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Effects of thyroid status on the characteristics of alpha1-, alpha2-, beta, imipramine and GABA receptors in the rat brain
Life Sciences, Vol. 48, pp. 659-666 Printed in the U.S.A.
Pergamon Press
EFFECTS OF THYROID STATUSON THE CHARACTERISTICS OF ALPHAI-, ALPHA2-, BETA, IMIPRAMINE AND GABARECEPTORS IN THE RAT BRAIN Maurizio Sandrini, Donatella Marrama, Anna Valeria Vergoni and Alfio Bertolini Institute of Pharmacology, University of Modena, Via G. Campi 287, 41100 Modena, Italy (Received in final form December II, 1990)
Summary The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alphal, alphaz, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [~H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [~H]prazosin, [~H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [~H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status. Thyroid hormones play an important role in regulating both peripheral and central noradrenergic function (1-4). In experimental models, hypo- and hyperthyroidism respectively decreased and enhanced noradrenaline synthesis and/or turnover in the brain (5-?). I t has also been suggested (8) that thyroid hormones primarily influence receptor sensitivity: indeed, changes in pre- and post-synaptic noradrenergic receptors have been demonstrated in dysthyroid states. Postsynaptic changes included increase in the number of beta- adrenoceptors in the corpus striatum but not in the cortex (9) and increased locomotor responses to alpha-adrenergic agonists in reserpinized mice (5,7) induced by hyperthyroidism, and a decrease in the number of alpha2 and beta brain adrenoceptors induced by hypothyroidism (9-11). These experimental data are in agreement with the clinical observation that T3 potentiates the activity of t r i c y c l i c antidepressants (12-16). Presynaptic alpha2-adrenoceptor function is increased in hyperthyroidism and suppressed in hypothyroidism (9). Moreover, i t seems reasonable to expect that brain imipramine- and GABA binding sites may be altered in dysthyroid states, because hypo- and hyperthyroidism are typically associated with several kinds of mental and behavioral disorders (13,17,18). However, available experimental data are very limited. 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
660
Dysthyroidlsm and Receptors
Vol. 48, No. 7, 1991
The present study was aimed at investigating the effect of experimentally-altered thyroid status (hypo- or hyperthyroidism) on the characteristics of adrenoceptors (alphal, alpha2 and beta), GABA-receptors, and imipramine receptors in different, discrete areas of the rat brain. Methods Adult male rats of a Wistar strain (Morini, S.Polo d'Enza, Reggio nell'Emilia, I t a l y ) , weighing 150-200 g at the beginning of the experiment, were used. They were housed five per cage, under controlled conditions of temperature (22±0.2 °C) and humidity (60%), on a 12 h light/dark cycle (lights on 0700) and with free access to food and water. Hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 ~g/kg of L-triiodothyronine (T~, sodium salt, Sigma Chemical Co., MO, USA) (9). Hypothyroidism was induced by administering propylthiouracil (PTU, Sigma Chemical Co., MO, USA), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks (19). Control rats were daily treated with s.c. injection of T3 solvent (1 ml/kg) and with 2 ml water p.o. Rats were killed by decapitation 2 h after the last treatment, under ether anesthesia. Rats were exsanguinated and blood collected. Each sample was centrifuged at 3,000 rpm for 10 min and serum was immediately separated. T o t a l serum L-tetraiodotyronine (T4) was determined by radioimmunoassay. The brains were removed and immediately placed on an ice-cold surface. The brain regions (hypothalamus, hippocampus and cerebral cortex) were quickly dissected and stored at -20 °C prior to membrane preparation. Alphas- and beta-adrenoceptors were evaluated according to the method of Mogilnicka and Nielsen (20) using [3H]prazosin and [~H]dihydroalprenolol, respectively, as ligands. Alpha2-adrenoceptors were evaluated according to the method of Dickinson et al. (21) using [3H]yohimbine as ligand. Imipramine binding sites were evaluated essentially according to the method of Raisman et al. (22): briefly, brain tissues were homogenized in 20 ml of ice-cold buffer (50 mM Tris HCl, 120 mM NaCl, 5 mM KCl, pH 7.4) using a Polytron homogenizer (setting 6, 15 s); after centrifugation (48,000 x 4) for 10 min at 4 °C, the pellet was resuspended and centrifuged twice in an equal volume (20 ml) of the same ice-cold buffer. The final pellet was resuspended in the same buffer to give a final protein concentration of 400-700 pg/ml. Protein was determined by the method of Lowry et al. (23) using bovine serum albumin as standard. GABA receptors were evaluated according to the method of Enna and Snyder (24) using [~H]muscimol as ligand. Radioligands were obtained from New England Nuclear (Firenze, Italy) and had the following specific activities: [~H]prazosin, 82.0 Ci/mmol; [3H]dihydroalprenolol, 60.0 Ci/mmol; [3H]yohimbine, 87.1Ci/mmol; [3H]imipramine, 73.8 Ci/mmol; [~H]muscimol, 25.3 Ci/mmol. For [~H]dihydroalprenolol, specific binding was defined as that displaced by 10 ~M (±) propranolol and ranged from 50 to 70%. For [3H]prazosin and [~H]yohimbine, specific binding was defined as that displaced by 10 ~M phentofamine and ranged from 70 to 75%. For [3H]imipramine and [~]muscimol, specific
Vol. 48, No. 7, 1991
Dysthyroldism and Receptors
661
binding was defined as that displaced by 100 pM imipramine and 1 mM GABA respectively. The following concentrations were used: [~H]dihydroalprenolol from 0.065 to 4.18 riM; [3H]prazosin from 0.037 to 1.2 nM; [~H]yohimbine from 0.15 to 5.00 nM; [3H]imipramine from 0.15 to 5 nM; [3H]muscimol from 7.25 to 240 nM. The results of saturation experiments were analyzed according to Scatchard (25). The equilibrium dissociation constant (K~) and maximum number of binding sites (Bmax) were calculated individually for each sample using 6-7 concentrations of labelled drugs. All assays were done in t r i p l i c a t e . The resulting dissociation constants and binding capacities were summarized as arithmetic means and were compared using the Scheff~ procedure following an analysis of variance. Results Effects of T3 and PTU treatment on thyroid state in rats. Our experimental treatment schedule was able to alter the thyroid status of rats as shown by the the values of serum T4 that were, 5.1±0.3 pg/dl - I , >25 pg/dl - I , and 1.1±0.06 pg/dl - I for saline-, T3-, and PTU-treated rats, respectively (P
662
Dysthyroidlsm and Receptors
Vol. 48, No. 7, 1991
TABLE I Effects of Thyroid Status on alphal-, alpha2- and beta-Receptors in Membrane of Rat Cerebral Cortex. Treatment Li gand
[~H]prazosin
[~H]yohimbi ne
[~H]di hydroal prenol ol
Vehi cl e Bma× Kd Bma~ Kd
Bmax Kd
T~
PTU
205.0 + 4.1
215.3 ± 5.0
0.3 ± 0.1
0.3 ± 0.1
368.2 ± 8.2
349.3 ± 7.8
3.7 + 0.2
3.6 ± 0.2
3.5 ± 0.2
229.3 ± 7 . 6 *
145.0 ± 5 . 6 *
2.0 ± 0.2
2.2 ± 0.2
180.1± 6.2 2.2 ± 0.2
168.0 + 5.0 * 0.3 ± 0.1 282.1 ± 7.0 *
In adult male rats, hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 pg/kg of L-triiodothyronine (T3) and hypothyroidism was induced by administering propyltiouracil (PTU), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks. Control rats were daily treated with s.c. injection of T~ solvent (1 ml/kg) and with 2 ml water p.o. Bm,x is expressed as fmol/mg protein, Kd is expressed as nM. Data are means±S.E.M, from groups of 12 rats. Alphal- and beta adrenoceptors were evaluated using [3H]prazosin and [3H]dihydroalprenolol according to the method of Mogilnicka and Nielsen (16) as ligands, respectively. Alpha2-adrenoceptors were evaluated according to the method of Dickinson et al. (4) using [3H]yohimbine as ligand. * P
(-19.4%) (ANOVA, F=11.81, DF=2,11, P
Vol. 48, No. 7, 1991
Dysthyroidlsm and Receptors
663
