92
Episodic Secretion of Thyroxine (T4) and Triiodothyronine (T3) from Porcine Thyroid Fragments in Vitro W. A. Scherbaum, N. G. Morgenthaler and W. G. Rossmanith * Abteilung Innere Medizin I, Medizinische Klinik und Poliklinik und *Abteilung Frauenheilkunde und Geburtshilfe, Universitat Ulm, Ulm, Germany
Pulsatile hormone secretion has been recognized for all tropic hormones including thyrotropin (TSH) both in vivo and in vitro (Rossmanith, Mortola, Laughlin and Yen 1988; Rossmanith, Yen and Rasmussen 1989). The possibility of an autonomous pulsatile activity of the thyroid gland has not thus far been considered. An in vivo approach to solve this question is not appropriate due to the long biological half-life of thyroid hormones, the high binding capacity of plasma proteins, and the shift of hormone between different compartments (Greenspan, Klibanski, Schoenfeld and Ridgway 1986). We therefore utilized an in vitro system and perfused isolated thyroid fragments containing dispersed follicles, which are considered the smallest functional entities of the thyroid gland.
Results 7 perifusions were analysed for T4, and 6 for T3. Hormone pulses were detected in all the perifusions. The mean fraction concentration of T4 was 64.5 + 18.2 ng/ml, and for T3 1.0 ±0.17 ng/ml. The pulse periodicity of T4 was 49.2 ±7.7, and for T3 38.7 ± 2.3 min/pulse. Under basal conditions, the pulse amplitude of T4 was 16.8 + 4.1, and for T3 0.26 + 0.04 ng/ml (see Figures 1A and B). No significant pulses were detected in the control experiments. As shown in Figures 2A and B, the release amplitudes of both, T3 and T4 appeared to increase in response to TSH stimulation while the pulse frequency remained unchanged.
Materials and Methods Porcine thyroid follicles were obtained from thyroid glands and put on ice. Tissue was placed in medium 199 containing 0.05% BSA and 10000 U/l penicillin/streptomycin (Sigma, Munich, Germany). It was minced with scissors and razor blades and pressed through a nylon mesh of 2.5 mm diameter. Approximately 500 mg of tissue were placed in a perifusion column of 2.5 ml volume and perfused with oxygenated (95% O2 and 5% CO2) medium 199 at a constant flow rate of 100 (ul/min. The temperature of the column was kept constant at 38.5 °C by a recirculating water bath. The effluent was collected on ice at 4—6 min intervals for up to 420 min. For the stimulation tests, bovine thyrotropin (TSH) (Sigma) was used at a concentration of 10 mU/ml. To evaluate for experimental variability, a control run was performed with medium containing triiodothyronine (T3) and thyroxine (T4) at concentrations equivalent to those observed under experimental conditions. At the end of each test, samples were frozen and kept at — 20 °C until further testing. In all samples T3 and T4 were determined by commercial radioimmunoassays (Henning, Berlin, Germany). In these assays the lower limit of detection for T3 was 0.2 ng/ml, and for T4 it was 2.5 ng/ml. A computerized version of the PULSAR pulse detection program (Merriam and Wachter 1982) searched for significant secretory episodes within the hormone data series. The criteria of defining pulses were based upon the standard deviation of the control perifusion. To be designated a pulse, the concentrations of five consecutive fractions had to exceed 2,1.5,1.2,1 and 0.8 times the standard deviation over the smoothed baseline which was composed of 1/3 of the perifusion time.
Horm. metab. Res.23(1991)92-93 © Georg Thieme Verlag Stuttgart -New York
Fig. 1 Triiodothyronine (T3 - (A)), and thyroxine (T4 - (B)), concentrations in the perifusion fractions of isolated porcine thyroid fragments. Asterisks (*) indicate significant secretory episodes defined by the PULSAR program.
Received: 9 Nov. 1990
Accepted: 13 Nov. 1990
Downloaded by: National University of Singapore. Copyrighted material.
