Short Communications 494
Autoradiographic Evidence of Opioid Binding Sites in the Thyroid Gland M. Freire-Garabal, J. L. Balboa, M. J. Nunez, M. T. Castano and A. Belmonte Department of Pharmacology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
Previous investigations showed a direct stimulatory effect of opiates on the thyroid function. Tal, Koranyi, Kovacs and Endroczi (1984) found that single doses of morphine injection produced a significant rise of the serum T3 and T4 levels, with a concomitant suppression of the pituitary TSH secretion. It was also observed that morphine induced a thyroxine release from the rat thyroid gland in vitro, and resulted in an accumultion of cAMP in the thyroid tissue (Tal, Kovacs, Koranyi and Endroczi 1986). Nevertheless, the possible role of opiate receptors at target cell in the thyroid gland remains to be elucidated. In the present paper we studied the autoradiographic localization of [3H]Enkephalin in slides of thyroid tissue of saline, naloxone and morphine pretreated rats. Materials and Methods Male rats of the Sprague-Dawley strain, weighing 250—300 g (Interfauna Iberica S.A., Barcelona, Spain) were used. They were housed, six per cage, in a temperature (22—24 °C) and humidity controlled animal room, with an alternating light-dark cycle (lights on at 0600, lights off at 1800), with food (PANLAB Diet A.03) and water ad libitum. The animals were submitted to different treatments according to the group they were assigned to: Group A. 0.9% saline i.p., Group B. Naloxone (2 mg/kg i.p.) (Naloxone HC1, Abello Laboratories, Madrid, Spain), Group C. Morphine (1, 2, 4 and 8 mg/kg i.m.) (Morphine HC1, Sigma Chemical Co., St. Louis, MO, USA). The volume of all injections was 1 ml/kg body weight. One hour after drug administration, rats were intravenously injected with 0.3 ml of a solution prepared with 250 uCi of (20 Ci/mmol) [3H]Enkephalin ([Tyrosyl-3,5-H]Enkepha\m (5-Lleucine) (Amersham Co. England) in 10 ml of H2O and, 10 minutes later, they were sacrificed by decapitation. At time of death, the thyroid gland was quickly removed. The samples were fixed in a 10% neutral buffered formaldehyde solution for 24 hours and entirely embedded in paraffin. Serial sections 2—4 u, thick were cut, and placed on gelatin-coated slides. Slides were dipped in emulsion (LM-1. Amersham Co. England) and exposed for 45 days, and then developed and stained with hematoxylin-eosin. Several slides from each animal were quantified for autoradiographic grain density. At least three slides were selected as close as possible to each designated histological level, and four squares representing 500 u2, each were automatically counted (MIP. Microm SA, Spain). Background activity was determined over areas of the slide without tissue. The net counts were then averaged and reported with the standard error of the mean. Student's t-test was used to determine statistical significance between groups. Horm. metab. Res. 24 (1992) 494-495 © Georg Thieme VerlagStuttgart.New York
Results The autoradiographic grain density was examined in several parts of the thyroid gland and expressed as mean+lSD (Table 1). There was a significant drop in autoradiopgrahic grain density in rats pretreated with naloxone and morphine (Differences p < 0.05). Table 1 [ 3H]Enkephalin binding to the thyroid gland tissue of rats, expressed as the mean of grain counts/500 u2 for a representative 6 animals per group and percentage decreases in the naloxone and morphine treated rats. (*p < 0.05 compared with saline; *p < 0.01 compared with saline). Grain density (gr/500 u2)
%
Group
Treatment
A
Saline
54.1 ±8.7
B
Naloxone
20.3 ±5.9
-62.4
C
Morphine 1 mg/kg 2 mg/kg 4 mg/kg 8 mg/kg
41.2* ±9.5 32.5* ±4.8 28.2* ±5.9 23.6* ±2.9
-23.8 -39.9 -47.8 -56.3