TABLE II Effect of Thyroid Status on [3H]Imipramine Binding in Different Rat Brain Regions. Treatment Tissue
Vehicle B,,a,,
6 2 5 . 0 + 20.1
T3
PTU
6 8 5 . 8 + 24.7
4 6 8 . 2 ± 18.2 *
Cortex Kd
Hypothal amus
Hippocampus
3.9 ±
0.3
3.7 ±
0.3
4.0 +
0.4
Bmax
840.2 ± 3 1 . 5
881.3 _+ 3 0 . 6
680.1 _+ 23.6 *
Kd
4.6 ± 0.3
4.4 ± 0.2
4.9 ± 0.3
Bmix
720.1 _+ 2 5 . 2
745.3 _+ 2 3 . 5
970.0 ± 30.8 *
Kd
7.8 ±
0.4
7.6 ±
0.4
7.7 +
0.4
In adult male rats, hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 ~g/kg of L-triiodothyronine (T3) and hypothyroidism was induced by administering propyltiouracil (PTU), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks. Control rats were daily treated with s.c. injection of T3 solvent (1 ml/kg) and with 2 ml water p.o. Bmix is expressed as fmol/mg protein, Kd is expressed as nM. Data are means±S.E.M, from groups of 12 rats. * P
roidism caused a decrease in the number of alphas-, alpha2-, and beta-adrenoceptors. These d a t a are in agreement with those obtained by Gross et al. (10,11), who found a substantial reduction in cortical alphas-, alpha2andbeta-adrenoceptor density in adult hypothyroid rats. On the other hand, Atterwill et al. (9) found a reduction in the number of striatal and hypothalamic beta-adrenoceptors, a reduced function of presynaptic alpha2-adrenoceptors, but n~ change in the number of cortical beta-adrenoceptors, again in adult hypothyroid rats. However, while Gross et al. used a PTU treatment schedule (at least 4 weeks) similar to that adopted in our present study, Atterwill et al. administered PTU for only two weeks. In our hands, hyperthyroidism caused an increase in the number of cortical beta-adrenoceptors, while having no effect on alphal- and alpha2-adrenoceptors. These data again disagree with those of Atterwill et al. (9), who found that hyperthyroidism caused an increase in striatal beta-adrenoceptors, but no change in membranes from cerebral cortex. Others (10) found, on the other hand, an increase in alphal-adrenoceptors in membranes from cerebral cortex in hyperthyroid rats. In such a puzzling picture i t is perhaps worth remembering that hyperthyroidism greatly increases the number of beta-adrenoceptors in tissues outside the CNS (heart ventricle, atria and vas deferens of rats) (26,27). As far as [3H]imipramine binding sites are concerned, our present results show that their number is decreased in the cerebral cortex and hypothalamus and increased in the hippocampus of hypothyroid rats, while i t is not changed in any of these brain areas in hyperthyroid rats. I t is pertinent to recall that several mental and behavioral symptoms occurring in human hypothyroidism are also
664
Dysthyroidism and Receptors
Vol. 48, No. 7, 1991
140. Bmax
Kd
• CONTROLS
4.22 • 0.05
33.76*-2.7
• T3
320i0.04¢k
33.89~:2.8
0 PTU
4.12 ±0.04
34.75:!:2.7
120
100 c c
0 E
6O
I10 4O
20J
\
1
\ \
2
SB
3
(pmolmg
FIG.
0
4
5
prot)
1
Scatchard plot obtained by linear regression representing the saturation binding of [3H]muscimol in cortex membranes of controls, T3- and PTU-treated rats, as described in the text. The values are the means of four experiments. P
characteristic of endogenous depression, where the number of brain imipramine receptors is decreased. Our data are, however, at variance with those of Vaccarl (2), who found that the number of cortical imipramine binding sites was decreased by a chronic antithyroid treatment only when i t was administered for the f i r s t 30 days of l i f e , not when i t was performed in mature rats. Yet, Biassoni and Vaccari (28) found that the antithyroid drugs methimazole and PTU increase the binding of [~H]imipramine to rat brain membranes. However, their data were obtained from "in v i t r o " experiments, and the increased binding of [3H]imipramine involved the low- rather that the high-affinity compartment for this ligand; our present data, on the other hand, were obtained "in vivo" by evaluating only the high-affinity compartment. As far as cortical GABA binding sites are concerned, our results show that their number is not modified in hypothyroid rats, while i t is s i g n i f i cantly reduced in hyperthyroid rats.
Vol. 48, No. 7, 1991
Dysthyroidism and Receptors
665
Finally, i t should be stressed that our concurrently-performed "in vitro" experiments allow us to exclude the possibility that the binding of alphal, alpha2, beta and imipramine ligands may have been influenced by the possible presence of T= or PTU in the membrane samples. In conclusion, the present study further support the view that thyroid hormones influence neurotransmission in the CNS. In particular, our findings show that hypothyroidism is associated with a decreased number of adrenoceptors (alphal, alpha2 and beta) and of imipramine receptors in the cerebral cortex, with a decreased number of imipramine receptors in the hypothalamus and with an increased number of imipramine receptors in the hippocampus, whereas hyperthyroidism is associated with an increased number of beta-adrenoceptors and with a decreased number of GABA receptors in the cerebral cortex. Acknowledgements This work was supported in part by grants from "Ministero dell'UniversitA e della Ricerca Scientifica e Tecnologica" and from "Consiglio Nazionale delle Ricerche", Roma. References 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18.
W. EMLEN, D.S. SEGAL and A.J. MANDELL, Science 175 79-82 (1972). A. VACCARI, Br. J. Pharmacol. 84 ?73-?78 (1985). R.S. WILLIAMS and R.J. LEFKOWITZ J.H., Molecular Basis of Thyroid Hormone Action, J.H. Oppenheimer and H.H. Samuels (eds), 325-349, Academic Press, New York (1983). L.T. WILLIAMS, R.J. LEFKOWITZ, A.M. WATANABE, D.R. HATHAWAYand H.R. BESCH, J. Biol. Chem. 252 278?-2789 (1977). G. ENGSTROM, T.H. SVENSSONand B. WALDECK, Brain Res. 77 471-483 (1974). J . H . JACOBI, G. MUELLER and R.J. WURTMAN, Endocrinology 97 1332-1335 (1975). U. STROMBOM, T.H. SVENSSON, D.M. JACKSON and J. ENGSTROM, J. Neural Transm. 41 73-92 (1977). A.J. PRANGE, J.L. MEEK and M.A. LIPTON, L i f e Sci. 9 901-907 (1970). C.K. ATTERWILL, S.J. BUNN, D.J. ATKINSON, S.L. SMITH and D.J. HEAL, J. Neural Transm. 59 43-55 (1984). G. GROSS, O.E. BRODDE and H.J. SCHUMANN, Naunyn-Schmiedeberg's Arch. Pharmacol. 316 45-50 (1981). G. GROSS, and H.J. SCHUMANN, J. Pharm. Pharmacol. 33 552-554 (1981). P.G. ETTIGI, G.M. BROWN, and J.A. SEGGIE, Psychosom. Med. 41 203-208 (1979). G.R. GEWIRTZ,' D. MALASPINA, J.A. HATTERER, S. FEUREISEN, D. KLEIN and J.M. GORMAN, Am. J. Psychiatry 145 1012-1014 (1988). A. PILC and K.G. LLOYD, L i f e Sci. 35 2149-2154 (1984). D. WHEATLEY, Arch. Gen. P s i c h i a t r y 26 229-233 (1972). I.C. WILSON, A.J. PRANGE and P.P. LARA, The t h y r o i d axis~ drugs and behavior, A.J. Prange (ed), 49-62, Raven Press, New York (1974). R.H. GERNER, L. FAIRBANKS, G.M. ANDERSON, J.G. YOUNG, M. SCHEININ, M. LINNOILA, T.A. HARE, B.A. SHAYWITZ and D.J. COHEN, Am. J. Psychiatry 141 1533-1540 (1984). A.J. PRANGE, J.C. GARBUTT and P.T. LOOSEN, PsEchopharmacology: the t h i r d
666
19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Dysthyroldism and Receptors
Vol. 48, No. 7, 1991
9eneration of progress, H.Y. Meltzer (ed), 629-653, Raven Press, New York (1987). A.J. PATEL, A. RABIE, P.D. LEWIS and R. BALAZS, Brain Res. 104 33-48 (1976). E. MOGILNICKA and M. NIELSEN, Eur. J. Pharmaco]. 12___11107-111 (1986). K.E.J. DICKINSON, R.M. McKERNAN, C.M.N. MILES, K.S. LEYS and P.S. SEVER Eur. J. Pharmaco]. 120 285-293 (1986). R. RAISRAN, M.S. BRILEY, and S.Z. LANGER, Eur. J. Pharmaco]. 61 373-380 (1980). O.H. LOWRY, N.J. ROSENBROUGH, A.L. FARRand R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). S.J. ENNAand S.H. SNYDER, Mol. Pharmacol. 13 442-453 (1977). G. SCATCHARD, Ann. N.Y. Acad. Sci. 51 660-672 (1949). T. CIARALDI and G.V. MARINETTI, Biochem. Biophys. Res. Comm. 74 984-991 (1977). A.W. FOX, E.N. JUBERG, J.M. MAY, R.D. JOHNSON, P.W. ABEL and K.P. MINNEMAN, J. Pharm. Exp. Ther. 235 715-723 (1985). R. BIASSONI and A. VACCARI, Br. J. Pharmacol. 85 (1985) 447-456.