Introduction
Episodic Secretion of T4 and T3 From Porcine Thyroid Fragments in Vitro
Horm. metab. Res. 23 (1991)
93
Discussion Perifusing thyroid fragments in vitro, we were able to demonstrate an episodic release of T3 as well as T4. By means of the established PULSAR pulse detecting algorithm, clear secretory episodes were identified approximately every 50 minutes for T4 and every 40 minutes for T3. By contrast, no significant pulses were detected in the control perifusion. This observation indicates that the pulses identified truly represented changes in the secretory activity rather than random fluctuations. Furthermore, it is important to mention that these pulses occurred in the absence of a TSH stimulatory influence. The fact that our system responded to TSH stimulation by an increase of T3 and T4 indicates that the cell integrity was preserved under our experimental conditions. The release frequencies of T3 and T4 secretion did not change in response to TSH stimulation, suggesting that the release pattern is an inherent feature of these cells. These results are in support of the idea that the episodic secretory activity is a general release pattern of all endocrine glands, a mechanism that may economize the transmission of signals (Romijn, Adriaanse, Brabant, Prank, Endert and Wiersinga 1990). Acknowledgements We like to thank Prof. Dr. Drs. h. c. E. F. Pfeiffer and Prof. Dr. C. Lauritzen for their continuous support.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sche 225/3-2, to W. A. S. and Ro 657/2-1, to W.G.R.). References Greenspan, S. L., A. Klibanski, D. Schoenfeld, E. C. Ridgway: J. Clin. Endocrinol. Metab. 63:661-668 (1986) Merriam, G. R., K. W. Wachter: Am. J. Physiol. 243: E310-E318 (1982) Romijn, J. A., R. Adriaanse, G. Brabant, K. Prank, E. Endert, W. M. Wiersinga:!. Clin. Endocrinol. Metab. 70:1631-1636 (1990) Rossmanith, W. G., J. F. Mortola, G. A. Laughlin, S. S. C. Yen: J. Clin. Endocrinol. Metab. 67:560-564(1988) Rossmanith, W. G., S. S. C. Yen, D. D. Rasmussen: J. Neuroendocrinous!-94(1989)
Requests for reprints should be addressed to: Prof. Dr. W. A. Scherbaum Abteilung Innere Medizin I Medizinische Klinik und Poliklinik der Universitat Ulm Robert-Koch-StraBe 8 D-7900 Ulm (Germany)
Downloaded by: National University of Singapore. Copyrighted material.
Fig 2 Triiodothyronine (T3 - (A)) and thyroxine (T4 (B)) concentrations in the perifusion fractions during basal and TSH-stimulated conditions. Asterisks (*) indicate significant secretory episodes defined by the PULSAR program.
Episodic Secretion of Thyroxine (T4) and Triiodothyronine (T3) from Porcine Thyroid Fragments in Vitro W. A. Scherbaum, N. G. Morgenthaler and W. G. Rossmanith * Abteilung Innere Medizin I, Medizinische Klinik und Poliklinik und *Abteilung Frauenheilkunde und Geburtshilfe, Universitat Ulm, Ulm, Germany
Pulsatile hormone secretion has been recognized for all tropic hormones including thyrotropin (TSH) both in vivo and in vitro (Rossmanith, Mortola, Laughlin and Yen 1988; Rossmanith, Yen and Rasmussen 1989). The possibility of an autonomous pulsatile activity of the thyroid gland has not thus far been considered. An in vivo approach to solve this question is not appropriate due to the long biological half-life of thyroid hormones, the high binding capacity of plasma proteins, and the shift of hormone between different compartments (Greenspan, Klibanski, Schoenfeld and Ridgway 1986). We therefore utilized an in vitro system and perfused isolated thyroid fragments containing dispersed follicles, which are considered the smallest functional entities of the thyroid gland.
Results 7 perifusions were analysed for T4, and 6 for T3. Hormone pulses were detected in all the perifusions. The mean fraction concentration of T4 was 64.5 + 18.2 ng/ml, and for T3 1.0 ±0.17 ng/ml. The pulse periodicity of T4 was 49.2 ±7.7, and for T3 38.7 ± 2.3 min/pulse. Under basal conditions, the pulse amplitude of T4 was 16.8 + 4.1, and for T3 0.26 + 0.04 ng/ml (see Figures 1A and B). No significant pulses were detected in the control experiments. As shown in Figures 2A and B, the release amplitudes of both, T3 and T4 appeared to increase in response to TSH stimulation while the pulse frequency remained unchanged.
