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High-affinity 3H-imipramine binding in rat cerebral cortex
European Journal o f Pharmacology, 54 (1979) 307--308 © Elsevier/North-Holland Biomedical Press
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Rapid communication H I G H - A F F I N I T Y 3H-IMIPRAMINE BINDING IN RAT CEREBRAL CORTEX R. RAISMAN, M. BRILEY and S.Z. LANGER Synthdlabo, L.E.R.S., Department o f Biology, 58 rue de la Glacidre, 75013 Paris, France
Received 29 Januray 1979, accepted 30 January 1979 Tricyclic antidepressants are p o t e n t inhibitors of neuronal bioamine uptake (Glowinski and Axelrod, 1964). In addition they act directly upon muscarinic (Snyder and Yamamura, 1977) and histaminic (Green and Maayani, 1977) receptors. It is n o t clear, however, if these actions modulate the primary or secondary effects of these drugs. The tricyclic antidepressant, 3H-amitriptyline, was shown to bind predominantly to histamine H2 and muscarinic receptors (Rehavi and Sokolovsky, 1978) in rat brain. On the other hand low.affinity 3H-imipramine binding to rat brain synaptosomes (Kd = about 10 pM) has recently been demonstrated (O'Brien et al., 1978). In the present study we demonstrate the presence of specific high-affinity binding sites for 3H-imipramine in rat cerebral cortex. Male Sprague-Dawley rats ( 1 8 0 - - 2 5 0 g ) were decapitated and their brain immediately removed and dissected. The cortex was homogenised (Virtis 45 homogeniser) in 50 volumes of ice-cold buffer (50 mM Tris/ HC1, pH 7.4, 120 mM NaC1, 5 mM KC1) and centrifuged at 30,000 × g for 10 min. This washing procedure was repeated twice. The final pellet was resuspended in 33.3 volumes of the same buffer. 3H-imipramine binding was determined b y incubating aliquots of the membrane suspension (final concentration 30 mg original w e t weight/ml) with 3H-imipramine (29.8 Ci/ mmole, N.E.N. Chemicals) and other drugs, where appropriate, in a final volume of 250 pl for 60 min at 0°C. After incubation 100 pl of the incubation medium was then diluted
into 5 ml of ice-cold buffer and rapidly filtered under vacuum through Whatman G F / F glassfibre filters. The filters were washed with 3 × 5 ml ice-cold buffer, dried and the radioactivity counted in toluene, PPO (5 g/l}, POPOP (0.1 g/l). Specific binding was defined as that displaced b y 100 pM desipramine and represented 60% of the total binding at 5 nM 3H-imipramine. The specific binding of 3H-imipramine was saturable giving a linear Scatchard plot (r = 0.86) indicating a single population of binding sites. The apparent dissociation constant, Kd, was 6.8 + 1.0 nM (mean +- S.E.M., n = 4) with a maximal binding, Bmax, of 22.7 + 5.7 pmole/g tissue. The specific binding of 3H-imipramine was displaced by low concentrations of other tricyclic antidepressants such as amitryptiline (ICs0 15 nM), desipramine (ICs0 18 nM) and chlorimipramine (ICs0 25 nM). Atypical antidepressants displaced at much higher concentrations (iprindol, ICs0 5 . 5 p M ; mianserine, ICs0 2 0 p M ) . Noradrenaline displaced less than 30% at 100 pM whereas serotonin displaced 50% at 1 pM. Thus the specificity of this high-affinity 3H-imipramine binding does not appear to be identical to that of any receptor so far studied. It would therefore seem possible that this binding site for tricyclic antidepressants may be linked to their pharmacological site of action. The biological relevance of this binding site is demonstrated b y the observation that chronic treatment with desipramine results in a substantial decrease in 3H-imipramine binding in the rat cerebral cortex (Raisman, Briley
308
and Langer, unpublished observations). The study of high-affinity 3H-imipramine binding may thus offer a new approach to the study of depression and the search for new antidepressant drugs.
References Glowinski, J. and J. Axelrod, 1964, Inhibition of uptake of tritiated noradrenaline in the intact rat brain b y imipramine and related compounds, Nature 204, 1318.
Green, J.P. and S. Maayani, 1977, Tricyclic antidepressant drugs block histamine H2 receptor in brain, Nature 269, 163. O'Brien, R.A., N.M. Spirt and W.D. Horst, 1978, Antipressant receptor bindingin brain, Neurosci. Abstr. 4, 1366. Rehavi, M. and M. Sokolovsky, 1978, Multiple binding sites of tricyclic antidepressant drugs to mammalian brain receptors, Brain Res. 149, 525. Snyder, S.H. and H.I. Yamamura, 1977, Antidepressants and the muscarinic acetylcholine receptor, Arch. Gen. Psychiat. 34,236.