Life Sciences, Vol. 48, pp. 659-666 Printed in the U.S.A.
Pergamon Press
EFFECTS OF THYROID STATUSON THE CHARACTERISTICS OF ALPHAI-, ALPHA2-, BETA, IMIPRAMINE AND GABARECEPTORS IN THE RAT BRAIN Maurizio Sandrini, Donatella Marrama, Anna Valeria Vergoni and Alfio Bertolini Institute of Pharmacology, University of Modena, Via G. Campi 287, 41100 Modena, Italy (Received in final form December II, 1990)
Summary The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alphal, alphaz, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [~H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [~H]prazosin, [~H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [~H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status. Thyroid hormones play an important role in regulating both peripheral and central noradrenergic function (1-4). In experimental models, hypo- and hyperthyroidism respectively decreased and enhanced noradrenaline synthesis and/or turnover in the brain (5-?). I t has also been suggested (8) that thyroid hormones primarily influence receptor sensitivity: indeed, changes in pre- and post-synaptic noradrenergic receptors have been demonstrated in dysthyroid states. Postsynaptic changes included increase in the number of beta- adrenoceptors in the corpus striatum but not in the cortex (9) and increased locomotor responses to alpha-adrenergic agonists in reserpinized mice (5,7) induced by hyperthyroidism, and a decrease in the number of alpha2 and beta brain adrenoceptors induced by hypothyroidism (9-11). These experimental data are in agreement with the clinical observation that T3 potentiates the activity of t r i c y c l i c antidepressants (12-16). Presynaptic alpha2-adrenoceptor function is increased in hyperthyroidism and suppressed in hypothyroidism (9). Moreover, i t seems reasonable to expect that brain imipramine- and GABA binding sites may be altered in dysthyroid states, because hypo- and hyperthyroidism are typically associated with several kinds of mental and behavioral disorders (13,17,18). However, available experimental data are very limited. 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
660
Dysthyroidlsm and Receptors
Vol. 48, No. 7, 1991
The present study was aimed at investigating the effect of experimentally-altered thyroid status (hypo- or hyperthyroidism) on the characteristics of adrenoceptors (alphal, alpha2 and beta), GABA-receptors, and imipramine receptors in different, discrete areas of the rat brain. Methods Adult male rats of a Wistar strain (Morini, S.Polo d'Enza, Reggio nell'Emilia, I t a l y ) , weighing 150-200 g at the beginning of the experiment, were used. They were housed five per cage, under controlled conditions of temperature (22±0.2 °C) and humidity (60%), on a 12 h light/dark cycle (lights on 0700) and with free access to food and water. Hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 ~g/kg of L-triiodothyronine (T~, sodium salt, Sigma Chemical Co., MO, USA) (9). Hypothyroidism was induced by administering propylthiouracil (PTU, Sigma Chemical Co., MO, USA), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks (19). Control rats were daily treated with s.c. injection of T3 solvent (1 ml/kg) and with 2 ml water p.o. Rats were killed by decapitation 2 h after the last treatment, under ether anesthesia. Rats were exsanguinated and blood collected. Each sample was centrifuged at 3,000 rpm for 10 min and serum was immediately separated. T o t a l serum L-tetraiodotyronine (T4) was determined by radioimmunoassay. The brains were removed and immediately placed on an ice-cold surface. The brain regions (hypothalamus, hippocampus and cerebral cortex) were quickly dissected and stored at -20 °C prior to membrane preparation. Alphas- and beta-adrenoceptors were evaluated according to the method of Mogilnicka and Nielsen (20) using [3H]prazosin and [~H]dihydroalprenolol, respectively, as ligands. Alpha2-adrenoceptors were evaluated according to the method of Dickinson et al. (21) using [3H]yohimbine as ligand. Imipramine binding sites were evaluated essentially according to the method of Raisman et al. (22): briefly, brain tissues were homogenized in 20 ml of ice-cold buffer (50 mM Tris HCl, 120 mM NaCl, 5 mM KCl, pH 7.4) using a Polytron homogenizer (setting 6, 15 s); after centrifugation (48,000 x 4) for 10 min at 4 °C, the pellet was resuspended and centrifuged twice in an equal volume (20 ml) of the same ice-cold buffer. The final pellet was resuspended in the same buffer to give a final protein concentration of 400-700 pg/ml. Protein was determined by the method of Lowry et al. (23) using bovine serum albumin as standard. GABA receptors were evaluated according to the method of Enna and Snyder (24) using [~H]muscimol as ligand. Radioligands were obtained from New England Nuclear (Firenze, Italy) and had the following specific activities: [~H]prazosin, 82.0 Ci/mmol; [3H]dihydroalprenolol, 60.0 Ci/mmol; [3H]yohimbine, 87.1Ci/mmol; [3H]imipramine, 73.8 Ci/mmol; [~H]muscimol, 25.3 Ci/mmol. For [~H]dihydroalprenolol, specific binding was defined as that displaced by 10 ~M (±) propranolol and ranged from 50 to 70%. For [3H]prazosin and [~H]yohimbine, specific binding was defined as that displaced by 10 ~M phentofamine and ranged from 70 to 75%. For [3H]imipramine and [~]muscimol, specific
Vol. 48, No. 7, 1991
Dysthyroldism and Receptors
661
binding was defined as that displaced by 100 pM imipramine and 1 mM GABA respectively. The following concentrations were used: [~H]dihydroalprenolol from 0.065 to 4.18 riM; [3H]prazosin from 0.037 to 1.2 nM; [~H]yohimbine from 0.15 to 5.00 nM; [3H]imipramine from 0.15 to 5 nM; [3H]muscimol from 7.25 to 240 nM. The results of saturation experiments were analyzed according to Scatchard (25). The equilibrium dissociation constant (K~) and maximum number of binding sites (Bmax) were calculated individually for each sample using 6-7 concentrations of labelled drugs. All assays were done in t r i p l i c a t e . The resulting dissociation constants and binding capacities were summarized as arithmetic means and were compared using the Scheff~ procedure following an analysis of variance. Results Effects of T3 and PTU treatment on thyroid state in rats. Our experimental treatment schedule was able to alter the thyroid status of rats as shown by the the values of serum T4 that were, 5.1±0.3 pg/dl - I , >25 pg/dl - I , and 1.1±0.06 pg/dl - I for saline-, T3-, and PTU-treated rats, respectively (P
662
Dysthyroidlsm and Receptors
Vol. 48, No. 7, 1991
