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(−)-deprenyl a selective mao ‘B’ inhibitor increases [3H]imipramine binding and decreases β-adrenergic receptor function
European Journal of Pharmacology, 89 (1983) 111-117
111
Elsevier Biomedical Press
( - ) - D E P R E N Y L A SELECTIVE MAO 'B' INHIBITOR INCREASES [3H]IMIPRAMINE BINDING AND DECREASES fl-ADRENERGIC RECEPTOR FUNCTION G A B R I E L L A ZSILLA **, M A R I A L. B A R B A C C I A *, OTTAVIO G A N D O L F I *, JOSEPH K N O L L ** and E R M I N I O COSTA *.t
* Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032, U.S.A., and ** Department of Pharmacology, Semmelweis University of Medicine, Budapest, Hungary Received 5 October 1982, revised MS received 3 January 1983, accepted 18 January 1983
G. ZSILLA, M.L. BARBACCIA, O. GANDOLFI, J. KNOLL and E. COSTA, (-)-Deprenyl a selective MAO 'B" inhibitor increases [3H]irnipramine binding and decreases fl-adrenergic receptor function, European J. Pharmacol. 89 (1983) 111-117. In rats, a seletive inhibition for 3 weeks of monoamineoxydase (MAO) type B elicited by daily doses of pargyline (2.5 pmol/kg) or (-)-deprenyl (1 pmol/kg) attenuated the NE dependent stimulation of cortical adenylate cyclase and reduced the number of brain recognition sites for fl-adrenergic receptor ligands. Similar actions were not elicited by a comparable dose regimen of (+)-amphetamine. Hence the inhibition of MAO B mimicks responses that are typically elicited by antidepressants. The molecular nature of the mechanisms involved cannot be understood, however, these mechanisms may not be identical for pargyline and (-)-deprenyl because this drug but not pargyline increased the number of [3H]imipramine recognition sites. Even high daily doses of pargyline (I00 #mol/kg, for 3 weeks) failed to change [3H]imipramine binding though they still down regulated fl-adrenergic recognition sites, the NE stimulation of adenylate cyclase and the Bmax of [3H]mianserin and [3H]spiroperidol binding. 5HT receptors
fl-Adrenergic receptors
Deprenyl
1. Introduction
Several lines of independent investigations indicate that typical and atypical antidepressants affect brain function by down regulating the binding sites for ligands of noradrenergic (Sulser et al., 1978; Kinnier et al., 1980; Brunello et al., 1982a) and serotonergic (5HT2) recognition sites (Peroutka and Snyder, 1980a, b; Blackshear and Sanders-Bush, 1982). Ultimately, antidepressants decrease the stimulus amplification elicited by norepinephrine (NE) on the adenylate cyclase coupled to fl-adrenergic receptors in rat brain (Vetulani and Sulser, 1975; Vetulani et al., 1976; Mishra et al., 1980; BruneUo et al., 1982a). The discovery that high affinity recognition sites for
* To whom all correspondence should be addressed.
MAO B
Adenylate cyclase
NE
Imipramine
antidepressant drugs are present in crude synaptic membranes (Raisman et al., 1979; Dumbrille-Ross et al., 1980; Rehavi et al., 1980; Palkovits et al., 1981; Peroutka and Snyder, 1981) and that their number can be modulated by treatment with antidepressants or by selective lesion of serotonergic neurons (Kinnier et al., 1980; Brunello et al., 1982b, c) has given us an additional dimension to study, at the molecular level, the modifications of brain function that mediate the beneficial effects of antidepressants. At least two kinds of antidepressant recognition sites are present in brain (Raisman et al., 1979; Dumbrille-Ross et al., 1980; Palkovits et al., 1981), which differ for anatomical location and effector specificity. One is located preferentially on 5HT axon terminals (Grob et al., 1981; Sette et al., 1981; Brunello et al., 1982c) and has the highest affinity for [3H]imipramine and the other is located postsynaptically to 5HT axon
112
terminals and has the highest affinity for [3H]mianserin (BruneUo et al., 1982c). Though we do not know which is the physiological effector for these two sites we suspect that the effector of the [3H]imipramine binding sites may be involved in the 5HT uptake regulation (Barbaccia et al., 1983) while that for the [3H]mianserin binding sites may function in 5HT receptor modulation (DumbrilleRoss et al., 1981; Brunello et al., 1982c; Barbaccia et al., 1983); both sites appear to be different from postsynaptic 5HT recognition sites (Barbaccia et al., 1983). The inhibition of monoamine oxidase (MAO) Type A relieves the symptoms of depression (Quitkin et al., 1979), down regulates serotonergic 5HT2 recognition sites (Peroutka and Snyder, 1980a) and noradrenergic receptor function. In this respect MAO inhibitors Type A resemble tricyclic antidepressants (Sulser et al., 1978). It is debated whether the selective inhibition of MAO Type B relieves the symptoms of depression (Mann and Gershon, 1980; Knoll, 1981a; Fowler et al., 1982; Knoll, 1982). The present report deals with experiments directed to ascertain whether (-)-deprenyl, a selective MAO Type B inhibitor (Knoll, 1981b) which was previously reported to affect dopaminergic turnover in rat brain striatum (Zsilla and Knoll, 1982), decreases the NE mediated signal amplification of adenylate cyclase or modifies the binding sites for typical and atypical antidepressants. The action of ( - )-deprenyl will be compared to that of pargyline which, in low doses also selectively inhibits MAO B (Neff and Fuentes, 1976).
