European Journal of Pharmacology 30 (1975) 159-163 © North-Holland Publishing Company

TRICYCLIC ANTIDEPRESSIVE DRUGS AND DOPAMINE-SENSITIVE ADENYLATE CYCLASE FROM RAT BRAIN STRIATUM Manfred E. KAROBATH* Department of Experimental Psychiatry, Psychiatrische Universiti~tsklinik, Universit~t Wien, Vienna, Austria

Received 25 June 1974, revised MS received 23 September 1974, accepted 18 October 1974 M.E. KAROBATH, Tricyclic antidepressive drugs and dopamine-sensitive adenylate cyclase from rat brain striatum, European J. Pharmacol. 30 (1975) 159-163. Tricyclic antidepressant drugs were examined as inhibitors of dopamine-sensitiveadenylate cyclase in a cell-free homogenate of rat brain striatum. Amitriptyline (Ki 0.17 ~M) and doxepin (Ki 0.24 ~M) were found to be potent inhibitors of dopamine-sensitive adenylate cyclase, chlorimipramine (Ki 0.59 tzM) and nortriptyline (Ki 0.50 #M) were moderate inhibitors and imipramine, desmethylimipramine, protriptyline and melitracene were weak inhibitors with Ki values higher than 1 #M. Adenylate cyclase Dopamine receptor

Striatum, rat Doxepin

Antidepressant drugs

1. Introduction Imipramine and other tricyclic antidepressant drugs have been found to be potent inhibitors of biogenic amine uptake (Glowinski and Axelrod, 1965; Murphy et al., 1970; Horn et al., 1971) and potentiation of biogenic amine effects is thought to be a major mechanism of action of these drugs. Their structure is similar to that of the neuroleptic phenothiazines and historically they have been developed from phenothiazines in the search for better antipsychotic drugs. Recently antipsychotic phenothiazines have been reported to be potent inhibitors of dopamine-sensitive adenylate cyclase (Clement-Cormier et al., 1974; Karobath and Leitich, 1974), an enzyme which is thought to closely resemble the dopamine receptor in the central nervous system (Kebabian et al., 1972). In these studies imipramine and desmethylimipramine have also been examined as non-antipsychotic control drugs and they were weak antago* Supported by 'Fonds zur F~Srderung der wissenschaftlichen Forschung in Osterreich', Projekt 1606.


nists of dopamine-sensitive adenylate cyclase (Clement-Cormier et al., 1974; Karobath and Leitich, 1974). These results, and the observation that amitriptyline also antagonizes dopamine-sensitive adenylate cyclase (Karobath, 1974) raised the question whether tricyclic antidepressant drugs as a group have the potential to inhibit an enzyme which is thought to resemble the dopamine receptor. The effects of eight antidepressant drugs on dopamine-sensitive adenylate cyclase were therefore examined and compared with the effects of neuroleptic drugs.

2. Materials and methods [a-a2P]-ATP and [8-3H]-cAMP were obtained from the Radiochemical Center Amersham and used without purification. Drugs were kindly donated as follows: chlorpromazine HC1, Smith, Kline and French, Philadelphia, Pa., U.S.A.; imipramine • HC1, chlorimipramine and doxepin, Ciba-Geigy A.G., Basel, Switzerland; droperidol, Janssen Pharmaceuticals Ltd. N.V. Beerse, Belgium; haloperidol, McNeil