TABLE I Effects of Thyroid Status on alphal-, alpha2- and beta-Receptors in Membrane of Rat Cerebral Cortex. Treatment Li gand
[~H]prazosin
[~H]yohimbi ne
[~H]di hydroal prenol ol
Vehi cl e Bma× Kd Bma~ Kd
Bmax Kd
T~
PTU
205.0 + 4.1
215.3 ± 5.0
0.3 ± 0.1
0.3 ± 0.1
368.2 ± 8.2
349.3 ± 7.8
3.7 + 0.2
3.6 ± 0.2
3.5 ± 0.2
229.3 ± 7 . 6 *
145.0 ± 5 . 6 *
2.0 ± 0.2
2.2 ± 0.2
180.1± 6.2 2.2 ± 0.2
168.0 + 5.0 * 0.3 ± 0.1 282.1 ± 7.0 *
In adult male rats, hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 pg/kg of L-triiodothyronine (T3) and hypothyroidism was induced by administering propyltiouracil (PTU), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks. Control rats were daily treated with s.c. injection of T~ solvent (1 ml/kg) and with 2 ml water p.o. Bm,x is expressed as fmol/mg protein, Kd is expressed as nM. Data are means±S.E.M, from groups of 12 rats. Alphal- and beta adrenoceptors were evaluated using [3H]prazosin and [3H]dihydroalprenolol according to the method of Mogilnicka and Nielsen (16) as ligands, respectively. Alpha2-adrenoceptors were evaluated according to the method of Dickinson et al. (4) using [3H]yohimbine as ligand. * P
(-19.4%) (ANOVA, F=11.81, DF=2,11, P
Vol. 48, No. 7, 1991
Dysthyroidlsm and Receptors
663
TABLE II Effect of Thyroid Status on [3H]Imipramine Binding in Different Rat Brain Regions. Treatment Tissue
Vehicle B,,a,,
6 2 5 . 0 + 20.1
T3
PTU
6 8 5 . 8 + 24.7
4 6 8 . 2 ± 18.2 *
Cortex Kd
Hypothal amus
Hippocampus
3.9 ±
0.3
3.7 ±
0.3
4.0 +
0.4
Bmax
840.2 ± 3 1 . 5
881.3 _+ 3 0 . 6
680.1 _+ 23.6 *
Kd
4.6 ± 0.3
4.4 ± 0.2
4.9 ± 0.3
Bmix
720.1 _+ 2 5 . 2
745.3 _+ 2 3 . 5
970.0 ± 30.8 *
Kd
7.8 ±
0.4
7.6 ±
0.4
7.7 +
0.4
In adult male rats, hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 ~g/kg of L-triiodothyronine (T3) and hypothyroidism was induced by administering propyltiouracil (PTU), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks. Control rats were daily treated with s.c. injection of T3 solvent (1 ml/kg) and with 2 ml water p.o. Bmix is expressed as fmol/mg protein, Kd is expressed as nM. Data are means±S.E.M, from groups of 12 rats. * P
roidism caused a decrease in the number of alphas-, alpha2-, and beta-adrenoceptors. These d a t a are in agreement with those obtained by Gross et al. (10,11), who found a substantial reduction in cortical alphas-, alpha2andbeta-adrenoceptor density in adult hypothyroid rats. On the other hand, Atterwill et al. (9) found a reduction in the number of striatal and hypothalamic beta-adrenoceptors, a reduced function of presynaptic alpha2-adrenoceptors, but n~ change in the number of cortical beta-adrenoceptors, again in adult hypothyroid rats. However, while Gross et al. used a PTU treatment schedule (at least 4 weeks) similar to that adopted in our present study, Atterwill et al. administered PTU for only two weeks. In our hands, hyperthyroidism caused an increase in the number of cortical beta-adrenoceptors, while having no effect on alphal- and alpha2-adrenoceptors. These data again disagree with those of Atterwill et al. (9), who found that hyperthyroidism caused an increase in striatal beta-adrenoceptors, but no change in membranes from cerebral cortex. Others (10) found, on the other hand, an increase in alphal-adrenoceptors in membranes from cerebral cortex in hyperthyroid rats. In such a puzzling picture i t is perhaps worth remembering that hyperthyroidism greatly increases the number of beta-adrenoceptors in tissues outside the CNS (heart ventricle, atria and vas deferens of rats) (26,27). As far as [3H]imipramine binding sites are concerned, our present results show that their number is decreased in the cerebral cortex and hypothalamus and increased in the hippocampus of hypothyroid rats, while i t is not changed in any of these brain areas in hyperthyroid rats. I t is pertinent to recall that several mental and behavioral symptoms occurring in human hypothyroidism are also
664
Dysthyroidism and Receptors
Vol. 48, No. 7, 1991
140. Bmax
Kd
• CONTROLS
4.22 • 0.05
33.76*-2.7
• T3
320i0.04¢k
33.89~:2.8
0 PTU
4.12 ±0.04
34.75:!:2.7
120
100 c c
0 E
6O
I10 4O
20J
\
1
\ \
2
SB
3
(pmolmg
FIG.
0
4
5
prot)
1
Scatchard plot obtained by linear regression representing the saturation binding of [3H]muscimol in cortex membranes of controls, T3- and PTU-treated rats, as described in the text. The values are the means of four experiments. P
characteristic of endogenous depression, where the number of brain imipramine receptors is decreased. Our data are, however, at variance with those of Vaccarl (2), who found that the number of cortical imipramine binding sites was decreased by a chronic antithyroid treatment only when i t was administered for the f i r s t 30 days of l i f e , not when i t was performed in mature rats. Yet, Biassoni and Vaccari (28) found that the antithyroid drugs methimazole and PTU increase the binding of [~H]imipramine to rat brain membranes. However, their data were obtained from "in v i t r o " experiments, and the increased binding of [3H]imipramine involved the low- rather that the high-affinity compartment for this ligand; our present data, on the other hand, were obtained "in vivo" by evaluating only the high-affinity compartment. As far as cortical GABA binding sites are concerned, our results show that their number is not modified in hypothyroid rats, while i t is s i g n i f i cantly reduced in hyperthyroid rats.
Vol. 48, No. 7, 1991
Dysthyroidism and Receptors
665
Finally, i t should be stressed that our concurrently-performed "in vitro" experiments allow us to exclude the possibility that the binding of alphal, alpha2, beta and imipramine ligands may have been influenced by the possible presence of T= or PTU in the membrane samples. In conclusion, the present study further support the view that thyroid hormones influence neurotransmission in the CNS. In particular, our findings show that hypothyroidism is associated with a decreased number of adrenoceptors (alphal, alpha2 and beta) and of imipramine receptors in the cerebral cortex, with a decreased number of imipramine receptors in the hypothalamus and with an increased number of imipramine receptors in the hippocampus, whereas hyperthyroidism is associated with an increased number of beta-adrenoceptors and with a decreased number of GABA receptors in the cerebral cortex. Acknowledgements This work was supported in part by grants from "Ministero dell'UniversitA e della Ricerca Scientifica e Tecnologica" and from "Consiglio Nazionale delle Ricerche", Roma. References 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18.
