Biología
Laboratorio de Bioquímica Nuclear-Depto. Nuclear Aplicada, Comisión Nacional de Energía Atómica, Av. del Libertador 8250, Buenos Aires
STUDIES ON THE MECHANISM OF ACTION OF POTASSIUM IODIDE ON THYROID PROTEIN BIOSYNTHESIS
By Mario A. Pisarev1) and Leonardo O. Aiello
ABSTRACT Potassium iodide (KI) has been shown to have an antigoitrogenic action and to inhibit in vivo thyroid protein biosynthesis. Beef thyroid slices were used to clarify further the mechanism of action of KI. Incubations were performed in Krebs-Ringer-bicarbonate (KRB) buffer under 95 % O2 and 5 % CO2. KI caused a slight decrease in the uptake of [3H]leucine by the tissue. When labelled leucine incorporation into protein was measured it was found that 10\m=-\6 m KI caused a marked inhibition. Increasing concentrations of KI up to 10\m=-\3 m did not further increase this inhibition. This effect of KI was reduced by simultaneous addition of 0.5 mm KClO4 or 1 mm methylmercaptoimidazole (MMI). In several experiments it was found that equimolar amounts of thyroxine (T4) or triiodothyronine (T3) were more potent than KI in inhibiting thyroid protein biosynthesis. In double labelled studies KI decreased [3H] leucine incorporation into thyroid soluble proteins and into immunoprecipitable thyroglobulin (Tg) while it did not modify that of [14C]galactosamine. When tissue specificity was examined, KI failed to alter [3H] leucine incorporation into proteins either in the liver or in the submaxillary gland. The present results indicate that intracellular KI is necessary to exert its effect on protein synthesis, and that this effect is mediated through a organic form of iodine, probably iodothyronines. This action of KI is specific for the thyroid gland.
Supported by grants of the CONICET and CNEA, Argentina. International Thyroid Conference, Boston, 1975
Presented at the 7th
•) Established investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Argentina.
298
Potassium iodide (KI) has been used for many years in the treatment of goitre and in the amelioration of Graves' disease symptoms. KI inhibits thyroid hor¬ mone formation (Wolff 8c Chaikoff 1948) and other thyroid parameters (see review by Wolff 1969). KI partially blocks the goitrogenic action of thyrotro¬ phin (TSH) in rats (Katakai et al. 1966) and of TSH, 3'5'cyclic adenosinemonophosphate (cyclic AMP) and 3'5'cyclic guanosinemonophosphate (cyclic GMP) in mice [Pisarev 8c Itoiz 1972). The antigoitrogenic action of KI has been correlated with its inhibitory effects on thyroid protein biosynthesis. How¬ ever, very little attention has been paid to the mechanism of action of KI on this parameter and the following studies were therefore performed.
MATERIALS AND METHODS Beef thyroid glands were obtained from a local slaughterhouse immediately after sacri¬ fice and carried to the laboratory in cold saline. The glands were dissected and freed from fat and connective tissue. Slices (40-100 mg) were obtained with a Stadie-Riggs microtome (A. Thomas, USA) and pre-incubated, usually for 30 min, in 2 ml of Krebs-Ringer-bicarbonate buffer (KRB) pH 7.2 at 37°C under an atmosphere of 95 % 02 and 5 °/o C02, with shaking. KI or other substances to be tested were included during the pre-incubation. At the end of this period a tracer dose (usually around 1 fiCi) of [3H]L-leucine [4,5-3H]leucine; 44.2 Ci/mmole; New England Nuclear, USA) was added and the incubation continued for another 60 min. In other studies incuba¬ tions were performed in the presence of [14C]D-galactosamine ([l-l4C]D-galactosamine; 34.6 mCi/mmole; New England Nuclear, USA), in addition to the [3H] leucine, during 120 min. At the end of the incubation the slices were washed thoroughly in either cold saline or KRB, blotted on filter paper, weighed, and homogenized with 1 ml of distilled water in all-glass Potter-Elvehjem type tissue homogeniser. To 0.3 ml of homogenate 0.5 ml of 20 °/o cold trichloroacetic acid, (TCA) was added, agitated and left overnight in the cold. After centrifuging at 3000 r. p. m. for 20 min (International refrigerated centrifuge) the precipitate was washed twice and digested with 1 ml of Protosol (New England Nuclear, USA) at 37°C. Aliquots of the homogenate were similarly treated. When the biosynthesis of soluble proteins was analyzed the thyroid slices were homogenized in 0.25 M sucrose in phosphate buffer pH 7.5. After centrifuging at 100 000 x g for 1 h (or 40 000 x g for 150 min) in a Spinco L2-65 centrifuge (Beckman Instr., USA) aliquots of the supernatant were examined for its total radioactivity, TCA precipitable and immunoprecipitable thyroglobulin (Tg). For this last procedure rabbit anti Tg serum and sheep anti-rabbit gamma globulin serum were added as described by Vassart 8c Dumont (1972). The precipitates were digested with Protosol and the radioactivity counted. The samples thus prepared were mixed with 10 ml of Ominifluor (New England Nuclear, USA) and counted in a Packard tri-carb liquid scintillation counter. In the double labelling studies appropriate corrections were made in order to determine the actual values of 3H and I4C counts. All samples were run in quadriplicate. Protein was determined in each sample in duplicate according to the method of Lowry et al. (1951). Statistical analysis was performed according to Student's ¿-test.
299
Table 1. Time-course inhibition of [3H]leucine
incorporation.