M.E. Karobath, Antidepressive drugs and dopamine receptor

Labs., Evansville, Ind., U.S.A.; nortriptyline, melithracene, Lundbeck and Co., Kobenhavn, Denmark; amitriptyline, protriptyline • HC1, Merck Sharp and Dohme, West Point, Pa., U.S.A.; desmethylimipramine • HC1, Lakeside Laboratories, Milwaukee, Wi., U.S.A. Assay of dopamine-stimulated adenylate cyclase was carried out with cell-free homogenates from rat brain striatum obtained from the brains of Sprague-Dawley rats weighing 2 0 0 - 2 5 0 g, as described previously (Kebabian et al., 1972). Thus rats were killed by decapitation and the striatum was rapidly dissected and homogenized in 25 vols. (w/v) of a hypotonic medium which contained: 2 mM TRIS-maleate buffer, pH 7.4 and 2 mM EGTA. 0.01 ml of this homogenate were added to each assay tube. The standard incubation medium (final volume 0.05 ml) contained (mM): TRIS-maleate buffer pH 7.3, 80; [a-32p]-ATP (about 1 × 10 6 cpm) 0.5; MgSO4 2.0; theophylline 10; unlabeled cAMP 0.15; EGTA 0.4; plus test substances. Reactions were initiated by the addition of [a-32P]-ATP and conducted for 150 sec in a shaking water bath at 30°C (Kebabian et al., 1972). The reactions were terminated by placing the tubes in a boiling water bath for 2 min. [8-3H] -cAMP (1 × 10 4 cpm) was then added as an internal standard to permit estimation of the recovery of cAMP after column chromatography, cAMP was then separated by chromatography on neutral aluminum oxide columns and counted by liquid scintillation spectrome-

try (Ramachandran, 1971). Recovery of 3H-cAMP was 90-100%. Blank values were obtained by incubation of the complete reaction mixtures with boiled homogenates and were less than 0.015% of added [~.32p] -ATP.

3. Results As previously described by Kebabian et al. (1972), 40/aM dopamine caused an increase in cAMP formation of about 100% over baseline values in the absence of added dopamine (table 1). Apomorphine in 10 /aM concentration was also found to stimulate dopamine-sensitive adenylate cyclase although the stimulation in our experiments was slightly less than that described previously (Kebabian et al., 1972). As shown in table 1, chlorpromazine and the tricyclic antidepressant drugs amitriptyline, chlorimipramine and imipramine were found to inhibit the stimulation by 40/aM dopamine or by 10/aM apomorphine. When the enzyme was stimulated by varying concentrations of dopamine (2.5 to 1,000 /aM) and the inhibitory effect of 1/aM amitriptyline was examined, the drug caused a shift in the dose-response curve to dopamine (fig. 1), and a double reciprocal plot of these data suggested a competitive inhibition of the response to dopamine. In order to compare the relative potency of tricyclic antidepressive drugs with that of neuroleptic

Table 1 Inhibition of dopamine-sensitive adenylate cyclase by drugs. Adenylate cyclase from rat brain striatum was stimulated by 40 tzM dopamine or by 10 ~M apomorphine. Basal activity in the absence of stimulating agents was 5.67 +-0.06 (n = 8) pmole cAMP per test tube. The control stimulation i.e. the increase in enzyme activity in the presence of 40 uM dopamine in the absence of any test substance was 5.52 -+0.16 (n = 7) pmole cAMP and is represented as 100%. The data are expressed as percentage of the control stimulation and represent mean values -+S.E.M. (n = number of observations). Drug (concentration)

None Chlorpromazine (1 uM) Amitriptyline (1 taM) Chlorimipramine (3 taM) Imipramine (10 uM) Dopamine (40 uM) * (n): number of observations.

Stimulation of cAMP formation in % of control stimulation 40 I~Mdopamine,

10 ~tM apomorphine

100 44.6 48.4 65.0 60.9 --

25.4 + 1.4 (8) 14.9 + 1.3 (4) 19.6 -+ 1.3 (4) 14.3 -+ 1.3 (4) 17.9 -+0.9 (4) 55.8 -+ 3.1 (8)

-+ 2.9 (7) * -+ 1.8 (4) + 2.9 (4) +-0.7 (4) -+0.7 (4)

M.E. Karobath, A ntidepressive drugs and dopamine receptor





Fig. 1. Effect of various concentrations of dopamine alone or in combination with 1 taM amitriptyline on adenylate cyclase activity in homogenates of rat brain striatum. In the absence of added dopamine and amitriptyline 3.0 -* 0.1 pmole cAMP were formed (hatched area). Results are mean values -+ S.E.M. (n = 4) pmole cAMP formed per assay tube. Incubation without amitriptyline (o); incubation in the presence of 1 #M amitriptyline (o).

