiments were performed in triplicate and replicated at least three times with similar results. Drugs were examined at 4 or more concentrations.
(a) Regional distribution Brain region
Specific binding in fmol/mg protein
Guinea pig Whole brain Striatum Frontal cortex Hypothalamus Cerebellum Midbrain Thalamus Brain stem Hippocampus Spinal cord Rat Whole brain
102 122 99 78 73 64 54 46 28 22 39
(b) Binding inhibitors Drug
Dynorphin-(1-13) DADLE Morphine
IC50 (nM) [3H] Tifluadom
[3H] DHM
[3H] DADLE
0.21 7 500 170
0.49 5.8 1.8
5.2 1.7 128
338
[3 H]tifluadom. Scatchard analysis indicated at least two components of binding with apparent dissociation constant K D = 0.43 nM and 5.6 nM with a maximal number of binding sites of 170 and 330 fmol/mg protein, respectively. Addition of 200 mM NaC1 decreased specific binding by more than 50%. The amount of specific tifluadom binding in guinea-pig brain was highest in striatum and frontal cortex and lowest in brain-stem and hippocampus (table 1). This distribution parallels roughly that described for benzomorphan receptors in the rat (Chang et al., 1981). The high specificity of [3H]tifluadom binding in comparison with [3H]DHM and [3H]DADLE binding was revealed by the inhibitory potencies of dynorphin, DADLE and morphine, respectively. While the latter two compounds showed relatively little difference in the IC50 values with [3H]DHM and [3H]DADLE binding, they yielded IC50 values which differed by more than 10000 fold for [3H]tifluadom binding (table 1). The ability of [3H]tifluadom to label sites associated with K-receptors provides a powerful tool to evaluate ~-receptor properties. Further studies with [3H]tifluadom may clarify the existence of the various classes of opiate receptor subpopulations.
Acknowledgements The author wishes to thank Dr. P.M. MOiler and Dr. N. FlOck for synthesizing [3H]tifluadom, as well as Mr. S. Henriot for his excellent technical assistance.
References Chang, K.-J., E. Hazum and P. Catrecasas, 1981, Novel opiate binding sites selective for benzomorphan drugs, Proc. Natl. Acad. Sci. U.S.A. 78, 4141. Chavkin, C., I.F. James and A. Goldstein, 1981, Dynorphin is a specific endogenous ligand of the K opioid receptor, Science 215, 413. Gillian, M.G.C., H.W. Kosterlitz and S.J. Patterson, 1980, Comparison of the binding characteristics of tritiated opiates and opioid peptides, Br. J. Pharmacol. 70, 481. Goodman, R.R. and S.H. Snyder, 1982, K Opiate receptors localized by autoradiography to deep layers of cerebral cortex: Relation to sedative effects, Proc. Natl. Acad. Sci. U.S.A. 79, 5703. ROmer, D., H.H. B0scher, R.C. Hill, R. Maurer, T.J. Petcher, H. Zeugner, W. Benson, E. Firmer, W. Milowski and P.W. Thies, 1982, Unexpected opioid activity in a known class of drug, Life Sci. 31, 1217.