Discussion In the present investigation, we observed a high grain density, which reflects the density of H]Enkephalin binding sites, of 84.2 ±4.8 grains/u2. , in control rats injected with saline. There is a significant decrease in autoradiographic grain density in most portions of the thyroid of rats pretreated with naloxone which indicates a specific binding of [ H ]Enkephalin to these sites. Morphine induced a dosedependent decrease in grain counts as expression of saturability. The inactive (+)-isomer of morphine or naloxone did not block the binding. Our results are in good agreement with those of Tal et al. (1984) who first observed that opiates may exert a short-term stimulatory effect on the thyroid gland. Such observations were also in accordance with those of Redding, Bowers and Schally 1966) who found that a single injection of morphine markedly increased 131 I release from the thyroid glands of mice. Later studies under in vitro conditions (Tal et al. 1986) showed that morphine induced the release of T4 and the accumulation of cAMP in the thyroid tissue. Moreover, the administration of naloxone did not change the T4 release, but prevented the morphine-induced changes and inhibited the accumulation of cAMP in the thyroid tissue. The latter data were interpreted as a result of the activation of a secretory mechanism at target organ level. Received: 13 Aug. 1991
Accepted: 29 June 1992 after revision
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Introduction
Horm. metab. Res. 24 (1992)
Opioid Binding Sites in Thyroid Gland Nevertheless, the possible existence of opiate receptors at target cell level in the thyroid gland remained to be elucidated. Although further studies must be performed, our data at present support the evidence of opioid binding sites in the thyroid gland, which may be involved in the short-term stimulatory effect of opiates on the thyroid gland of rats.
Acknowledgements
495
Requests for reprints should be addressed to: Dr. Manuel Freire-Garabal Huerfanas 19-2 15703 Santiago de Compostela Spain
We wish to express our gratitude to M. E. Vilarino and to J. A. Veira for their technical support and to G. Costoya and P. Fernandez-Vila for their bibliographic assistance.
Redding.T. W.,C. Y.Bowers, A. V. Schally: Acta Endocrinol. 51:391 — 399(1966) Tal, E. L. Koranyi, Z. S. Kovacs, E. Endroczi: Acta Endocrinol. 105: 511-514(1984) Tal, E., Z. S. Kovacs, L. Koranyi, E. Endroczi: Horm. Metab. Res. 18: 238-240 (1986)
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References
Autoradiographic Evidence of Opioid Binding Sites in the Thyroid Gland M. Freire-Garabal, J. L. Balboa, M. J. Nunez, M. T. Castano and A. Belmonte Department of Pharmacology, School of Medicine, University of Santiago de Compostela, Santiago de Compostela, Spain
Previous investigations showed a direct stimulatory effect of opiates on the thyroid function. Tal, Koranyi, Kovacs and Endroczi (1984) found that single doses of morphine injection produced a significant rise of the serum T3 and T4 levels, with a concomitant suppression of the pituitary TSH secretion. It was also observed that morphine induced a thyroxine release from the rat thyroid gland in vitro, and resulted in an accumultion of cAMP in the thyroid tissue (Tal, Kovacs, Koranyi and Endroczi 1986). Nevertheless, the possible role of opiate receptors at target cell in the thyroid gland remains to be elucidated. In the present paper we studied the autoradiographic localization of [3H]Enkephalin in slides of thyroid tissue of saline, naloxone and morphine pretreated rats. Materials and Methods Male rats of the Sprague-Dawley strain, weighing 250—300 g (Interfauna Iberica S.A., Barcelona, Spain) were used. They were housed, six per cage, in a temperature (22—24 °C) and humidity controlled animal room, with an alternating light-dark cycle (lights on at 0600, lights off at 1800), with food (PANLAB Diet A.03) and water ad libitum. The animals were submitted to different treatments according to the group they were assigned to: Group A. 0.9% saline i.p., Group B. Naloxone (2 mg/kg i.p.) (Naloxone HC1, Abello Laboratories, Madrid, Spain), Group C. Morphine (1, 2, 4 and 8 mg/kg i.m.) (Morphine HC1, Sigma Chemical Co., St. Louis, MO, USA). The volume of all injections was 1 ml/kg body weight. One hour after drug administration, rats were intravenously injected with 0.3 ml of a solution prepared with 250 uCi of (20 Ci/mmol) [3H]Enkephalin ([Tyrosyl-3,5-H]Enkepha\m (5-Lleucine) (Amersham Co. England) in 10 ml of H2O and, 10 minutes later, they were sacrificed by decapitation. At time of death, the thyroid gland was quickly removed. The samples were fixed in