I I000

Table 2 Effect of various drugs on dopamine-sensitive adenylate cyclase in homogenates of rat brain striatum. Concentrations of drugs causing 50% inhibition in cAMP accumulation produced by 40 tam dopamine in rat striatal homogenates were determined by incubating drugs in at least three concentrations in quadruplicate and by plotting the data on log probit paper. K i values were calculated from the relationship: Iso = Ki (1 + S/K m) (Cheng and Prusoff, 1973). Iso is the concentration of drug required to give 50% inhibition of the dopamine-stimulated increase in enzyme activity, S is the concentration (40 ~tM) of dopamine. The mean value for the concentrations of dopamine required to give half-maximal activation of the enzyme (K m) was 5 taM in these experiments. Iso (~M)

K I (/aM)

Tricyclic antidepressive drugs Amitriptyline Doxepin Nortriptyline Chlorimipramine Protriptyline Melithracene Imipramine Desmethylimipramine

1.6 2.1 4.5 5.3 11.2 12.3 15.1 >20

-+ 0.17 +- 0.3 +- 0.7 -+ (12 -+ 2.6 -+ 3.7 -+ 2.0

(3) * (3) (3) (3) (3) (3) (3)

0.17 0.24 0.50 0.59 1.24 1.37 1.51 >2

Neuroleptic drugs Chlorpromazine Haloperidol Droperidol * (n): number of observations.

0.6 -+ 0.0 (4) 0.8 + Q 2 (3) 3.6 -+ 0.3 (3)

0.06 0.09 0.40


M.E. Karobath, Antidepressive drugs and dopamine receptor

drugs, Is o values of the inhibition of the response to 40/aM dopamine were determined. From these results the Ki values were calculated using the method of Cheng and Prusoff (1973) (table 2).

4. Discussion The inhibition of dopamine-sensitive adenylate cyclase of rat brain striatal homogenates by tricyclic antidepressant drugs does not appear to be a general and specific property of these drugs, since their potency varies considerably. Amitriptyline and doxepin are much more potent than all other tricyclic antidepressant drugs. But when the potency of amitriptyline and doxepin is compared with that of other inhibito[s of dopamine-sensitive adenylate cyclase, they are less potent than the neuroleptic phenothiazines and about as potent as butyrophenones (table 2) (Clement-Cormier et al., 1974; Karobath and Leitich, 1974) or pimozide (Clement-Cormier et al., 1974). N-demethylation seems to decrease potency as inhibitor of dopamine-sensitive adenylate cyclase since the tertiary amines amitriptyline and imipramine are more potent than their secondary amine analogues and metabolites nortriptyline and desmethylimipramine. A similar observation was made by Miller and Iversen (1974), who found that the monoand di-desmethyl derivatives of chlorpromazine blocked the effect of dopamine to a lesser extent than did chlorpromazine. However, while chlorpromazine was previously found to be about half as potent as promazine (Clement-Cormier et al., 1974), we observed that the iminodibenzyl derivative chlorimipramine was morg potent than imipramine. Tricyclic antidepressant drugs are moderate to weak inhibitors of dopamine uptake into rat striatal synaptosomes (Horn et al., 1971). When this action is compared with their effect on dopamine-sensitive adenylate cyclase, tricyclic antidepressant drugs appear to be better inhibitors of dopamine-sensitive adenylate cyclase (table 2) than of dopamine uptake (Horn et al., 1971). Nevertheless, there appears to be a discrepancy between the potent effects of some tricyclic antidepressant drugs on dopamine-sensitive adenylate cyclase in vitro and the low or even absent antipsychotic or neuroleptic effects of these drugs in vivo (Da Prada

