Neuroscience Letters, 48 (1984) 217-221

Elsevier Scientific Publishers Ireland Ltd.

NSL 02805

MODIFICATION OF ~-ADRENERGIC RECEPTOR BINDING IN RAT BRAIN FOLLOWING THYROXINE ADMINISTRATION

217

AUTHUR S. PERUMAL, URIEL HALBREICH and AMIRAM I. BARKAI

New York State Psychiatric Institute and Department o f Psychiatry, Columbia University College o f Physicians and Surgeons, 722 W. 168th Street, New York, N Y 10032 (U.S.A.)

(Received January 10th, 1984; Revised version received March 28th, 1984; Accepted April 1 lth, 1984)

Key words: ~-adrenergic receptor - thyroxine - rat brain - cerebral cortex

The effect of repeated administration of thyroxine on ~-adrenergic receptor binding was studied in several brain regions in the rat using [3H]dihydroalprenolol as a ligand. Thyroxine treatment resulted in an increased density of ~-adrenergic receptors in the cerebral cortex while a decreased binding was found in subcortical tissue representing the thalamus, striatum and parts of the limbic system. There was no change in binding in the cerebellum or in the brainstem. The results indicate that thyroxine may regulate ~-adrenergic receptors in certain brain areas.

It has recent ly been d e m o n s t r a t e d tha t t hy ro id h o r m o n e s m a y regula te the dens i ty

o f B-adrenergic recep tors in m e m b r a n e s p r e p a r e d f rom d i f fe ren t t issues and tha t this

h o r m o n a l effect is t issue-specif ic . Thus while t r i i o d o t h y r o n i n e (T3) t r ea tmen t

resul ted in an increase in the B-adrenergic recep tor b ind ing in hear t t issue [15, 20],

such increases were not seen in lymphocy tes [20] or lung tissue [15]. S imi lar ly an

increase in the dens i ty o f /3 -ad rene rg ic receptors fo l lowing T3 admin i s t r a t i on was

obse rved in the submax i l l a ry g land [12]. These obse rva t ions have led to the

hypo thes i s tha t an increase in the ava i lab i l i ty o f t hy ro id h o r m o n e s p romotes

t r ansmiss ion in no rad rene rg i c pa thways , inc luding central nervous system

pa thways , by enhanc ing /3 -adrenerg ic recep tor func t ions [19]. Since dys func t ion o f

ca t echo lamine receptors has been pos tu la t ed in affect ive d i sorders [3, 4] several in,

ves t iga tors have used an t idepressan t drugs in c o m b i n a t i o n with T3 in an a t t empt to

ame l io ra t e depress ion in depress ive pat ients who had not r e s p o n d e d to t r ea tmen t

with the an t idepres san t drug a lone [7, 8]. How e ve r the effects o f thy ro id h o r m o n e s

on adrenergic recep tors in b ra in t issue has not yet been s tudied.

The present s tudy was des igned to invest igate the effect o f repet i t ive t r ea tmen t

with thyrox ine on ~-adrenerg ic r ecep to r b ind ing in several b ra in regions o f the rat .

The results indicate tha t thyrox ine t r ea tmen t is assoc ia ted with an increase in the

dens i ty o f B-adrenergic receptors in the cerebra l cor tex and with a decrease in recep-

to r b ind ing in the ' m i d r e g i o n ' a rea which includes the tha l amus and par t s o f the l im-

0304-3940/84/$ 03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd.

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bic system. No change was observed, however, in the cerebellum or in the brainstem.

Male Wistar rats weighing between 250 and 300 g were used. Food and water were supplied ad libitum. Animals were divided into two groups; 6 rats per group. One group was treated with sodium levothyroxine (T4 synthroid; Flint laboratories, Deerfield, IL) i.p., while the other group received saline injections and served as a control. The T4 regimen consisted of daily injections of 375 #g/kg for 7 consecutive days.

Animals were sacrificed by decapitation 2 h after the last injection. The brains were rapidly removed and dissected over ice to obtain the following regions: (a) cerebral cortex; (b) cerebellum; (c) pons and medulla, posterior to vertical plane 0 according to the stereotaxic atlas of Pellegrino et al. [10]; and (d) the remainder of the brain, which represented a combined thalamus-s t r ia tum-midbrain , defined here as 'midregion' . Freshly dissected brain regions were homogenized with 15-20 vols. of Tris-HC1 buffer (pH 7.0) in a Brinkman polytron for 30 s. The homogenate was then centrifuged at 48,000 g for 20 min. The supernatant was discarded and the pellet was rehomogenized for 15 s after resuspending in the same volume with the above-mentioned buffer. It was recentrifuged as above and the pellet was suspended in l0 vols. of the original wet weight of tissue in 50 mM Tris-HC1 (pH 8.0). It was polytronized briefly 0 - 2 s) to obtain a homogeneous suspension. All the above operations were carried out at 4°C. The tissue suspension was aliquoted into pre- labeled plastic tubes and stored at - 7 0 ° C until used.