W. EMLEN, D.S. SEGAL and A.J. MANDELL, Science 175 79-82 (1972). A. VACCARI, Br. J. Pharmacol. 84 ?73-?78 (1985). R.S. WILLIAMS and R.J. LEFKOWITZ J.H., Molecular Basis of Thyroid Hormone Action, J.H. Oppenheimer and H.H. Samuels (eds), 325-349, Academic Press, New York (1983). L.T. WILLIAMS, R.J. LEFKOWITZ, A.M. WATANABE, D.R. HATHAWAYand H.R. BESCH, J. Biol. Chem. 252 278?-2789 (1977). G. ENGSTROM, T.H. SVENSSONand B. WALDECK, Brain Res. 77 471-483 (1974). J . H . JACOBI, G. MUELLER and R.J. WURTMAN, Endocrinology 97 1332-1335 (1975). U. STROMBOM, T.H. SVENSSON, D.M. JACKSON and J. ENGSTROM, J. Neural Transm. 41 73-92 (1977). A.J. PRANGE, J.L. MEEK and M.A. LIPTON, L i f e Sci. 9 901-907 (1970). C.K. ATTERWILL, S.J. BUNN, D.J. ATKINSON, S.L. SMITH and D.J. HEAL, J. Neural Transm. 59 43-55 (1984). G. GROSS, O.E. BRODDE and H.J. SCHUMANN, Naunyn-Schmiedeberg's Arch. Pharmacol. 316 45-50 (1981). G. GROSS, and H.J. SCHUMANN, J. Pharm. Pharmacol. 33 552-554 (1981). P.G. ETTIGI, G.M. BROWN, and J.A. SEGGIE, Psychosom. Med. 41 203-208 (1979). G.R. GEWIRTZ,' D. MALASPINA, J.A. HATTERER, S. FEUREISEN, D. KLEIN and J.M. GORMAN, Am. J. Psychiatry 145 1012-1014 (1988). A. PILC and K.G. LLOYD, L i f e Sci. 35 2149-2154 (1984). D. WHEATLEY, Arch. Gen. P s i c h i a t r y 26 229-233 (1972). I.C. WILSON, A.J. PRANGE and P.P. LARA, The t h y r o i d axis~ drugs and behavior, A.J. Prange (ed), 49-62, Raven Press, New York (1974). R.H. GERNER, L. FAIRBANKS, G.M. ANDERSON, J.G. YOUNG, M. SCHEININ, M. LINNOILA, T.A. HARE, B.A. SHAYWITZ and D.J. COHEN, Am. J. Psychiatry 141 1533-1540 (1984). A.J. PRANGE, J.C. GARBUTT and P.T. LOOSEN, PsEchopharmacology: the t h i r d
666
19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Dysthyroldism and Receptors
Vol. 48, No. 7, 1991
9eneration of progress, H.Y. Meltzer (ed), 629-653, Raven Press, New York (1987). A.J. PATEL, A. RABIE, P.D. LEWIS and R. BALAZS, Brain Res. 104 33-48 (1976). E. MOGILNICKA and M. NIELSEN, Eur. J. Pharmaco]. 12___11107-111 (1986). K.E.J. DICKINSON, R.M. McKERNAN, C.M.N. MILES, K.S. LEYS and P.S. SEVER Eur. J. Pharmaco]. 120 285-293 (1986). R. RAISRAN, M.S. BRILEY, and S.Z. LANGER, Eur. J. Pharmaco]. 61 373-380 (1980). O.H. LOWRY, N.J. ROSENBROUGH, A.L. FARRand R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). S.J. ENNAand S.H. SNYDER, Mol. Pharmacol. 13 442-453 (1977). G. SCATCHARD, Ann. N.Y. Acad. Sci. 51 660-672 (1949). T. CIARALDI and G.V. MARINETTI, Biochem. Biophys. Res. Comm. 74 984-991 (1977). A.W. FOX, E.N. JUBERG, J.M. MAY, R.D. JOHNSON, P.W. ABEL and K.P. MINNEMAN, J. Pharm. Exp. Ther. 235 715-723 (1985). R. BIASSONI and A. VACCARI, Br. J. Pharmacol. 85 (1985) 447-456.
Pergamon Press
EFFECTS OF THYROID STATUSON THE CHARACTERISTICS OF ALPHAI-, ALPHA2-, BETA, IMIPRAMINE AND GABARECEPTORS IN THE RAT BRAIN Maurizio Sandrini, Donatella Marrama, Anna Valeria Vergoni and Alfio Bertolini Institute of Pharmacology, University of Modena, Via G. Campi 287, 41100 Modena, Italy (Received in final form December II, 1990)
Summary The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alphal, alphaz, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [~H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [~H]prazosin, [~H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [~H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status. Thyroid hormones play an important role in regulating both peripheral and central noradrenergic function (1-4). In experimental models, hypo- and hyperthyroidism respectively decreased and enhanced noradrenaline synthesis and/or turnover in the brain (5-?). I t has also been suggested (8) that thyroid hormones primarily influence receptor sensitivity: indeed, changes in pre- and post-synaptic noradrenergic receptors have been demonstrated in dysthyroid states. Postsynaptic changes included increase in the number of beta- adrenoceptors in the corpus striatum but not in the cortex (9) and increased locomotor responses to alpha-adrenergic agonists in reserpinized mice (5,7) induced by hyperthyroidism, and a decrease in the number of alpha2 and beta brain adrenoceptors induced by hypothyroidism (9-11). These experimental data are in agreement with the clinical observation that T3 potentiates the activity of t r i c y c l i c antidepressants (12-16). Presynaptic alpha2-adrenoceptor function is increased in hyperthyroidism and suppressed in hypothyroidism (9). Moreover, i t seems reasonable to expect that brain imipramine- and GABA binding sites may be altered in dysthyroid states, because hypo- and hyperthyroidism are typically associated with several kinds of mental and behavioral disorders (13,17,18). However, available experimental data are very limited. 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
660
Dysthyroidlsm and Receptors
Vol. 48, No. 7, 1991
The present study was aimed at investigating the effect of experimentally-altered thyroid status (hypo- or hyperthyroidism) on the characteristics of adrenoceptors (alphal, alpha2 and beta), GABA-receptors, and imipramine receptors in different, discrete areas of the rat brain. Methods Adult male rats of a Wistar strain (Morini, S.Polo d'Enza, Reggio nell'Emilia, I t a l y ) , weighing 150-200 g at the beginning of the experiment, were used. They were housed five per cage, under controlled conditions of temperature (22±0.2 °C) and humidity (60%), on a 12 h light/dark cycle (lights on 0700) and with free access to food and water. Hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 ~g/kg of L-triiodothyronine (T~, sodium salt, Sigma Chemical Co., MO, USA) (9). Hypothyroidism was induced by administering propylthiouracil (PTU, Sigma Chemical Co., MO, USA), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks (19). Control rats were daily treated with s.c. injection of T3 solvent (1 ml/kg) and with 2 ml water p.o. Rats were killed by decapitation 2 h after the last treatment, under ether anesthesia. Rats were exsanguinated and blood collected. Each sample was centrifuged at 3,000 rpm for 10 min and serum was immediately separated. T o t a l serum L-tetraiodotyronine (T4) was determined by radioimmunoassay. The brains were removed and immediately placed on an ice-cold surface. The brain regions (hypothalamus, hippocampus and cerebral cortex) were quickly dissected and stored at -20 °C prior to membrane preparation. Alphas- and beta-adrenoceptors were evaluated according to the method of Mogilnicka and Nielsen (20) using [3H]prazosin and [~H]dihydroalprenolol, respectively, as ligands. Alpha2-adrenoceptors were evaluated according to the method of Dickinson et al. (21) using [3H]yohimbine as ligand. Imipramine binding sites were evaluated essentially according to the method of Raisman et al. (22): briefly, brain tissues were homogenized in 20 ml of ice-cold buffer (50 mM Tris HCl, 120 mM NaCl, 5 mM KCl, pH 7.4) using a Polytron homogenizer (setting 6, 15 s); after centrifugation (48,000 x 4) for 10 min at 4 °C, the pellet was resuspended and centrifuged twice in an equal volume (20 ml) of the same ice-cold buffer. The final pellet was resuspended in the same buffer to give a final protein concentration of 400-700 pg/ml. Protein was determined by the method of Lowry et al. (23) using bovine serum albumin as standard. GABA receptors were evaluated according to the method of Enna and Snyder (24) using [~H]muscimol as ligand. Radioligands were obtained from New England Nuclear (Firenze, Italy) and had the following specific activities: [~H]prazosin, 82.0 Ci/mmol; [3H]dihydroalprenolol, 60.0 Ci/mmol; [3H]yohimbine, 87.1Ci/mmol; [3H]imipramine, 73.8 Ci/mmol; [~H]muscimol, 25.3 Ci/mmol. For [~H]dihydroalprenolol, specific binding was defined as that displaced by 10 ~M (±) propranolol and ranged from 50 to 70%. For [3H]prazosin and [~H]yohimbine, specific binding was defined as that displaced by 10 ~M phentofamine and ranged from 70 to 75%. For [3H]imipramine and [~]muscimol, specific