and Pletscher, 1966). A similar discrepancy between the effects of drugs in vivo and their action on dopamine-sensitive adenylate cyclase in vitro has been observed in studies with antipsychotic drugs when phenothiazines were compared with butyrophenones and pimozid (Clement-Cormier et al., 1974; Karobath and Leitich, 1974). Based on the assumption that an intimate association exists between dopamine-sensitive adenylate cyclase and the dopamine receptor, several explanations have been offered to explain this discrepancy. These explanations mainly refer to factors which contribute to different availability of drugs at the dopamine receptor such as differences in aquaeous solubility, drug distribution, metabolism and excretion (Clement-Cormier et al., 1974). Furthermore, pharmacologic actions unrelated to the effects of drugs on the dopamine receptor, such as central antimuscarinic properties, could also antagonize the biochemical and functional effects of drugs on the dopamine receptor in striatal tissue (Miller and Hiley, 1974). It has recently been shown that tricyclic antidepressant drugs can inhibit norepinephrine-induced accumulation of cAMP in slices of rat brain cortex (Palmer, 1974). These observations and the present data seem to suggest that some tricyclic antidepressant drugs have the potential to inhibit enzymes which are thought to closely resemble receptors of catecholamines in the central nervous system.

Acknowledgements The excellent technical assistance of H. Leitich is gratefully acknowledged.

References Cheng, Y. and W.H. Prusoff, 1973, Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (Iso) of an enzymatic reaction, Biochem. Pharmacol. 22, 3099. Clement-Cormier, Y.C., J.W. Kebabian, G.L. Petzold and P. Greengard, 1974, Dopamine-sensitive adenylate cyclase in mammalian brain: a possible site of action of anti-psychotic drugs, Proc. Natl. Acad. Sci. U.S.A. 71, 1113. DaPrada, M. and A. Pletscher, 1966, On the mechanism of chlorpromazine-induced changes in cerebral homovanillic acid levels, J. Pharm. Pharmacol. 18,628.

M.E. Karobath, A n tidepressive drugs and dopamine receptor Glowinski, J. and J. Axelrod, 1965, Effect of drugs on the uptake, release and metabolism of aH-norepinephrine in the rat brain, J. Pharmacol. Exptl. Therap. 149, 43. Horn, A.S., J.T. Coyle and S.H. Snyder, 1971, Catecholamine uptake by synaptosomes from rat brain, Mol. Pharmacol. 7, 66. Karobath, M., 1974, Trizyklisehe antidepressive Substanzen und dopamin-sensitive Adenylcyclase aus dem Nucleus caudatus der Ratte, Drug Res. 24, 1019. Karobath, M. and H. Leitich, 1974, Antipsychotic drugs and dopamine-stimulated adenylate cyclase prepared from corpus striatum of rat brain, Proc. Natl. Acad. Sci. U.S.A. 71, 2915. Kebabian, J.W., G.L. Petzold and P. Greengard, 1972, Dopamine-sensitive adenylate cyclase in caudate nucleus of rat brain and its similarity to the 'dopamine receptor', Proc. Natl. Acad. Sci. U.S.A. 69, 2145.


Miller, R.J. and C.R. Hiley, 1974, Antimuscarinic properties of neuroleptics and drug-induccd parkinsonism, Nature 248, 596. Miller, R.J. and L.L. lversen, 1974, Effect of chlorpromazine and some of its metabolites on the dopamine-sensitive adenylate cyclase of rat brain striatum, J. Pharm. Pharmacol. 26,142. Mtirphy, D.L., R.W. Colburn, J.M. Davis and W.E. Bunney, 1970, lmipramine and lithium effects on biogenic amine transport in depressed and manic-depressed patients, Amer. J. Psychiat. 127, 115. Palmer, G.C., 1974, Inhibitory actions of tricyclic antidepressants on rat brain adenylate cyclase-cyclic AMP, Trans. Amer. Soc. Neurochem. 5,151 Abstract. Ramachandran, J., 1971, A new and simple method for separation of adenosine 3(5,-cyclic monophosphate from other nucleotides and its use in the assay of adenyl cyclase, Anal. Biochem. 43, 227.

Tricyclic antidepressive drugs and dopamine-sensitive adenylate cyclase from rat brain striatum.

Tricyclic antidepressant drugs were examined as inhibitors of dopamine-sensitive adenylate cyclase in a cell-free homogenate of rat brain striatum. Am...
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