pared for radioreceptor binding studies. [3H]Imipramine binding assays were carried out according to Kinnier et al. (1980). The nonspecific binding was determined by incubating the samples in presence of 10 #M desmethylimipramine. The crude synaptic membranes suspension ( - 10 mg of tissue wet weight/ml) was incubated with different concentrations of [3H]imipramine in a range between 0.5 and 10 nM. [3H]Mianserin binding assays were performed according to Dumbrille-Ross et al. (1981). The nonspecific binding was defined by carrying out the assay in presence of 10 #M cold mianserin. The so-called 5HT2 recognition sites, labeled by [3H]spiroperidol were studied according to the procedures described by Peroutka and Snyder (1980b). The nonspecific binding was defined by incubating the samples in presence of 1 #M ketanserin (Leysen et al., 1982) or 10 #M LSD. The fl-adrenergic recognition sites were studied according to the procedures described by Bylund and Snyder (1976) using [ 3H]dihydroalprenolol ([3H]DHA) as the specific ligand. The nonspecific binding was defined in presence of 1 #M dihydroalprenolol. Modification of binding sites characteristics were analyzed according to the method of Scatchard (1949). The basal cyclic AMP accumulation and that elicited by NE was measured in the incubation medium of minces (300 by 300 #m) prepared from fresh frontal cortices, according to the procedures described by Blumberg et al. (1976). The protein concentration was measured according to the procedures described by Lowry et al. (1951) using bovine serum albumin as a standard. Student's t-test was used to analyze the data.
2. Materials and methods 3. Results
Male Sprague-Dawley rats from Zivic-Miller (150 g) were used in all the experiments. The animals were daily injected s.c. with 1 #mol/kg of either (-)-deprenyl or (+)-amphetamine and either with 2.5 or 100 pmol/kg i.p., of pargyline for 21 days. The rats were decapitated 24-36 h after the last drug injection. After the brain was rapidly extracted from the skulls, the brain structures were dissected on ice and kept frozen at - 7 0 ° C until crude synaptic membranes were pre-
Daily injections of (-)-deprenyl (1 #mol/kg s.c.) repeated during three weeks changed the kinetic characteristics of the [3H]imipramine recognition sites located in crude synaptic membranes prepared from rat frontal cortex and hippocampus. In the hippocampus, as shown by the Bmax value (table 1), the number of [3Hlimipramine recognition sites was increased without any significant change in the apparent K D value. The
113 TABLE 1 Kinetic characteristics of [ 3H]imipramine specific binding to crude synaptic membranes prepared from cortex or hippocampus of rats repeatedly injected with ( - )-deprenyl, ( + )-amphetamine and pargyline. Each Scatchard analysis was performed by plotting the data obtained in binding assays with 8 different concentrations of ligand (from 0.5 to 10 nM); each experiment was repeated at least three times. Treatment
Cortical membranes
Hippocampal membranes
KD
KD (nM)
Bmax ( f m o l / m g prot)
Bmax ( f m o l / m g prot)
(nM) Saline ( - )-Deprenyl ( 1 / z m o l / k g s.c., 21 days) ( + )-Amphetamine (1/~ m o t / k g s.c., 21 days) Pargyline (2.5/~mol/kg i.p. 21 days) Pargyline (100/~ m o l / k g i.p., 21 days)
5.3 + 0.7
650 + 100
5.2 + 0.7
560 + 50
8.2 + 1.5
1065 + 150 a
7.0 + 0.5
800 + 90 a
4.5 + 0.6
720 + 90
5.1 +0.5
690-t- 80
6.9 + 0.5
650 + 85
a p < 0.01 when compared to values obtained in saline-treated rats.
Table 1 also shows that (+)-amphetamine, a structural analogue and a possible metabolite of deprenyl, given daily for three weeks at a dose regimen similar to that of (-)-deprenyl, failed to
increase in Bmax was greater in cortex than in hippocampus; in fig. 1 is represented a typical Scatchard plot obtained by the analysis of [3H]imipramine binding data from cortical membranes.
!,000
J
if"
A
300t {
'~.
=
E
-
\,
°
200 -- ~
O
"~
20o [ ~ .~
,00
,t
.
_
/
e~
=
"~
~
0
,o
[~H]-imipromine (nM)
• ~ ~ •
I 500
Deprenyl
KD=6"SnM
n• Bmax --~...~mlaOxo0,mo, / mg protein
",J 1000
I "-,. 1500
I 2000
Bound (fmol/rag protein) Fig. 1. Repeated injections of ( - ) - d e p r e n y l increase the Bmax of [3H]imipramine specific binding in crude synaptic membranes prepared from rat frontal cortex. ( - ) - D e p r e n y l was injected s.c. for 21 days at the dose of 1/Lmol/kg. The animals were killed 2 4 - 3 6 h after the last injection. This graph is representative of one experiment done in triplicate. The difference in the Bmax value for [ 3H]imipramine specific binding between rats treated with ( - )-deprenyl or saline was significant (P < 0.01). A similar experiment was repeated three times, and similar results were obtained.
114 TABLE 2 Kinetic characteristics of [3H]mianserin and [3H]spiroperidol specific binding to crude synaptic membranes prepared from brain cortex of rats repeatedly injected with ( - ) - d e p r e n y l o r pargyline. [3HlMianserin and [3H]spiroperidol specific bindings were calculated by subtracting from the total binding the values obtained in presence of l0 # M cold mianserin and 10 /~M LSD, respectively. Each value represents the mean + S.E.M. of at least six separate determinations. Treatment
....