Vol. 48, No. 7, 1991
Dysthyroldism and Receptors
661
binding was defined as that displaced by 100 pM imipramine and 1 mM GABA respectively. The following concentrations were used: [~H]dihydroalprenolol from 0.065 to 4.18 riM; [3H]prazosin from 0.037 to 1.2 nM; [~H]yohimbine from 0.15 to 5.00 nM; [3H]imipramine from 0.15 to 5 nM; [3H]muscimol from 7.25 to 240 nM. The results of saturation experiments were analyzed according to Scatchard (25). The equilibrium dissociation constant (K~) and maximum number of binding sites (Bmax) were calculated individually for each sample using 6-7 concentrations of labelled drugs. All assays were done in t r i p l i c a t e . The resulting dissociation constants and binding capacities were summarized as arithmetic means and were compared using the Scheff~ procedure following an analysis of variance. Results Effects of T3 and PTU treatment on thyroid state in rats. Our experimental treatment schedule was able to alter the thyroid status of rats as shown by the the values of serum T4 that were, 5.1±0.3 pg/dl - I , >25 pg/dl - I , and 1.1±0.06 pg/dl - I for saline-, T3-, and PTU-treated rats, respectively (P
662
Dysthyroidlsm and Receptors
Vol. 48, No. 7, 1991
TABLE I Effects of Thyroid Status on alphal-, alpha2- and beta-Receptors in Membrane of Rat Cerebral Cortex. Treatment Li gand
[~H]prazosin
[~H]yohimbi ne
[~H]di hydroal prenol ol
Vehi cl e Bma× Kd Bma~ Kd
Bmax Kd
T~
PTU
205.0 + 4.1
215.3 ± 5.0
0.3 ± 0.1
0.3 ± 0.1
368.2 ± 8.2
349.3 ± 7.8
3.7 + 0.2
3.6 ± 0.2
3.5 ± 0.2
229.3 ± 7 . 6 *
145.0 ± 5 . 6 *
2.0 ± 0.2
2.2 ± 0.2
180.1± 6.2 2.2 ± 0.2
168.0 + 5.0 * 0.3 ± 0.1 282.1 ± 7.0 *
In adult male rats, hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 pg/kg of L-triiodothyronine (T3) and hypothyroidism was induced by administering propyltiouracil (PTU), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks. Control rats were daily treated with s.c. injection of T~ solvent (1 ml/kg) and with 2 ml water p.o. Bm,x is expressed as fmol/mg protein, Kd is expressed as nM. Data are means±S.E.M, from groups of 12 rats. Alphal- and beta adrenoceptors were evaluated using [3H]prazosin and [3H]dihydroalprenolol according to the method of Mogilnicka and Nielsen (16) as ligands, respectively. Alpha2-adrenoceptors were evaluated according to the method of Dickinson et al. (4) using [3H]yohimbine as ligand. * P
(-19.4%) (ANOVA, F=11.81, DF=2,11, P
Vol. 48, No. 7, 1991
Dysthyroidlsm and Receptors
663
TABLE II Effect of Thyroid Status on [3H]Imipramine Binding in Different Rat Brain Regions. Treatment Tissue
Vehicle B,,a,,
6 2 5 . 0 + 20.1
T3
PTU
6 8 5 . 8 + 24.7
4 6 8 . 2 ± 18.2 *
Cortex Kd
Hypothal amus
Hippocampus
3.9 ±
0.3
3.7 ±
0.3
4.0 +
0.4
Bmax
840.2 ± 3 1 . 5
881.3 _+ 3 0 . 6
680.1 _+ 23.6 *
Kd
4.6 ± 0.3
4.4 ± 0.2
4.9 ± 0.3
Bmix
720.1 _+ 2 5 . 2
745.3 _+ 2 3 . 5
970.0 ± 30.8 *
Kd
7.8 ±
0.4
7.6 ±
0.4
7.7 +
0.4
In adult male rats, hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 ~g/kg of L-triiodothyronine (T3) and hypothyroidism was induced by administering propyltiouracil (PTU), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks. Control rats were daily treated with s.c. injection of T3 solvent (1 ml/kg) and with 2 ml water p.o. Bmix is expressed as fmol/mg protein, Kd is expressed as nM. Data are means±S.E.M, from groups of 12 rats. * P
roidism caused a decrease in the number of alphas-, alpha2-, and beta-adrenoceptors. These d a t a are in agreement with those obtained by Gross et al. (10,11), who found a substantial reduction in cortical alphas-, alpha2andbeta-adrenoceptor density in adult hypothyroid rats. On the other hand, Atterwill et al. (9) found a reduction in the number of striatal and hypothalamic beta-adrenoceptors, a reduced function of presynaptic alpha2-adrenoceptors, but n~ change in the number of cortical beta-adrenoceptors, again in adult hypothyroid rats. However, while Gross et al. used a PTU treatment schedule (at least 4 weeks) similar to that adopted in our present study, Atterwill et al. administered PTU for only two weeks. In our hands, hyperthyroidism caused an increase in the number of cortical beta-adrenoceptors, while having no effect on alphal- and alpha2-adrenoceptors. These data again disagree with those of Atterwill et al. (9), who found that hyperthyroidism caused an increase in striatal beta-adrenoceptors, but no change in membranes from cerebral cortex. Others (10) found, on the other hand, an increase in alphal-adrenoceptors in membranes from cerebral cortex in hyperthyroid rats. In such a puzzling picture i t is perhaps worth remembering that hyperthyroidism greatly increases the number of beta-adrenoceptors in tissues outside the CNS (heart ventricle, atria and vas deferens of rats) (26,27). As far as [3H]imipramine binding sites are concerned, our present results show that their number is decreased in the cerebral cortex and hypothalamus and increased in the hippocampus of hypothyroid rats, while i t is not changed in any of these brain areas in hyperthyroid rats. I t is pertinent to recall that several mental and behavioral symptoms occurring in human hypothyroidism are also
664
Dysthyroidism and Receptors
Vol. 48, No. 7, 1991
140. Bmax
Kd
• CONTROLS
4.22 • 0.05
33.76*-2.7
• T3
320i0.04¢k
33.89~:2.8
0 PTU
4.12 ±0.04
34.75:!:2.7
120
100 c c
0 E
6O
I10 4O
20J
\
1
\ \
2
SB
3
(pmolmg
FIG.
0
4
5
prot)
1
Scatchard plot obtained by linear regression representing the saturation binding of [3H]muscimol in cortex membranes of controls, T3- and PTU-treated rats, as described in the text. The values are the means of four experiments. P
characteristic of endogenous depression, where the number of brain imipramine receptors is decreased. Our data are, however, at variance with those of Vaccarl (2), who found that the number of cortical imipramine binding sites was decreased by a chronic antithyroid treatment only when i t was administered for the f i r s t 30 days of l i f e , not when i t was performed in mature rats. Yet, Biassoni and Vaccari (28) found that the antithyroid drugs methimazole and PTU increase the binding of [~H]imipramine to rat brain membranes. However, their data were obtained from "in v i t r o " experiments, and the increased binding of [3H]imipramine involved the low- rather that the high-affinity compartment for this ligand; our present data, on the other hand, were obtained "in vivo" by evaluating only the high-affinity compartment. As far as cortical GABA binding sites are concerned, our results show that their number is not modified in hypothyroid rats, while i t is s i g n i f i cantly reduced in hyperthyroid rats.
Vol. 48, No. 7, 1991
Dysthyroidism and Receptors
665
Finally, i t should be stressed that our concurrently-performed "in vitro" experiments allow us to exclude the possibility that the binding of alphal, alpha2, beta and imipramine ligands may have been influenced by the possible presence of T= or PTU in the membrane samples. In conclusion, the present study further support the view that thyroid hormones influence neurotransmission in the CNS. In particular, our findings show that hypothyroidism is associated with a decreased number of adrenoceptors (alphal, alpha2 and beta) and of imipramine receptors in the cerebral cortex, with a decreased number of imipramine receptors in the hypothalamus and with an increased number of imipramine receptors in the hippocampus, whereas hyperthyroidism is associated with an increased number of beta-adrenoceptors and with a decreased number of GABA receptors in the cerebral cortex. Acknowledgements This work was supported in part by grants from "Ministero dell'UniversitA e della Ricerca Scientifica e Tecnologica" and from "Consiglio Nazionale delle Ricerche", Roma. References 1. 2. 3.
4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.
18.