/3-Adrenergic receptor assay was carried out essentially according to Bylund and Snyder [5]. The frozen tissue samples were thawed quickly and centrifuged at 48,000 g to sediment the membranes. The pellets were resuspended in fresh 50 mM Tris- HCI buffer (pH 8.0) in the same volume and used for the assay. The assay tube con- tained 6 mg tissue, 50 mM Tris-HC1 buffer (pH 8.0) and [3Hl-dihydroalprenolol (DHA, 3 nM) in a final volume of 1 ml. The incubation was carried out at room temperature for 20 min. At the end of incubation samples were returned to ice and 4 ml of ice-cold 50 mM Tris-HCl (pH 8.0) was added. It was then filtered through Whatman G F / B filter discs under vacuum and the washing was repeated 3 more times with ice-cold buffer. The filter discs were transferred to counting vials and 10 ml of scintillation fluid (ACS Amersham) was added. The radioactivity was counted in a Beckman Scintillation Counter. The non-specific binding was measured in the presence of excess amount of alprenolol (20 ~M). Each assay was carried out in triplicate.

When a change in ligand binding at a concentration of 3 nM was observed, the binding assay was repeated with 5 different ligand concentrations with pooled tissue and the results then analyzed by a Scatchard plot [16] to determine whether the observed change in binding was due to a change in the density of binding sites (Bmax), a change in affinity (Kd), or a change in both.

Animals treated with the T4 preparat ion for 7 consecutive days showed a signifi-

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TABLE I

B-ADRENERGIC BINDING IN SEVERAL BRAIN REGIONS IN CONTROL AND THYROXINE

TREATED RATS

*P<0.05; **P<0.01 (two-tailed Student's t-test).

Brain region Specific binding (pmol/g tissue)

Control Thyroxine % Change

Cortex 4.1:1:0.1 5.5 _ 0.1 + 34**

'Midregion' 4.3 _-!- 0.1 3.5 + 0.3 - 19*

Cerebellum 3.0+0.1 3.1 _+0.2 + 3

Pons-medulla 3.7 ± 0.2 3.8 _+ 0.3 + 2

cant increase in/~-adrenergic binding in cerebral cortex (+ 34070) and a decreased binding in the 'midregion' tissue ( - 19070) compared to controls, whereas no signifi- cant changes were found in the cerebellum or in the pons-medulla regions (Table I). Saturation experiments revealed that the observed changes in binding were due to a change in the density of binding sites (Bmax) with no apparent change in binding affinity values. The Bmax and Ka values for cortex membranes of T'4-treated rats were 7.9 pmol /g tissue and 1.63 nM, compared with 6.2 pmol /g and 1.56 nM in the con- trols (Fig. 1). It should be pointed out that neither T3 nor T4 in the concentration

I o m

0 2 4 6 8

P i c o m o l e s / G m . tissue

Fig. 1. Effect of thyroxine on/3-adrenergic receptors: cortical tissue from control and thyroxine-treated rats was assayed as described in the methods sections. Concentrations of [3HldihydrOalprenolol ranged from 0.5 to 4 nM. e - - - e , Control (Bmax 6.2 pmol/g tissue); and q~---o, thyroxine-treated (Bmx 7.8 pmol /g tissue).

220

range of 0.5 to 5/~M, had any significant effect on in vitro [3H]DHA binding. Hence the effect of T4 may not be due to its direct action on receptors in vivo. Although changes in ~receptor number are sometimes associated with concomitant changes in receptor-mediated function [21], it remains to be established that the increase in ~-receptors observed here is linked to changes in catecholamine-related biochemical or physiological properties.

These results demonstrate for the first time that T4 administration to rats may induce changes in the number of B-adrenergic binding sites in certain brain areas, while the number of binding sites on other areas remains unchanged. Thus the T4 effect on cerebral B-adrenergic receptors appears to be region-specific and is pro- bably dependent upon the availability of thyroxine receptors in the 'responding' brain region.

The present results also indicate that T4 may regulate B-adrenergic receptors in brain and provide a possible explanation to previous observations that the therapeutic effects of antidepressant drugs could be potentiated, in otherwise non- responding depressed patients, by the combined administration of thyroid hormone preparations [7, 8, 13]. Several investigators have previously shown that most anti- depressant agents, including electroconvulsive shock treatment (ECT), induce down-regulation of B-adrenergic receptors in the rat brain [1, 2, 6, 9, 11, 17, 18]. There is also evidence that repeated ECT may influence the level of thyrotropin releasing hormone in certain brain regions [14], thereby implying that thyroid func- tions may be involved in the mechanisms which contribute to the antidepressant ef- fect of ECT.

This study was supported by HRC Grant 1715 and NIMH Grant 33690. The skillful technical assistance of George Cannova and Spencer Erman is gratefully acknowledged.

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