TCA
precipitable radioactivity DPM/fig protein
Treatment
Control Kl 10-4 m Control Kl 10-4 M Pre-incubation time slices + 1
519 245 743 454
30 30 60 60
was
P
Laboratorio de Bioquímica Nuclear-Depto. Nuclear Aplicada, Comisión Nacional de Energía Atómica, Av. del Libertador 8250, Buenos Aires
STUDIES ON THE MECHANISM OF ACTION OF POTASSIUM IODIDE ON THYROID PROTEIN BIOSYNTHESIS
By Mario A. Pisarev1) and Leonardo O. Aiello
ABSTRACT Potassium iodide (KI) has been shown to have an antigoitrogenic action and to inhibit in vivo thyroid protein biosynthesis. Beef thyroid slices were used to clarify further the mechanism of action of KI. Incubations were performed in Krebs-Ringer-bicarbonate (KRB) buffer under 95 % O2 and 5 % CO2. KI caused a slight decrease in the uptake of [3H]leucine by the tissue. When labelled leucine incorporation into protein was measured it was found that 10\m=-\6 m KI caused a marked inhibition. Increasing concentrations of KI up to 10\m=-\3 m did not further increase this inhibition. This effect of KI was reduced by simultaneous addition of 0.5 mm KClO4 or 1 mm methylmercaptoimidazole (MMI). In several experiments it was found that equimolar amounts of thyroxine (T4) or triiodothyronine (T3) were more potent than KI in inhibiting thyroid protein biosynthesis. In double labelled studies KI decreased [3H] leucine incorporation into thyroid soluble proteins and into immunoprecipitable thyroglobulin (Tg) while it did not modify that of [14C]galactosamine. When tissue specificity was examined, KI failed to alter [3H] leucine incorporation into proteins either in the liver or in the submaxillary gland. The present results indicate that intracellular KI is necessary to exert its effect on protein synthesis, and that this effect is mediated through a organic form of iodine, probably iodothyronines. This action of KI is specific for the thyroid gland.
Supported by grants of the CONICET and CNEA, Argentina. International Thyroid Conference, Boston, 1975
Presented at the 7th
•) Established investigator of the Consejo Nacional de Investigaciones Científicas y Técnicas
(CONICET), Argentina.
298
Potassium iodide (KI) has been used for many years in the treatment of goitre and in the amelioration of Graves' disease symptoms. KI inhibits thyroid hor¬ mone formation (Wolff 8c Chaikoff 1948) and other thyroid parameters (see review by Wolff 1969). KI partially blocks the goitrogenic action of thyrotro¬ phin (TSH) in rats (Katakai et al. 1966) and of TSH, 3'5'cyclic adenosinemonophosphate (cyclic AMP) and 3'5'cyclic guanosinemonophosphate (cyclic GMP) in mice [Pisarev 8c Itoiz 1972). The antigoitrogenic action of KI has been correlated with its inhibitory effects on thyroid protein biosynthesis. How¬ ever, very little attention has been paid to the mechanism of action of KI on this parameter and the following studies were therefore performed.
MATERIALS AND METHODS Beef thyroid glands were obtained from a local slaughterhouse immediately after sacri¬ fice and carried to the laboratory in cold saline. The glands were dissected and freed from fat and connective tissue. Slices (40-100 mg) were obtained with a Stadie-Riggs microtome (A. Thomas, USA) and pre-incubated, usually for 30 min, in 2 ml of Krebs-Ringer-bicarbonate buffer (KRB) pH 7.2 at 37°C under an atmosphere of 95 % 02 and 5 °/o C02, with shaking. KI or other substances to be tested were included during the pre-incubation. At the end of this period a tracer dose (usually around 1 fiCi) of [3H]L-leucine [4,5-3H]leucine; 44.2 Ci/mmole; New England Nuclear, USA) was added and the incubation continued for another 60 min. In other studies incuba¬ tions were performed in the presence of [14C]D-galactosamine ([l-l4C]D-galactosamine; 34.6 mCi/mmole; New England Nuclear, USA), in addition to the [3H] leucine, during 120 min. At the end of the incubation the slices were washed thoroughly in either cold saline or KRB, blotted on filter paper, weighed, and homogenized with 1 ml of distilled water in all-glass Potter-Elvehjem type tissue homogeniser. To 0.3 ml of homogenate 0.5 ml of 20 °/o cold trichloroacetic acid, (TCA) was added, agitated and left overnight in the cold. After centrifuging at 3000 r. p. m. for 20 min (International refrigerated centrifuge) the precipitate was washed twice and digested with 1 ml of Protosol (New England Nuclear, USA) at 37°C. Aliquots of the homogenate were similarly treated. When the biosynthesis of soluble proteins was analyzed the thyroid slices were homogenized in 0.25 M sucrose in phosphate buffer pH 7.5. After centrifuging at 100 000 x g for 1 h (or 40 000 x g for 150 min) in a Spinco L2-65 centrifuge (Beckman Instr., USA) aliquots of the supernatant were examined for its total radioactivity, TCA precipitable and immunoprecipitable thyroglobulin (Tg). For this last procedure rabbit anti Tg serum and sheep anti-rabbit gamma globulin serum were added as described by Vassart 8c Dumont (1972). The precipitates were digested with Protosol and the radioactivity counted. The samples thus prepared were mixed with 10 ml of Ominifluor (New England Nuclear, USA) and counted in a Packard tri-carb liquid scintillation counter. In the double labelling studies appropriate corrections were made in order to determine the actual values of 3H and I4C counts. All samples were run in quadriplicate. Protein was determined in each sample in duplicate according to the method of Lowry et al. (1951). Statistical analysis was performed according to Student's ¿-test.
299
Table 1. Time-course inhibition of [3H]leucine
incorporation.
TCA
precipitable radioactivity DPM/fig protein
Treatment
Control Kl 10-4 m Control Kl 10-4 M Pre-incubation time slices + 1
519 245 743 454
30 30 60 60
was
P
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