W. EMLEN, D.S. SEGAL and A.J. MANDELL, Science 175 79-82 (1972). A. VACCARI, Br. J. Pharmacol. 84 ?73-?78 (1985). R.S. WILLIAMS and R.J. LEFKOWITZ J.H., Molecular Basis of Thyroid Hormone Action, J.H. Oppenheimer and H.H. Samuels (eds), 325-349, Academic Press, New York (1983). L.T. WILLIAMS, R.J. LEFKOWITZ, A.M. WATANABE, D.R. HATHAWAYand H.R. BESCH, J. Biol. Chem. 252 278?-2789 (1977). G. ENGSTROM, T.H. SVENSSONand B. WALDECK, Brain Res. 77 471-483 (1974). J . H . JACOBI, G. MUELLER and R.J. WURTMAN, Endocrinology 97 1332-1335 (1975). U. STROMBOM, T.H. SVENSSON, D.M. JACKSON and J. ENGSTROM, J. Neural Transm. 41 73-92 (1977). A.J. PRANGE, J.L. MEEK and M.A. LIPTON, L i f e Sci. 9 901-907 (1970). C.K. ATTERWILL, S.J. BUNN, D.J. ATKINSON, S.L. SMITH and D.J. HEAL, J. Neural Transm. 59 43-55 (1984). G. GROSS, O.E. BRODDE and H.J. SCHUMANN, Naunyn-Schmiedeberg's Arch. Pharmacol. 316 45-50 (1981). G. GROSS, and H.J. SCHUMANN, J. Pharm. Pharmacol. 33 552-554 (1981). P.G. ETTIGI, G.M. BROWN, and J.A. SEGGIE, Psychosom. Med. 41 203-208 (1979). G.R. GEWIRTZ,' D. MALASPINA, J.A. HATTERER, S. FEUREISEN, D. KLEIN and J.M. GORMAN, Am. J. Psychiatry 145 1012-1014 (1988). A. PILC and K.G. LLOYD, L i f e Sci. 35 2149-2154 (1984). D. WHEATLEY, Arch. Gen. P s i c h i a t r y 26 229-233 (1972). I.C. WILSON, A.J. PRANGE and P.P. LARA, The t h y r o i d axis~ drugs and behavior, A.J. Prange (ed), 49-62, Raven Press, New York (1974). R.H. GERNER, L. FAIRBANKS, G.M. ANDERSON, J.G. YOUNG, M. SCHEININ, M. LINNOILA, T.A. HARE, B.A. SHAYWITZ and D.J. COHEN, Am. J. Psychiatry 141 1533-1540 (1984). A.J. PRANGE, J.C. GARBUTT and P.T. LOOSEN, PsEchopharmacology: the t h i r d
666
19. 20. 21. 22. 23. 24. 25. 26. 27. 28.
Dysthyroldism and Receptors
Vol. 48, No. 7, 1991
9eneration of progress, H.Y. Meltzer (ed), 629-653, Raven Press, New York (1987). A.J. PATEL, A. RABIE, P.D. LEWIS and R. BALAZS, Brain Res. 104 33-48 (1976). E. MOGILNICKA and M. NIELSEN, Eur. J. Pharmaco]. 12___11107-111 (1986). K.E.J. DICKINSON, R.M. McKERNAN, C.M.N. MILES, K.S. LEYS and P.S. SEVER Eur. J. Pharmaco]. 120 285-293 (1986). R. RAISRAN, M.S. BRILEY, and S.Z. LANGER, Eur. J. Pharmaco]. 61 373-380 (1980). O.H. LOWRY, N.J. ROSENBROUGH, A.L. FARRand R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). S.J. ENNAand S.H. SNYDER, Mol. Pharmacol. 13 442-453 (1977). G. SCATCHARD, Ann. N.Y. Acad. Sci. 51 660-672 (1949). T. CIARALDI and G.V. MARINETTI, Biochem. Biophys. Res. Comm. 74 984-991 (1977). A.W. FOX, E.N. JUBERG, J.M. MAY, R.D. JOHNSON, P.W. ABEL and K.P. MINNEMAN, J. Pharm. Exp. Ther. 235 715-723 (1985). R. BIASSONI and A. VACCARI, Br. J. Pharmacol. 85 (1985) 447-456.
Life Sciences, Vol. 48, pp. 659-666 Printed in the U.S.A.
Pergamon Press
EFFECTS OF THYROID STATUSON THE CHARACTERISTICS OF ALPHAI-, ALPHA2-, BETA, IMIPRAMINE AND GABARECEPTORS IN THE RAT BRAIN Maurizio Sandrini, Donatella Marrama, Anna Valeria Vergoni and Alfio Bertolini Institute of Pharmacology, University of Modena, Via G. Campi 287, 41100 Modena, Italy (Received in final form December II, 1990)
Summary The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alphal, alphaz, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [~H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [~H]prazosin, [~H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [~H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status. Thyroid hormones play an important role in regulating both peripheral and central noradrenergic function (1-4). In experimental models, hypo- and hyperthyroidism respectively decreased and enhanced noradrenaline synthesis and/or turnover in the brain (5-?). I t has also been suggested (8) that thyroid hormones primarily influence receptor sensitivity: indeed, changes in pre- and post-synaptic noradrenergic receptors have been demonstrated in dysthyroid states. Postsynaptic changes included increase in the number of beta- adrenoceptors in the corpus striatum but not in the cortex (9) and increased locomotor responses to alpha-adrenergic agonists in reserpinized mice (5,7) induced by hyperthyroidism, and a decrease in the number of alpha2 and beta brain adrenoceptors induced by hypothyroidism (9-11). These experimental data are in agreement with the clinical observation that T3 potentiates the activity of t r i c y c l i c antidepressants (12-16). Presynaptic alpha2-adrenoceptor function is increased in hyperthyroidism and suppressed in hypothyroidism (9). Moreover, i t seems reasonable to expect that brain imipramine- and GABA binding sites may be altered in dysthyroid states, because hypo- and hyperthyroidism are typically associated with several kinds of mental and behavioral disorders (13,17,18). However, available experimental data are very limited. 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
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The present study was aimed at investigating the effect of experimentally-altered thyroid status (hypo- or hyperthyroidism) on the characteristics of adrenoceptors (alphal, alpha2 and beta), GABA-receptors, and imipramine receptors in different, discrete areas of the rat brain. Methods Adult male rats of a Wistar strain (Morini, S.Polo d'Enza, Reggio nell'Emilia, I t a l y ) , weighing 150-200 g at the beginning of the experiment, were used. They were housed five per cage, under controlled conditions of temperature (22±0.2 °C) and humidity (60%), on a 12 h light/dark cycle (lights on 0700) and with free access to food and water. Hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 ~g/kg of L-triiodothyronine (T~, sodium salt, Sigma Chemical Co., MO, USA) (9). Hypothyroidism was induced by administering propylthiouracil (PTU, Sigma Chemical Co., MO, USA), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks (19). Control rats were daily treated with s.c. injection of T3 solvent (1 ml/kg) and with 2 ml water p.o. Rats were killed by decapitation 2 h after the last treatment, under ether anesthesia. Rats were exsanguinated and blood collected. Each sample was centrifuged at 3,000 rpm for 10 min and serum was immediately separated. T o t a l serum L-tetraiodotyronine (T4) was determined by radioimmunoassay. The brains were removed and immediately placed on an ice-cold surface. The brain regions (hypothalamus, hippocampus and cerebral cortex) were quickly dissected and stored at -20 °C prior to membrane preparation. Alphas- and beta-adrenoceptors were evaluated according to the method of Mogilnicka and Nielsen (20) using [3H]prazosin and [~H]dihydroalprenolol, respectively, as ligands. Alpha2-adrenoceptors were evaluated according to the method of Dickinson et al. (21) using [3H]yohimbine as ligand. Imipramine binding sites were evaluated essentially according to the method of Raisman et al. (22): briefly, brain tissues were homogenized in 20 ml of ice-cold buffer (50 mM Tris HCl, 120 mM NaCl, 5 mM KCl, pH 7.4) using a Polytron homogenizer (setting 6, 15 s); after centrifugation (48,000 x 4) for 10 min at 4 °C, the pellet was resuspended and centrifuged twice in an equal volume (20 ml) of the same ice-cold buffer. The final pellet was resuspended in the same buffer to give a final protein concentration of 400-700 pg/ml. Protein was determined by the method of Lowry et al. (23) using bovine serum albumin as standard. GABA receptors were evaluated according to the method of Enna and Snyder (24) using [~H]muscimol as ligand. Radioligands were obtained from New England Nuclear (Firenze, Italy) and had the following specific activities: [~H]prazosin, 82.0 Ci/mmol; [3H]dihydroalprenolol, 60.0 Ci/mmol; [3H]yohimbine, 87.1Ci/mmol; [3H]imipramine, 73.8 Ci/mmol; [~H]muscimol, 25.3 Ci/mmol. For [~H]dihydroalprenolol, specific binding was defined as that displaced by 10 ~M (±) propranolol and ranged from 50 to 70%. For [3H]prazosin and [~H]yohimbine, specific binding was defined as that displaced by 10 ~M phentofamine and ranged from 70 to 75%. For [3H]imipramine and [~]muscimol, specific