[ 3H]Mianserin
Saline ( - )-Deprenyl (i # m o l / k g s.c., 21 days) Pargyline (2.5 # mol/kg i.p., 21 days) Pargyline (100/~mol/kg i.p., 21 days)
[ 3H]Spiroperidol
Ko (nM)
Bma× ( f m o l / m g prot)
KD (nM)
Braax (fmol/mg prot)
2.0 ± 0.5
281 ± 47
0.42 ± 0.12
98 _+ 16
2.2+0.3
270+40
0.51 ±0.11
87± 13
1.8 + 0.3
264 4- 30
0.70 ± 0.2
101 ± 15
3.7 ± 0.7
195 + 15 a
0.80 ± 0.31
59 ± 8 ~
a p < 0.05 when compared to values obtained in saline treated rats.
induce any change in the characteristics of [ 3 H ] imipramine binding to cortical crude synaptic membranes. Pargyline; given repeatedly for three weeks at two different doses, failed to change [3H]imipramine binding. Table 1 shows that in rats receiving either 2.5 #mol/kg, of pargyline to inhibit MAO B selectively or 100 /tmol/kg, of pargyline to block MAO A and B (Neff and Fuentes, 1976), neither the Bmax nor the K o of [3H]imipramine binding is changed. The specific binding of [3H]mianserin, an atypical antidepressant, measured in crude synaptic membranes
prepared from cortices of rats receiving repeated (-)-deprenyl injections as described above was not modified (table 2). Moreover, in the same membrane preparations we could not detect any changes in 5HT2 recognition sites (table 2). The 5HT 2 recognition sites remained unchanged also after daily doses of pargyline (2.5 /~mol/kg) repeated for 3 weeks. In contrast, with higher doses of pargyline (100 #mol/kg) that block MAO Type A and B the Bmax of both [3H]mianserin and 5HT 2 binding sites was reduced (table 2). Since (-)-deprenyl attenuates the symptoms of
TABLE 3 NE-stimulated cAMP accumulation in rat brain cortical slices and [3H]dihydroalprenolol ([3H]DHA) specific binding to crude synaptic membranes prepared from cortex of rats repeatedly injected with ( - )-deprenyl or pargyline, n = number of animals used for the determination of the basal and NE-stimulated cAMP accumulation. The assay was carried out separately for each animal, always in triplicate. Treatment
(n)
cAMP accumulation ( p m o l / m g prot per 15 min) Basal
Saline ( -- )-Deprenyl (l # m o l / k g s.c., 21 days) Pargyline (2.5/~mol/kg i.p., 21 days) Pargyline (100/~mol/kg i.p., 21 days)
NE stimulation (% increase)
+ 50 i~M N E
[3H]DHA spec. binding KD Bmax (nM)
( f m o l / m g prot)
(18)
8.4+0.4
15.8+0.8 b
88 b
2.9_+0.4
200+ 5
(8)
8.0+0.5
10.3-1-0.6 a
29 a
2.9-t-0.5
149_+ 15 a
(5)
12.1 +2.5
15.3±1.3 ~
26 a
2.0+0.5
150±13 ~
(5)
11.1 + 1.5
14.4 +0.9 a
30 a
2.7 ±0.3
160 ± 10 a
a p < 0.05 when compared to saline treated animals. b p < 0.01 when compared to basal values.
115 depression (Knoll, 1981a), we studied whether similarly to other antidepressants it could change the number of fl-adrenergic recognition sites and the signal amplification elicited by NE on adenylate cyclase. As shown in table 3, the stimulation of cyclic AMP accumulation elicited by NE in cortical slices prepared from rats receiving daily injections of (-)-deprenyl (1 #mol/kg s.c.) for three weeks was reduced. This reduction is similar to that observed in slices from cortices of rats receiving daily injections of pargyline of either 2.5 or 100 #mol/kg i.p., for three weeks. As shown in table 3, pargyline, no matter which dose was used, tends to increase the basal cAMP accumulation in cortical slices. Table 3 also indicates that the desensitization of the cAMP generating system toward NE stimulation elicited by (-)-deprenyl and pargyline either 2.5 or 100 #mol/kg was related to a decrease in number of fl-adrenergic recognition sites. All the treatments, in fact, elicited a decrease in the number of [3H]DHA specific binding sites by about 25%.
4. Discussion
A small dose of (-)-deprenyl (1 #mol/kg s.c.) or of pargyline (2.5/~mol/kg i.p.) given daily for three weeks was reported to inhibit by 80% or better the activity of MAO Type B while the activity of MAO Type A remains substantially unchanged (Neff and Fuentes, 1976; Campbell et al., 1979; Ekstedt et al., 1979). In contrast, 100 #mol/kg i.p. of pargyline given daily for three weeks causes an almost complete inhibition in the activity of both Type A and B enzyme (Campbell et al., 1979; Neff and Fuentes, 1976). Repeated (-)-deprenyl injections (1 #mol/kg) but not repeated pargyline injections (2.5 or 100 #mol/kg increase the Bin,x of [3H]imipramine binding to membranes of cortex and hippocampus. This difference can be explained by assuming that the action of (-)-deprenyl on [3H]imipramine binding is independent from the inhibition of MAO A and B or from the selective inhibition of MAO Type B, because even 2.5 #mol/kg i.p. of pargyline given daily for 3 weeks, which like (-)-deprenyl selectively inhibits MAO Type B (Neff and
Fuentes, 1976; Campbell et al., 1979) fails to change the binding of [3H]imipramine. From the experiments reported in table 1 we can exclude that the increase in Bin,x of [3H]imipramine binding is due to the accumulation of amphetamine which is formed as a metabolite of (-)-deprenyl. To evaluate the specificity of the modification of the recognition sites for [3H]imipramine elicited by (-)-deprenyl we have investigated whether (-)-deprenyl and both doses of pargyline can modify the characteristics of the recognition sites for [3H]mianserin or for [3H]spiroperidol, two sites that have been considered related to the function of 5HT2 receptors (Peroutka and Snyder, 1980a, b; 1981). The results obtained indicate that only pargyline (100 #mol/kg) reduces the Bma x of [3H]mianserin and of [3H]spiroperidol binding. This ligand labels many recognition sites in addition to 5HT2, including those for dopamine (Kebabian and Calne, 1979). However, since we used LSD to displace the specifically