Drug Development Research 2:403-406 (1982)

Role of Beta-Adrenergic Receptors in the Mechanism of Action of Second-Generation Antidepressants Vimala H. Sethy and Douglas W. Harris

CNS Research, The Upjohn Company, Kalamazoo, Michigan

ABSTRACT

Sethy, V. H., and D. W. Harris: Role of beta-adrenergic receptors in the mechanism of action of second-generation antidepressants. Drug Dev. Res. 2403-406, 1982.

The effect of chronic intravenous infusion of desrnethylimiprarnine (DMI), amitriptyline, mianserin, bupropion, alprazolam, or diazepam on the density of beta-adrenergic receptors in the rat cerebral cortex has been investigated in this study. DMI and amitriptyline signif- icantly decreased the density of beta-adrenergic receptors. Mainserin, bupropion, alpra- zolam, and diazepam had no significant effect on the density of beta-adrenergic receptors. Results of this study indicate that mechanisms other than beta-adrenergic receptors may be involved in the antidepressant activity of these drugs.

Key words: beta-adrenerglc receptors, antidepressants, secondgeneratlon

INTRODUCTION Chronic treatment with tricyclic antidepressants and monoamine oxidase inhibitors decreases

the number of beta-adrenergic receptors in the rat brain [Banerjee et al., 1977; Sellinger-Barnette et al., 1980; Sethy and Harris, 19811. Both the tricyclic antidepressants and monoamine-oxidase inhibitors facilitate noradrenergic activity either by blocking the uptake of norepinephrine or by blocking the metabolism of monoamines [Sulser et al., 19781. Mianserin, bupropion and alprazolam have been found to be antidepressants in humans [Shopsin, 1980; Fabre and McLendon, 19801 but have no, or little, activity on either the uptake of norepinephrine and serotonin or on monoamine

Received February 23, 1982; accepted February 26, 1982.

Address reprint requests to Vimala H. Sethy, M.D., Ph.D., CNS Research, The Upjohn Company, Kalamazoo, MI49001.

0272-4391/82/0204-0403$01.50 0 1982 Alan R. Liss, Inc.

404 Sethy and Harris

oxidase [Shopsin, 19801. It is not known with certainty whether beta-adrenergic receptors are involved in the mechanism of action of second-generation antidepressants like mianserin, bupropion, and alprazolam. This report presents the results of chronic treatment with second-generation and tricyclic antidepressants on the density of beta-adrenergic receptors in the cerebral cortex of rats.

MATERIALS AND METHODS

Drug Treatment Female rats weighing 250-300 g were implanted with polyethylene cannulae in the jugular

vein according to the method of Weeks [1972]. DMI (10 mgkg/d), amitriptyline, mianserin (each 30 mgkgld), or bupropion (100 mgkgld) was infused continuously through a venous cannula for 7 d in a volume of 4.5 ml/d. Control rats received an equal volume of vehicle (0.9% sodium chloride).

Alprazolam and diazepam were dissolved in 95% ethyl alcohol. These drugs (30 mg/kg/d each) were infused chronically through intravenous cannulae for 15 min every hr on the hr for 30 d in a volume of 1.11 ml/d. Control rats received an equal volume of 95% ethyl alcohol.

The administration of each drug was stopped 24 hr prior to decapitation of the animals. After decapitation the brain was quickly removed from the skull, The cerebral cortex was dissected out by the method of Glowinski and Iversen [ 19661 and frozen at - 70°C until assayed.

High-Affinity Receptor Binding Beta-adrenergic receptor binding assays were carried out as described previously [Sethy and

Harris, 19811 using [3H]-dihydroalprenolol ([3H]-DHA) as a ligand. On the day of the assay, tissue was weighed, thawed in 25 ml of cold (4°C) 50 niM Tris-HCl buffer, pH 8.0, and homogenized using a Brinkman Polytron PCU-2-110 homogenizer for 30 sec at setting no. 6. The homogenate was centrifuged at 39,000 X g for 10 min. The pellet was washed twice by resuspension and recentrifugation, as described above. The final pellet was resuspended in 100 vol of the same buffer.

Measurement of t3H]-DHA binding was performed by incubating 1.0 ml aliquots of the membrane preparation with 0.1 ml of [3H]-DHA (specific activity 49.1 Ci/mmol) to give a final concentration ranging from 0.15-1.5 nM, 0.1 ml of distilled water or dl-propranolol (10 wM) and 0.8 ml of 50 mM Tris-HC1 buffer, pH 8.0, to give a final volume of 2.0 ml. The samples were incubated for 30 min at 25°C. The incubation was terminated by filtering the samples, under vacuum, through a Whatman GF/B glass fiber filter disc, 3.7 cm in diameter. Each tube was rinsed

TABLE 1. Effect of Chronic Intravenous Infusion of Antidepressant Drugs on Specific [3H]- DHA Binding to the Cerebral Cortex

~ ~

Duration of Bmax:a fmoVmg Drug Dose (mglkgld) infusion id) protein Kd:a nM

Saline DMI Amitriptyline Saline Mianserin Saline Bupropion Ethyl alcohol (95%) Diazepam Alprazolam