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binding was defined as that displaced by 100 pM imipramine and 1 mM GABA respectively. The following concentrations were used: [~H]dihydroalprenolol from 0.065 to 4.18 riM; [3H]prazosin from 0.037 to 1.2 nM; [~H]yohimbine from 0.15 to 5.00 nM; [3H]imipramine from 0.15 to 5 nM; [3H]muscimol from 7.25 to 240 nM. The results of saturation experiments were analyzed according to Scatchard (25). The equilibrium dissociation constant (K~) and maximum number of binding sites (Bmax) were calculated individually for each sample using 6-7 concentrations of labelled drugs. All assays were done in t r i p l i c a t e . The resulting dissociation constants and binding capacities were summarized as arithmetic means and were compared using the Scheff~ procedure following an analysis of variance. Results Effects of T3 and PTU treatment on thyroid state in rats. Our experimental treatment schedule was able to alter the thyroid status of rats as shown by the the values of serum T4 that were, 5.1±0.3 pg/dl - I , >25 pg/dl - I , and 1.1±0.06 pg/dl - I for saline-, T3-, and PTU-treated rats, respectively (P
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TABLE I Effects of Thyroid Status on alphal-, alpha2- and beta-Receptors in Membrane of Rat Cerebral Cortex. Treatment Li gand
[~H]prazosin
[~H]yohimbi ne
[~H]di hydroal prenol ol
Vehi cl e Bma× Kd Bma~ Kd
Bmax Kd
T~
PTU
205.0 + 4.1
215.3 ± 5.0
0.3 ± 0.1
0.3 ± 0.1
368.2 ± 8.2
349.3 ± 7.8
3.7 + 0.2
3.6 ± 0.2
3.5 ± 0.2
229.3 ± 7 . 6 *
145.0 ± 5 . 6 *
2.0 ± 0.2
2.2 ± 0.2
180.1± 6.2 2.2 ± 0.2
168.0 + 5.0 * 0.3 ± 0.1 282.1 ± 7.0 *
In adult male rats, hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 pg/kg of L-triiodothyronine (T3) and hypothyroidism was induced by administering propyltiouracil (PTU), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks. Control rats were daily treated with s.c. injection of T~ solvent (1 ml/kg) and with 2 ml water p.o. Bm,x is expressed as fmol/mg protein, Kd is expressed as nM. Data are means±S.E.M, from groups of 12 rats. Alphal- and beta adrenoceptors were evaluated using [3H]prazosin and [3H]dihydroalprenolol according to the method of Mogilnicka and Nielsen (16) as ligands, respectively. Alpha2-adrenoceptors were evaluated according to the method of Dickinson et al. (4) using [3H]yohimbine as ligand. * P
(-19.4%) (ANOVA, F=11.81, DF=2,11, P
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TABLE II Effect of Thyroid Status on [3H]Imipramine Binding in Different Rat Brain Regions. Treatment Tissue
Vehicle B,,a,,
6 2 5 . 0 + 20.1
T3
PTU
6 8 5 . 8 + 24.7
4 6 8 . 2 ± 18.2 *
Cortex Kd
Hypothal amus
Hippocampus
3.9 ±
0.3
3.7 ±
0.3
4.0 +
0.4
Bmax
840.2 ± 3 1 . 5
881.3 _+ 3 0 . 6
680.1 _+ 23.6 *
Kd
4.6 ± 0.3
4.4 ± 0.2
4.9 ± 0.3
Bmix
720.1 _+ 2 5 . 2
745.3 _+ 2 3 . 5
970.0 ± 30.8 *
Kd
7.8 ±
0.4
7.6 ±
0.4
7.7 +
0.4
In adult male rats, hyperthyroidism was induced by daily subcutaneous (s.c.) injection for 7 days with 100 ~g/kg of L-triiodothyronine (T3) and hypothyroidism was induced by administering propyltiouracil (PTU), 50 mg/rat/day in 2 ml water, by stomach tube (p.o.) for 5 weeks. Control rats were daily treated with s.c. injection of T3 solvent (1 ml/kg) and with 2 ml water p.o. Bmix is expressed as fmol/mg protein, Kd is expressed as nM. Data are means±S.E.M, from groups of 12 rats. * P
roidism caused a decrease in the number of alphas-, alpha2-, and beta-adrenoceptors. These d a t a are in agreement with those obtained by Gross et al. (10,11), who found a substantial reduction in cortical alphas-, alpha2andbeta-adrenoceptor density in adult hypothyroid rats. On the other hand, Atterwill et al. (9) found a reduction in the number of striatal and hypothalamic beta-adrenoceptors, a reduced function of presynaptic alpha2-adrenoceptors, but n~ change in the number of cortical beta-adrenoceptors, again in adult hypothyroid rats. However, while Gross et al. used a PTU treatment schedule (at least 4 weeks) similar to that adopted in our present study, Atterwill et al. administered PTU for only two weeks. In our hands, hyperthyroidism caused an increase in the number of cortical beta-adrenoceptors, while having no effect on alphal- and alpha2-adrenoceptors. These data again disagree with those of Atterwill et al. (9), who found that hyperthyroidism caused an increase in striatal beta-adrenoceptors, but no change in membranes from cerebral cortex. Others (10) found, on the other hand, an increase in alphal-adrenoceptors in membranes from cerebral cortex in hyperthyroid rats. In such a puzzling picture i t is perhaps worth remembering that hyperthyroidism greatly increases the number of beta-adrenoceptors in tissues outside the CNS (heart ventricle, atria and vas deferens of rats) (26,27). As far as [3H]imipramine binding sites are concerned, our present results show that their number is decreased in the cerebral cortex and hypothalamus and increased in the hippocampus of hypothyroid rats, while i t is not changed in any of these brain areas in hyperthyroid rats. I t is pertinent to recall that several mental and behavioral symptoms occurring in human hypothyroidism are also
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140. Bmax
Kd
• CONTROLS
4.22 • 0.05
33.76*-2.7
• T3
320i0.04¢k
33.89~:2.8
0 PTU
4.12 ±0.04
34.75:!:2.7
120
100 c c
0 E
6O
I10 4O
20J
\
1
\ \
2
SB
3
(pmolmg
FIG.
0
4
5
prot)
1
Scatchard plot obtained by linear regression representing the saturation binding of [3H]muscimol in cortex membranes of controls, T3- and PTU-treated rats, as described in the text. The values are the means of four experiments. P
characteristic of endogenous depression, where the number of brain imipramine receptors is decreased. Our data are, however, at variance with those of Vaccarl (2), who found that the number of cortical imipramine binding sites was decreased by a chronic antithyroid treatment only when i t was administered for the f i r s t 30 days of l i f e , not when i t was performed in mature rats. Yet, Biassoni and Vaccari (28) found that the antithyroid drugs methimazole and PTU increase the binding of [~H]imipramine to rat brain membranes. However, their data were obtained from "in v i t r o " experiments, and the increased binding of [3H]imipramine involved the low- rather that the high-affinity compartment for this ligand; our present data, on the other hand, were obtained "in vivo" by evaluating only the high-affinity compartment. As far as cortical GABA binding sites are concerned, our results show that their number is not modified in hypothyroid rats, while i t is s i g n i f i cantly reduced in hyperthyroid rats.
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Finally, i t should be stressed that our concurrently-performed "in vitro" experiments allow us to exclude the possibility that the binding of alphal, alpha2, beta and imipramine ligands may have been influenced by the possible presence of T= or PTU in the membrane samples. In conclusion, the present study further support the view that thyroid hormones influence neurotransmission in the CNS. In particular, our findings show that hypothyroidism is associated with a decreased number of adrenoceptors (alphal, alpha2 and beta) and of imipramine receptors in the cerebral cortex, with a decreased number of imipramine receptors in the hypothalamus and with an increased number of imipramine receptors in the hippocampus, whereas hyperthyroidism is associated with an increased number of beta-adrenoceptors and with a decreased number of GABA receptors in the cerebral cortex. Acknowledgements This work was supported in part by grants from "Ministero dell'UniversitA e della Ricerca Scientifica e Tecnologica" and from "Consiglio Nazionale delle Ricerche", Roma. References 1. 2. 3.
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