bound [3H]spiroperidol, we are measuring 5HT2 recognition sites, selectively. At present it is not understood whether [3H]mianserin and [3H]spiroperidol bind to the same 5HT2 receptors because the destruction of 5HT axons increased the Bmax of [3H]mianserin binding measured in presence or in absence of mepyzamine (Brunello et al., 1982c) without changing that of 5HT2 recognition sites (Barbaccia et al., 1983). This indicates that despite the similarity in the behavior of [3H]mianserin and [3H]spiroperidol binding shown in table 2, the two recognition sites cannot be considered to be identical. Since pargyline (2.5 #mol/kg) and (-)-deprenyl (1 #mol/kg) affect the recognition sites for [3H]imipramine in a completely different fashion, we have investigated whether they modify the fladrenergic receptor in a similar fashion. The data reported in table 3 show that small doses of pargyline and (-)-deprenyl despite the different action on the recognition sites for [3H]imipramine down regulate the recognition sites for fl-adrenergic ligands and decrease the signal amplification elicited by NE on the adenylate cyclase activity. Since the presence of 5HT axons is an absolute requirement for the desensitization of fl-adrenergic receptors caused by repeated daily injections of imipramine and DMI (Brunello et al., 1982a) but
116
not for that caused by a similar treatment with mianserin (Barbaccia et al., 1983) we could assume that small doses of (-)-deprenyl or pargyline act on 5HT axons by a mechanism dependent on MAO type B inhibition. Indeed the action of (-)-deprenyl and pargyline is not identical because of the difference in their action on [3H]imipramine recognition sites which are located on 5HT axons. This difference cannot be explained at this time; perhaps it may be clarified when the endogenous effector for these sites is known. In contrast, high doses of pargyline affect additional brain mechanisms as revealed by the down regulation of [3H]mianserin and [3H]spiroperidol binding sites; for instance these high doses of pargyline could down regulate fl-adrenergic recognition sites and the signal amplification of NE receptors by increasing the amount of NE reaching NE recognition sites. The question then arises as to whether the [3H]imipramine binding reflects the recognition site for an endogenous ligand which might be stored either in 5HT neurons or in another axon innervating the 5HT axons and thereby modulating the 5HT uptake system (Barbaccia et al., 1983). So far our results are compatible with the view that (-)-deprenyl may cause an inhibition of the release of this hypothetical effector and thereby cause supersensitivity of its recognition sites.
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tion of typical and atypical antidepressant recognition sites in rat brain, in: Typical and Atypical Antidepressants from Molecular Mechanisms to Clinical Practice, eds. E. Costa and G. Racagni (Raven Press, New York) p. 179. Brunello, N., D.-M. Chuang and E. Costa, 1982c, Different synaptic location of mianserin and imipramine binding sites, Science 215, 1112. Bylund, D.B. and S.H. Snyder, 1976, Beta adrenergic receptor binding in membrane preparations from mammalian brain, Mol. Pharmacol. 12, 568. Campbell, I.C., D.L. Murphy, D.W. Gallager and J.F. Tallman, 1979, Neurotransmitter-related adaptation in the central nervous system following chronic monoamine oxidase inhibition, in: Monoamine Oxidase: Structure, Function and Altered Function, eds. T.P. Singer, R.W. Von Korff and D.L. Murphy (Academic Press, New York) p. 517. " Dumbrille-Ross, A., S.W. Tang and D.V. Coscina, 1981, Differential binding of [3H]imipramine and [3H]-mianserin in rat cerebral cortex, Life Sci. 29, 2049. Dumbrille-Ross, A., S.W. Tang and P. Seeman, 1980, High affinity binding of [3H]mianserin to rat cerebral cortex, European J. Pharmacol. 68, 395. Ekstedt, B., Magyar, K. and Knoll, J., 1979, Does the B form of selective monoamine oxidase inhibitor lose selectivity by long term treatment?, Biochem. Pharmacol. 28, 919. Fowler, C.J., K.F. Tipton, A.V.P. MacKay and M.B.H. Youdim, 1982, Human platelet monoamine oxidase - A useful enzyme in the study of psychiatric disorders, Neuroscience 7, 1577. Grob, G., M. Gothert, M.-P. Ender and H.-J. Schumann, 1981, [3H]-Imipramine binding sites in the rat brain. Selective localization on serotonergic neurones, Naunyn-Schmiedeb. Arch. Pharmacol. 317, 310. Kebabian, J. and D. Calne, 1979, Multiple categories of dopamine receptors, Nature 277, 93. Kinnier, W.J., D.M. Chuang and E. Costa, 1980, Down regulation of dihydroaiprenolol and imipramine binding sites in brain of rats repeatedly treated with imipramine, European J. Pharmacol. 67, 289. Knoll, J., 1981a, Further experimental support to the concept that (-)deprenyl facilitates dopaminergic neurotransmission in the brain, in: Monoamine Oxidase: Basic and Clinical Frontiers, eds. K. Kamijo, E. Usdin and T. Nagatsu (Excerpta Medica, Amsterdam-Oxford-Princeton) p. 230. Knoll, J., 1981b, The pharmacology of selective MAO inhibitors, in: Monoamine Oxidase Inhibitors, eds. M.B.H. Youdim and E.S. Paykel (John Wiley and Sons, Ltd.) p. 45. Knoll, J., 1982, Selective inhibition of B type monoamine oxidase in the brain: A drug strategy to improve the quality of life in senescence, in: Strategy in Drug Research, ed. J.A. Keverling-Buisman (Elsevier Scientific Publishing Company, Amsterdam) p. 107. Leysen, J.E., J.E. Niemegeers, J.M. Van Nueten and P.M. Laduron, 1982, [3H]-Ketanserin (R41468), a selective [3H]ligand for serotonin 2 receptor binding sites. Binding properties, brain distribution and functional role, Mol. Pharmacol. 21,301.