a 10% neutral buffered formaldehyde solution for 24 hours and entirely embedded in paraffin. Serial sections 2—4 u, thick were cut, and placed on gelatin-coated slides. Slides were dipped in emulsion (LM-1. Amersham Co. England) and exposed for 45 days, and then developed and stained with hematoxylin-eosin. Several slides from each animal were quantified for autoradiographic grain density. At least three slides were selected as close as possible to each designated histological level, and four squares representing 500 u2, each were automatically counted (MIP. Microm SA, Spain). Background activity was determined over areas of the slide without tissue. The net counts were then averaged and reported with the standard error of the mean. Student's t-test was used to determine statistical significance between groups. Horm. metab. Res. 24 (1992) 494-495 © Georg Thieme VerlagStuttgart.New York
Results The autoradiographic grain density was examined in several parts of the thyroid gland and expressed as mean+lSD (Table 1). There was a significant drop in autoradiopgrahic grain density in rats pretreated with naloxone and morphine (Differences p < 0.05). Table 1 [ 3H]Enkephalin binding to the thyroid gland tissue of rats, expressed as the mean of grain counts/500 u2 for a representative 6 animals per group and percentage decreases in the naloxone and morphine treated rats. (*p < 0.05 compared with saline; *p < 0.01 compared with saline). Grain density (gr/500 u2)
%
Group
Treatment
A
Saline
54.1 ±8.7
B
Naloxone
20.3 ±5.9
-62.4
C
Morphine 1 mg/kg 2 mg/kg 4 mg/kg 8 mg/kg
41.2* ±9.5 32.5* ±4.8 28.2* ±5.9 23.6* ±2.9
-23.8 -39.9 -47.8 -56.3
Discussion In the present investigation, we observed a high grain density, which reflects the density of H]Enkephalin binding sites, of 84.2 ±4.8 grains/u2. , in control rats injected with saline. There is a significant decrease in autoradiographic grain density in most portions of the thyroid of rats pretreated with naloxone which indicates a specific binding of [ H ]Enkephalin to these sites. Morphine induced a dosedependent decrease in grain counts as expression of saturability. The inactive (+)-isomer of morphine or naloxone did not block the binding. Our results are in good agreement with those of Tal et al. (1984) who first observed that opiates may exert a short-term stimulatory effect on the thyroid gland. Such observations were also in accordance with those of Redding, Bowers and Schally 1966) who found that a single injection of morphine markedly increased 131 I release from the thyroid glands of mice. Later studies under in vitro conditions (Tal et al. 1986) showed that morphine induced the release of T4 and the accumulation of cAMP in the thyroid tissue. Moreover, the administration of naloxone did not change the T4 release, but prevented the morphine-induced changes and inhibited the accumulation of cAMP in the thyroid tissue. The latter data were interpreted as a result of the activation of a secretory mechanism at target organ level. Received: 13 Aug. 1991
Accepted: 29 June 1992 after revision
Downloaded by: National University of Singapore. Copyrighted material.
Introduction
Horm. metab. Res. 24 (1992)
Opioid Binding Sites in Thyroid Gland Nevertheless, the possible existence of opiate receptors at target cell level in the thyroid gland remained to be elucidated. Although further studies must be performed, our data at present support the evidence of opioid binding sites in the thyroid gland, which may be involved in the short-term stimulatory effect of opiates on the thyroid gland of rats.
Acknowledgements
495
Requests for reprints should be addressed to: Dr. Manuel Freire-Garabal Huerfanas 19-2 15703 Santiago de Compostela Spain
We wish to express our gratitude to M. E. Vilarino and to J. A. Veira for their technical support and to G. Costoya and P. Fernandez-Vila for their bibliographic assistance.
Redding.T. W.,C. Y.Bowers, A. V. Schally: Acta Endocrinol. 51:391 — 399(1966) Tal, E. L. Koranyi, Z. S. Kovacs, E. Endroczi: Acta Endocrinol. 105: 511-514(1984) Tal, E., Z. S. Kovacs, L. Koranyi, E. Endroczi: Horm. Metab. Res. 18: 238-240 (1986)
Downloaded by: National University of Singapore. Copyrighted material.
References