307
Rapid communication H I G H - A F F I N I T Y 3H-IMIPRAMINE BINDING IN RAT CEREBRAL CORTEX R. RAISMAN, M. BRILEY and S.Z. LANGER Synthdlabo, L.E.R.S., Department o f Biology, 58 rue de la Glacidre, 75013 Paris, France
Received 29 Januray 1979, accepted 30 January 1979 Tricyclic antidepressants are p o t e n t inhibitors of neuronal bioamine uptake (Glowinski and Axelrod, 1964). In addition they act directly upon muscarinic (Snyder and Yamamura, 1977) and histaminic (Green and Maayani, 1977) receptors. It is n o t clear, however, if these actions modulate the primary or secondary effects of these drugs. The tricyclic antidepressant, 3H-amitriptyline, was shown to bind predominantly to histamine H2 and muscarinic receptors (Rehavi and Sokolovsky, 1978) in rat brain. On the other hand low.affinity 3H-imipramine binding to rat brain synaptosomes (Kd = about 10 pM) has recently been demonstrated (O'Brien et al., 1978). In the present study we demonstrate the presence of specific high-affinity binding sites for 3H-imipramine in rat cerebral cortex. Male Sprague-Dawley rats ( 1 8 0 - - 2 5 0 g ) were decapitated and their brain immediately removed and dissected. The cortex was homogenised (Virtis 45 homogeniser) in 50 volumes of ice-cold buffer (50 mM Tris/ HC1, pH 7.4, 120 mM NaC1, 5 mM KC1) and centrifuged at 30,000 × g for 10 min. This washing procedure was repeated twice. The final pellet was resuspended in 33.3 volumes of the same buffer. 3H-imipramine binding was determined b y incubating aliquots of the membrane suspension (final concentration 30 mg original w e t weight/ml) with 3H-imipramine (29.8 Ci/ mmole, N.E.N. Chemicals) and other drugs, where appropriate, in a final volume of 250 pl for 60 min at 0°C. After incubation 100 pl of the incubation medium was then diluted
into 5 ml of ice-cold buffer and rapidly filtered under vacuum through Whatman G F / F glassfibre filters. The filters were washed with 3 × 5 ml ice-cold buffer, dried and the radioactivity counted in toluene, PPO (5 g/l}, POPOP (0.1 g/l). Specific binding was defined as that displaced b y 100 pM desipramine and represented 60% of the total binding at 5 nM 3H-imipramine. The specific binding of 3H-imipramine was saturable giving a linear Scatchard plot (r = 0.86) indicating a single population of binding sites. The apparent dissociation constant, Kd, was 6.8 + 1.0 nM (mean +- S.E.M., n = 4) with a maximal binding, Bmax, of 22.7 + 5.7 pmole/g tissue. The specific binding of 3H-imipramine was displaced by low concentrations of other tricyclic antidepressants such as amitryptiline (ICs0 15 nM), desipramine (ICs0 18 nM) and chlorimipramine (ICs0 25 nM). Atypical antidepressants displaced at much higher concentrations (iprindol, ICs0 5 . 5 p M ; mianserine, ICs0 2 0 p M ) . Noradrenaline displaced less than 30% at 100 pM whereas serotonin displaced 50% at 1 pM. Thus the specificity of this high-affinity 3H-imipramine binding does not appear to be identical to that of any receptor so far studied. It would therefore seem possible that this binding site for tricyclic antidepressants may be linked to their pharmacological site of action. The biological relevance of this binding site is demonstrated b y the observation that chronic treatment with desipramine results in a substantial decrease in 3H-imipramine binding in the rat cerebral cortex (Raisman, Briley
308
and Langer, unpublished observations). The study of high-affinity 3H-imipramine binding may thus offer a new approach to the study of depression and the search for new antidepressant drugs.
References Glowinski, J. and J. Axelrod, 1964, Inhibition of uptake of tritiated noradrenaline in the intact rat brain b y imipramine and related compounds, Nature 204, 1318.
Green, J.P. and S. Maayani, 1977, Tricyclic antidepressant drugs block histamine H2 receptor in brain, Nature 269, 163. O'Brien, R.A., N.M. Spirt and W.D. Horst, 1978, Antipressant receptor bindingin brain, Neurosci. Abstr. 4, 1366. Rehavi, M. and M. Sokolovsky, 1978, Multiple binding sites of tricyclic antidepressant drugs to mammalian brain receptors, Brain Res. 149, 525. Snyder, S.H. and H.I. Yamamura, 1977, Antidepressants and the muscarinic acetylcholine receptor, Arch. Gen. Psychiat. 34,236.