i17 Lowry, O.H., M.J. Rosebrough, A.C. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. Mann, J. and S. Gershon, 1980, (l)-Deprenyl, a selective monoamine oxidase type B inhibitor in endogenous depression, Life Sci. 26, 877. Mishra, R., A. Janowsky and F. Sulser, 1980, Action of mianserin and zimelidine on the norepinephrine receptor coupled adenylate cyclase system in brain: Subsensitivity without reduction in fl-adrenergic receptor binding, Neuropharmacology 19, 983. Neff, N.H. and J.A. Fuentes, 1976, The use of selective monoamine oxidase inhibitor drugs for evaluating pharmacological and physiological mechanisms, in: Monoamine Oxidase and Its Inhibition, Ciba Foundation Symposium 39, Elsevier Excerpta Medica (North-Holland, Amsterdam) p. 163. Palkovits, M., R. Raisman, M. Briley and S.Z. Langer, 1981, Regional distribution of [3 H]imipramine in rat brain, Brain Res. 210, 493. Peroutka, S.J. and S.H. Snyder, 1980a, Long-term antidepressant treatment decreases spiroperidol labeled serotonin receptor binding, Science 210, 88. Peroutka, S.J. and S.H. Snyder, 1980b, Regulation of serotonin 2 (SHT) 2) receptors labeled with [3 H]-spiroperidol by chronic treatment with the antidepressant amitryptiline, J. Pharmacol. Exp. Ther. 215, 582. Peroutka, S.J. and S.H. Snyder, 1981, [3H]-Mianserin: Differential labeling of serotonin 2 and histamine I receptors in rat brain, J. Pharmacol. Exp. Ther. 216, 142.
Quitkin, F., A. Rigkin and D.F. Klein, 1979, Monoamine oxidase inhibitors. A review of antidepressant effectiveness, Arch. Gen. Psychiatry 36, 749. Rehavi, M., S.M. Paul, P. Skolnick and F.K. Goodwin, 1980, Demonstration of specific high affinity binding sites for [3H]imipramine in human brain, Life Sci. 26, 2273. Raisman, R., M.S. Briley and S.Z. Langer, 1979, Specific tricyclic antidepressant binding sites in rat brain, Nature 281, 148. Scatchard, G., 1949, The attraction of proteins for small molecules and ions, Ann. N.Y. Acad. Sci. 51,660. Sette, M., R. Raisman, M. Briley and S.Z. Langer, 1981, Localization of tricyclic antidepressant binding sites on serotonin nerve terminals, J. Neurochem. 37, 40. Sulser, F., J. Vetulani and P.L. Mobley, 1978, Mode of action of antidepressant drugs, Biochem. Pharmacol. 27, 257. Vetulani, J., R.J. Stawarz, J.V. Dingell and F. Sulser, 1976, A possible common mechanism of action of antidepressant treatments. Reduction in the sensitivity of the noradrenergic cyclic AMP generating system in the rat limbic forebrain, Nannyn-Schmiedeb. Arch. Pharmacol. 293, 109. Vetulani, J. and F. Sulser, 1975, Action of various antidepressant treatment reduces reactivity of noradrenergic cyclic AMP generating system in limbic forebrain, Nature 257, 495. Zsilla, G. and J. Knoll, 1982, The action of (-)deprenyl on monoamine turnover rate in rat brain, in: Typical and Atipycal Antidepressants: Molecular Mechanisms, eds. E. Costa and G. Racagni (Raven Press, New York) p. 211.
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Elsevier Biomedical Press
( - ) - D E P R E N Y L A SELECTIVE MAO 'B' INHIBITOR INCREASES [3H]IMIPRAMINE BINDING AND DECREASES fl-ADRENERGIC RECEPTOR FUNCTION G A B R I E L L A ZSILLA **, M A R I A L. B A R B A C C I A *, OTTAVIO G A N D O L F I *, JOSEPH K N O L L ** and E R M I N I O COSTA *.t
* Laboratory of Preclinical Pharmacology, National Institute of Mental Health, Saint Elizabeths Hospital, Washington, D.C. 20032, U.S.A., and ** Department of Pharmacology, Semmelweis University of Medicine, Budapest, Hungary Received 5 October 1982, revised MS received 3 January 1983, accepted 18 January 1983
G. ZSILLA, M.L. BARBACCIA, O. GANDOLFI, J. KNOLL and E. COSTA, (-)-Deprenyl a selective MAO 'B" inhibitor increases [3H]irnipramine binding and decreases fl-adrenergic receptor function, European J. Pharmacol. 89 (1983) 111-117. In rats, a seletive inhibition for 3 weeks of monoamineoxydase (MAO) type B elicited by daily doses of pargyline (2.5 pmol/kg) or (-)-deprenyl (1 pmol/kg) attenuated the NE dependent stimulation of cortical adenylate cyclase and reduced the number of brain recognition sites for fl-adrenergic receptor ligands. Similar actions were not elicited by a comparable dose regimen of (+)-amphetamine. Hence the inhibition of MAO B mimicks responses that are typically elicited by antidepressants. The molecular nature of the mechanisms involved cannot be understood, however, these mechanisms may not be identical for pargyline and (-)-deprenyl because this drug but not pargyline increased the number of [3H]imipramine recognition sites. Even high daily doses of pargyline (I00 #mol/kg, for 3 weeks) failed to change [3H]imipramine binding though they still down regulated fl-adrenergic recognition sites, the NE stimulation of adenylate cyclase and the Bmax of [3H]mianserin and [3H]spiroperidol binding. 5HT receptors
fl-Adrenergic receptors
Deprenyl
1. Introduction
Several lines of independent investigations indicate that typical and atypical antidepressants affect brain function by down regulating the binding sites for ligands of noradrenergic (Sulser et al., 1978; Kinnier et al., 1980; Brunello et al., 1982a) and serotonergic (5HT2) recognition sites (Peroutka and Snyder, 1980a, b; Blackshear and Sanders-Bush, 1982). Ultimately, antidepressants decrease the stimulus amplification elicited by norepinephrine (NE) on the adenylate cyclase coupled to fl-adrenergic receptors in rat brain (Vetulani and Sulser, 1975; Vetulani et al., 1976; Mishra et al., 1980; BruneUo et al., 1982a). The discovery that high affinity recognition sites for
* To whom all correspondence should be addressed.