I 10 7 30 I

I 30 7

I 100 I

30 30 30 30 30

-

-

-

-

132 2 5 0.61 f 0.04 0.52 2 0.04

110 f 4' 0.58 f 0.03 135 2 9 0.55 f 0.04 130 2 8 0.58 2 0.02 134 f I 0.52 2 0.05

82 f 3b

142 5 3 0.63 2 0.03 133 2 8 0.61 ? 0.05 131 2 6 0.53 t 0.06 119 f 8 0.49 * 0.04

"Each observation is the mean f SE of four experiments. bP<O.OO1. cP<o.OO1

Beta-Adrenergic Receptors and Antidepressants 405

with 5.0 ml of cold buffer, which was also filtered and finally the filter disc was washed three times using 5.0 ml of buffer for each wash. The filter paper was placed in a scintillation vial containing 15 ml of Amersham Searle ACSR cocktail. The radioactivity was counted by liquid scintillation spectrometry.

Specific binding was defined as the total binding minus binding in the presence of 10 pM dl-alprenolol.

Proteins were determined by the method of Lowry et a1 [1951].

Chemical and Drugs

Trizma base was purchased from Sigma Chemical Company (St. Louis, Missouri). All the antidepressants were obtained from the pharmaceutical company of origin. New England Nuclear (Boston, Massachusetts) was the source for [3H]-DHA.

Analysis of Data

by Scatchard analysis of the data. Statistical analysis was done using the paired t-test.

RESULTS

The maximum number of binding sites (B-) and dissociation constant (&) were determined

Chronic (7-d) intravenous infusion of DMI (10 mglkgld) and amitriptyline (30 mglkgld) significantly decreased the B,, of beta-adrenergic receptors in the cerebral cortex. Chronic treatment with these drugs had no significant effect on & of [3H]-DHA binding to the cortical membrane preparation.

Mianserin, alprazolam, and diazepam (30 mg/kg/d each), and bupropion (100 mg/kg/d) had no significant effect on either B,, or K d (Table 1).

Rats infused chronically (7 d) with DMI (10 mgikgld), amitriptyline, mianserin, alprazolam, and diazepam (30 mg/kg/d each), or bupropion (100 mg/kg/d), appeared healthy with respect to gain in weight, spontaneous motor activity, and muscle tone (as felt during handling).

DISCUSSION Chronic intravenous infusion of tricyclic antidepressants DMI and amitriptyline significantly

reduced the number of beta-adrenergic receptors in the cerebral cortex, as has been reported previously [Banerjee et al, 1977; Sellinger-Bamette et al, 19801. However, chronic treatment with second-generation antidepressants like mianserin, bupropion, and alprazolam had no significant effect on the density of beta-adrenergic receptors.

Mianserin has been shown to be an antidepressant in humans [Montgomery, 19801. Chronic treatment of rats with this compound did not alter the density of beta-adrenergic receptors in the cerebral cortex. This is consistent with the observations reported earlier [Clements-Jewery , 1978; Sellinger-Bamette et al., 1980; Mishra et al., 19801. Mianserin is a presynaptic alpha-adrenergic (alpha2)-receptor blocker [Fludder and Leonard, 1979a,b]. Blockade of alpha2 receptors may in- crease the release of norepinephrine from presynaptic nerve endings, leading to facilitation of postsynaptic adrenergic activity. Like cocaine and amphetamine [Sethy and Harris, 19811, which increase adrenergic activity [Sulser et al., 19781, mianserin failed to alter the B, of beta-adrenergic receptors.

Chronic treatment with bupropion, a clinically effective antidepressant [Fabre and McLendon, 19781, had no significant effect on the density of beta-adrenergic receptors in the cerebral cortex of rats. Similarly, Fems et al. [1981] did not find a significant change in beta-adrenergic receptors after chronic treatment with bupropion, 25 mg/kg/d intraperitoneally for 6 w. However, chronic administration of maximally tolerated doses (20-80 mg/kg t.i.d. intrapentoneally) has been dem- onstrated to produce beta-adrenergic subsensitivity [Ferris et al., 1981; Sellinger-Barnette et al., 19801. According to Ferris et al. [1981], this effect may not be related to the antidepressant activity of bupropion.

Hare [1974] reported beneficial effects of diazepam in neurotic depression. The use of benzodiazepines in the treatment of depression still remains controversial. Alprazolam, a tnazo-

4Q6 Sethy and Harris

lobenzodiazepine, has been found to have therapeutic value in the treatment of depression [Fabre and McLendon, 19801. Chronic treatment with both alprazolam and diazepam failed to alter the density of beta-adrenergic receptors. Similar results have been reported with diazepam [Sellinger- Rarnette et al., 19801.

The lack of effect of chronic treatment with mianserin, bupropion, and alprazolam on the H, of beta-adrenergic receptors indicates that these drugs may mediate their antidepressant activity by mechanisms other than those involving beta-adrenergic receptors.

ACKNOWLEDGMENTS

The authors wish to thank the following companies for supplying the drugs used in this study: Burroughs Wellcome (bupropion), CibaGeigy (DMI), Merck (amitriptyline), and Organon (mianserin) . REFERENCES Hanerjee, S.P., Kung, L.S., Riggi, S.J. and Chanda, S.K.: Development of beta-adrenergic receptor subsen-

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