Materials and Methods Porcine thyroid follicles were obtained from thyroid glands and put on ice. Tissue was placed in medium 199 containing 0.05% BSA and 10000 U/l penicillin/streptomycin (Sigma, Munich, Germany). It was minced with scissors and razor blades and pressed through a nylon mesh of 2.5 mm diameter. Approximately 500 mg of tissue were placed in a perifusion column of 2.5 ml volume and perfused with oxygenated (95% O2 and 5% CO2) medium 199 at a constant flow rate of 100 (ul/min. The temperature of the column was kept constant at 38.5 °C by a recirculating water bath. The effluent was collected on ice at 4—6 min intervals for up to 420 min. For the stimulation tests, bovine thyrotropin (TSH) (Sigma) was used at a concentration of 10 mU/ml. To evaluate for experimental variability, a control run was performed with medium containing triiodothyronine (T3) and thyroxine (T4) at concentrations equivalent to those observed under experimental conditions. At the end of each test, samples were frozen and kept at — 20 °C until further testing. In all samples T3 and T4 were determined by commercial radioimmunoassays (Henning, Berlin, Germany). In these assays the lower limit of detection for T3 was 0.2 ng/ml, and for T4 it was 2.5 ng/ml. A computerized version of the PULSAR pulse detection program (Merriam and Wachter 1982) searched for significant secretory episodes within the hormone data series. The criteria of defining pulses were based upon the standard deviation of the control perifusion. To be designated a pulse, the concentrations of five consecutive fractions had to exceed 2,1.5,1.2,1 and 0.8 times the standard deviation over the smoothed baseline which was composed of 1/3 of the perifusion time.
Horm. metab. Res.23(1991)92-93 © Georg Thieme Verlag Stuttgart -New York
Fig. 1 Triiodothyronine (T3 - (A)), and thyroxine (T4 - (B)), concentrations in the perifusion fractions of isolated porcine thyroid fragments. Asterisks (*) indicate significant secretory episodes defined by the PULSAR program.
Received: 9 Nov. 1990
Accepted: 13 Nov. 1990
Downloaded by: National University of Singapore. Copyrighted material.
Introduction
Episodic Secretion of T4 and T3 From Porcine Thyroid Fragments in Vitro
Horm. metab. Res. 23 (1991)
93
Discussion Perifusing thyroid fragments in vitro, we were able to demonstrate an episodic release of T3 as well as T4. By means of the established PULSAR pulse detecting algorithm, clear secretory episodes were identified approximately every 50 minutes for T4 and every 40 minutes for T3. By contrast, no significant pulses were detected in the control perifusion. This observation indicates that the pulses identified truly represented changes in the secretory activity rather than random fluctuations. Furthermore, it is important to mention that these pulses occurred in the absence of a TSH stimulatory influence. The fact that our system responded to TSH stimulation by an increase of T3 and T4 indicates that the cell integrity was preserved under our experimental conditions. The release frequencies of T3 and T4 secretion did not change in response to TSH stimulation, suggesting that the release pattern is an inherent feature of these cells. These results are in support of the idea that the episodic secretory activity is a general release pattern of all endocrine glands, a mechanism that may economize the transmission of signals (Romijn, Adriaanse, Brabant, Prank, Endert and Wiersinga 1990). Acknowledgements We like to thank Prof. Dr. Drs. h. c. E. F. Pfeiffer and Prof. Dr. C. Lauritzen for their continuous support.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sche 225/3-2, to W. A. S. and Ro 657/2-1, to W.G.R.). References Greenspan, S. L., A. Klibanski, D. Schoenfeld, E. C. Ridgway: J. Clin. Endocrinol. Metab. 63:661-668 (1986) Merriam, G. R., K. W. Wachter: Am. J. Physiol. 243: E310-E318 (1982) Romijn, J. A., R. Adriaanse, G. Brabant, K. Prank, E. Endert, W. M. Wiersinga:!. Clin. Endocrinol. Metab. 70:1631-1636 (1990) Rossmanith, W. G., J. F. Mortola, G. A. Laughlin, S. S. C. Yen: J. Clin. Endocrinol. Metab. 67:560-564(1988) Rossmanith, W. G., S. S. C. Yen, D. D. Rasmussen: J. Neuroendocrinous!-94(1989)
Requests for reprints should be addressed to: Prof. Dr. W. A. Scherbaum Abteilung Innere Medizin I Medizinische Klinik und Poliklinik der Universitat Ulm Robert-Koch-StraBe 8 D-7900 Ulm (Germany)
Downloaded by: National University of Singapore. Copyrighted material.
Fig 2 Triiodothyronine (T3 - (A)) and thyroxine (T4 (B)) concentrations in the perifusion fractions during basal and TSH-stimulated conditions. Asterisks (*) indicate significant secretory episodes defined by the PULSAR program.