MAO B
Adenylate cyclase
NE
Imipramine
antidepressant drugs are present in crude synaptic membranes (Raisman et al., 1979; Dumbrille-Ross et al., 1980; Rehavi et al., 1980; Palkovits et al., 1981; Peroutka and Snyder, 1981) and that their number can be modulated by treatment with antidepressants or by selective lesion of serotonergic neurons (Kinnier et al., 1980; Brunello et al., 1982b, c) has given us an additional dimension to study, at the molecular level, the modifications of brain function that mediate the beneficial effects of antidepressants. At least two kinds of antidepressant recognition sites are present in brain (Raisman et al., 1979; Dumbrille-Ross et al., 1980; Palkovits et al., 1981), which differ for anatomical location and effector specificity. One is located preferentially on 5HT axon terminals (Grob et al., 1981; Sette et al., 1981; Brunello et al., 1982c) and has the highest affinity for [3H]imipramine and the other is located postsynaptically to 5HT axon
112
terminals and has the highest affinity for [3H]mianserin (BruneUo et al., 1982c). Though we do not know which is the physiological effector for these two sites we suspect that the effector of the [3H]imipramine binding sites may be involved in the 5HT uptake regulation (Barbaccia et al., 1983) while that for the [3H]mianserin binding sites may function in 5HT receptor modulation (DumbrilleRoss et al., 1981; Brunello et al., 1982c; Barbaccia et al., 1983); both sites appear to be different from postsynaptic 5HT recognition sites (Barbaccia et al., 1983). The inhibition of monoamine oxidase (MAO) Type A relieves the symptoms of depression (Quitkin et al., 1979), down regulates serotonergic 5HT2 recognition sites (Peroutka and Snyder, 1980a) and noradrenergic receptor function. In this respect MAO inhibitors Type A resemble tricyclic antidepressants (Sulser et al., 1978). It is debated whether the selective inhibition of MAO Type B relieves the symptoms of depression (Mann and Gershon, 1980; Knoll, 1981a; Fowler et al., 1982; Knoll, 1982). The present report deals with experiments directed to ascertain whether (-)-deprenyl, a selective MAO Type B inhibitor (Knoll, 1981b) which was previously reported to affect dopaminergic turnover in rat brain striatum (Zsilla and Knoll, 1982), decreases the NE mediated signal amplification of adenylate cyclase or modifies the binding sites for typical and atypical antidepressants. The action of ( - )-deprenyl will be compared to that of pargyline which, in low doses also selectively inhibits MAO B (Neff and Fuentes, 1976).
pared for radioreceptor binding studies. [3H]Imipramine binding assays were carried out according to Kinnier et al. (1980). The nonspecific binding was determined by incubating the samples in presence of 10 #M desmethylimipramine. The crude synaptic membranes suspension ( - 10 mg of tissue wet weight/ml) was incubated with different concentrations of [3H]imipramine in a range between 0.5 and 10 nM. [3H]Mianserin binding assays were performed according to Dumbrille-Ross et al. (1981). The nonspecific binding was defined by carrying out the assay in presence of 10 #M cold mianserin. The so-called 5HT2 recognition sites, labeled by [3H]spiroperidol were studied according to the procedures described by Peroutka and Snyder (1980b). The nonspecific binding was defined by incubating the samples in presence of 1 #M ketanserin (Leysen et al., 1982) or 10 #M LSD. The fl-adrenergic recognition sites were studied according to the procedures described by Bylund and Snyder (1976) using [ 3H]dihydroalprenolol ([3H]DHA) as the specific ligand. The nonspecific binding was defined in presence of 1 #M dihydroalprenolol. Modification of binding sites characteristics were analyzed according to the method of Scatchard (1949). The basal cyclic AMP accumulation and that elicited by NE was measured in the incubation medium of minces (300 by 300 #m) prepared from fresh frontal cortices, according to the procedures described by Blumberg et al. (1976). The protein concentration was measured according to the procedures described by Lowry et al. (1951) using bovine serum albumin as a standard. Student's t-test was used to analyze the data.
2. Materials and methods 3. Results
Male Sprague-Dawley rats from Zivic-Miller (150 g) were used in all the experiments. The animals were daily injected s.c. with 1 #mol/kg of either (-)-deprenyl or (+)-amphetamine and either with 2.5 or 100 pmol/kg i.p., of pargyline for 21 days. The rats were decapitated 24-36 h after the last drug injection. After the brain was rapidly extracted from the skulls, the brain structures were dissected on ice and kept frozen at - 7 0 ° C until crude synaptic membranes were pre-
Daily injections of (-)-deprenyl (1 #mol/kg s.c.) repeated during three weeks changed the kinetic characteristics of the [3H]imipramine recognition sites located in crude synaptic membranes prepared from rat frontal cortex and hippocampus. In the hippocampus, as shown by the Bmax value (table 1), the number of [3Hlimipramine recognition sites was increased without any significant change in the apparent K D value. The
113 TABLE 1 Kinetic characteristics of [ 3H]imipramine specific binding to crude synaptic membranes prepared from cortex or hippocampus of rats repeatedly injected with ( - )-deprenyl, ( + )-amphetamine and pargyline. Each Scatchard analysis was performed by plotting the data obtained in binding assays with 8 different concentrations of ligand (from 0.5 to 10 nM); each experiment was repeated at least three times. Treatment
Cortical membranes
Hippocampal membranes
KD
KD (nM)
Bmax ( f m o l / m g prot)
Bmax ( f m o l / m g prot)
(nM) Saline ( - )-Deprenyl ( 1 / z m o l / k g s.c., 21 days) ( + )-Amphetamine (1/~ m o t / k g s.c., 21 days) Pargyline (2.5/~mol/kg i.p. 21 days) Pargyline (100/~ m o l / k g i.p., 21 days)
5.3 + 0.7
650 + 100
5.2 + 0.7
560 + 50
8.2 + 1.5
1065 + 150 a
7.0 + 0.5
800 + 90 a
4.5 + 0.6
720 + 90
5.1 +0.5
690-t- 80
6.9 + 0.5
650 + 85
a p < 0.01 when compared to values obtained in saline-treated rats.
Table 1 also shows that (+)-amphetamine, a structural analogue and a possible metabolite of deprenyl, given daily for three weeks at a dose regimen similar to that of (-)-deprenyl, failed to
increase in Bmax was greater in cortex than in hippocampus; in fig. 1 is represented a typical Scatchard plot obtained by the analysis of [3H]imipramine binding data from cortical membranes.
!,000
J
if"
A
300t {
'~.
=
E
-
\,
°
200 -- ~
O
"~
20o [ ~ .~
,00
,t
.
_
/
e~
=
"~
~
0
,o
[~H]-imipromine (nM)
• ~ ~ •
I 500
Deprenyl
KD=6"SnM
n• Bmax --~...~mlaOxo0,mo, / mg protein
",J 1000
I "-,. 1500
I 2000
Bound (fmol/rag protein) Fig. 1. Repeated injections of ( - ) - d e p r e n y l increase the Bmax of [3H]imipramine specific binding in crude synaptic membranes prepared from rat frontal cortex. ( - ) - D e p r e n y l was injected s.c. for 21 days at the dose of 1/Lmol/kg. The animals were killed 2 4 - 3 6 h after the last injection. This graph is representative of one experiment done in triplicate. The difference in the Bmax value for [ 3H]imipramine specific binding between rats treated with ( - )-deprenyl or saline was significant (P < 0.01). A similar experiment was repeated three times, and similar results were obtained.
114 TABLE 2 Kinetic characteristics of [3H]mianserin and [3H]spiroperidol specific binding to crude synaptic membranes prepared from brain cortex of rats repeatedly injected with ( - ) - d e p r e n y l o r pargyline. [3HlMianserin and [3H]spiroperidol specific bindings were calculated by subtracting from the total binding the values obtained in presence of l0 # M cold mianserin and 10 /~M LSD, respectively. Each value represents the mean + S.E.M. of at least six separate determinations. Treatment
....
[ 3H]Mianserin
Saline ( - )-Deprenyl (i # m o l / k g s.c., 21 days) Pargyline (2.5 # mol/kg i.p., 21 days) Pargyline (100/~mol/kg i.p., 21 days)
[ 3H]Spiroperidol
Ko (nM)
Bma× ( f m o l / m g prot)
KD (nM)
Braax (fmol/mg prot)
2.0 ± 0.5
281 ± 47
0.42 ± 0.12
98 _+ 16
2.2+0.3
270+40
0.51 ±0.11
87± 13
1.8 + 0.3
264 4- 30
0.70 ± 0.2
101 ± 15
3.7 ± 0.7
195 + 15 a
0.80 ± 0.31
59 ± 8 ~
a p < 0.05 when compared to values obtained in saline treated rats.
induce any change in the characteristics of [ 3 H ] imipramine binding to cortical crude synaptic membranes. Pargyline; given repeatedly for three weeks at two different doses, failed to change [3H]imipramine binding. Table 1 shows that in rats receiving either 2.5 #mol/kg, of pargyline to inhibit MAO B selectively or 100 /tmol/kg, of pargyline to block MAO A and B (Neff and Fuentes, 1976), neither the Bmax nor the K o of [3H]imipramine binding is changed. The specific binding of [3H]mianserin, an atypical antidepressant, measured in crude synaptic membranes
prepared from cortices of rats receiving repeated (-)-deprenyl injections as described above was not modified (table 2). Moreover, in the same membrane preparations we could not detect any changes in 5HT2 recognition sites (table 2). The 5HT 2 recognition sites remained unchanged also after daily doses of pargyline (2.5 /~mol/kg) repeated for 3 weeks. In contrast, with higher doses of pargyline (100 #mol/kg) that block MAO Type A and B the Bmax of both [3H]mianserin and 5HT 2 binding sites was reduced (table 2). Since (-)-deprenyl attenuates the symptoms of
TABLE 3 NE-stimulated cAMP accumulation in rat brain cortical slices and [3H]dihydroalprenolol ([3H]DHA) specific binding to crude synaptic membranes prepared from cortex of rats repeatedly injected with ( - )-deprenyl or pargyline, n = number of animals used for the determination of the basal and NE-stimulated cAMP accumulation. The assay was carried out separately for each animal, always in triplicate. Treatment
(n)
cAMP accumulation ( p m o l / m g prot per 15 min) Basal
Saline ( -- )-Deprenyl (l # m o l / k g s.c., 21 days) Pargyline (2.5/~mol/kg i.p., 21 days) Pargyline (100/~mol/kg i.p., 21 days)
NE stimulation (% increase)
+ 50 i~M N E
[3H]DHA spec. binding KD Bmax (nM)
( f m o l / m g prot)
(18)
8.4+0.4
15.8+0.8 b
88 b
2.9_+0.4
200+ 5
(8)
8.0+0.5
10.3-1-0.6 a
29 a
2.9-t-0.5
149_+ 15 a
(5)
12.1 +2.5
15.3±1.3 ~
26 a
2.0+0.5
150±13 ~
(5)
11.1 + 1.5
14.4 +0.9 a
30 a
2.7 ±0.3
160 ± 10 a
a p < 0.05 when compared to saline treated animals. b p < 0.01 when compared to basal values.
115 depression (Knoll, 1981a), we studied whether similarly to other antidepressants it could change the number of fl-adrenergic recognition sites and the signal amplification elicited by NE on adenylate cyclase. As shown in table 3, the stimulation of cyclic AMP accumulation elicited by NE in cortical slices prepared from rats receiving daily injections of (-)-deprenyl (1 #mol/kg s.c.) for three weeks was reduced. This reduction is similar to that observed in slices from cortices of rats receiving daily injections of pargyline of either 2.5 or 100 #mol/kg i.p., for three weeks. As shown in table 3, pargyline, no matter which dose was used, tends to increase the basal cAMP accumulation in cortical slices. Table 3 also indicates that the desensitization of the cAMP generating system toward NE stimulation elicited by (-)-deprenyl and pargyline either 2.5 or 100 #mol/kg was related to a decrease in number of fl-adrenergic recognition sites. All the treatments, in fact, elicited a decrease in the number of [3H]DHA specific binding sites by about 25%.
4. Discussion
A small dose of (-)-deprenyl (1 #mol/kg s.c.) or of pargyline (2.5/~mol/kg i.p.) given daily for three weeks was reported to inhibit by 80% or better the activity of MAO Type B while the activity of MAO Type A remains substantially unchanged (Neff and Fuentes, 1976; Campbell et al., 1979; Ekstedt et al., 1979). In contrast, 100 #mol/kg i.p. of pargyline given daily for three weeks causes an almost complete inhibition in the activity of both Type A and B enzyme (Campbell et al., 1979; Neff and Fuentes, 1976). Repeated (-)-deprenyl injections (1 #mol/kg) but not repeated pargyline injections (2.5 or 100 #mol/kg increase the Bin,x of [3H]imipramine binding to membranes of cortex and hippocampus. This difference can be explained by assuming that the action of (-)-deprenyl on [3H]imipramine binding is independent from the inhibition of MAO A and B or from the selective inhibition of MAO Type B, because even 2.5 #mol/kg i.p. of pargyline given daily for 3 weeks, which like (-)-deprenyl selectively inhibits MAO Type B (Neff and
Fuentes, 1976; Campbell et al., 1979) fails to change the binding of [3H]imipramine. From the experiments reported in table 1 we can exclude that the increase in Bin,x of [3H]imipramine binding is due to the accumulation of amphetamine which is formed as a metabolite of (-)-deprenyl. To evaluate the specificity of the modification of the recognition sites for [3H]imipramine elicited by (-)-deprenyl we have investigated whether (-)-deprenyl and both doses of pargyline can modify the characteristics of the recognition sites for [3H]mianserin or for [3H]spiroperidol, two sites that have been considered related to the function of 5HT2 receptors (Peroutka and Snyder, 1980a, b; 1981). The results obtained indicate that only pargyline (100 #mol/kg) reduces the Bma x of [3H]mianserin and of [3H]spiroperidol binding. This ligand labels many recognition sites in addition to 5HT2, including those for dopamine (Kebabian and Calne, 1979). However, since we used LSD to displace the specifically bound [3H]spiroperidol, we are measuring 5HT2 recognition sites, selectively. At present it is not understood whether [3H]mianserin and [3H]spiroperidol bind to the same 5HT2 receptors because the destruction of 5HT axons increased the Bmax of [3H]mianserin binding measured in presence or in absence of mepyzamine (Brunello et al., 1982c) without changing that of 5HT2 recognition sites (Barbaccia et al., 1983). This indicates that despite the similarity in the behavior of [3H]mianserin and [3H]spiroperidol binding shown in table 2, the two recognition sites cannot be considered to be identical. Since pargyline (2.5 #mol/kg) and (-)-deprenyl (1 #mol/kg) affect the recognition sites for [3H]imipramine in a completely different fashion, we have investigated whether they modify the fladrenergic receptor in a similar fashion. The data reported in table 3 show that small doses of pargyline and (-)-deprenyl despite the different action on the recognition sites for [3H]imipramine down regulate the recognition sites for fl-adrenergic ligands and decrease the signal amplification elicited by NE on the adenylate cyclase activity. Since the presence of 5HT axons is an absolute requirement for the desensitization of fl-adrenergic receptors caused by repeated daily injections of imipramine and DMI (Brunello et al., 1982a) but
116
not for that caused by a similar treatment with mianserin (Barbaccia et al., 1983) we could assume that small doses of (-)-deprenyl or pargyline act on 5HT axons by a mechanism dependent on MAO type B inhibition. Indeed the action of (-)-deprenyl and pargyline is not identical because of the difference in their action on [3H]imipramine recognition sites which are located on 5HT axons. This difference cannot be explained at this time; perhaps it may be clarified when the endogenous effector for these sites is known. In contrast, high doses of pargyline affect additional brain mechanisms as revealed by the down regulation of [3H]mianserin and [3H]spiroperidol binding sites; for instance these high doses of pargyline could down regulate fl-adrenergic recognition sites and the signal amplification of NE receptors by increasing the amount of NE reaching NE recognition sites. The question then arises as to whether the [3H]imipramine binding reflects the recognition site for an endogenous ligand which might be stored either in 5HT neurons or in another axon innervating the 5HT axons and thereby modulating the 5HT uptake system (Barbaccia et al., 1983). So far our results are compatible with the view that (-)-deprenyl may cause an inhibition of the release of this hypothetical effector and thereby cause supersensitivity of its recognition sites.
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