Influence of thyroid autoantibodies on thyroid cellular growth in vitro

Porcine thyroid follicle cells, cultured in suspension, were employed to investigate the effects of immunoglobulin preparations from patients with colloid goitre, Graves' disease or Hashimoto's thyroiditis on thyroid growth in vitro. Epidermal growth factor (EGF, 19 ng/ml) was used as a reference for maximum growth stimulation and produced a 9-fold increase in [3H]thymidine incorporation. Immunoglobulins (1000 micrograms/ml) were found to increase [3H]thymidine incorporation compared to control: from 10 normal individuals 32 +/- 4% (mean +/- SEM, % of EGF response), from 10 patients with colloid goitre 26 +/- 4% (not significantly different from normal), from 10 patients with Graves' disease 19 +/- 3% (P less than 0.05) and from 15 patients with Hashimoto's thyroiditis 11 +/- 2% (P less than 0.001). No patient immunoglobulin preparation showed activity greater than that of normal individuals. The lower growth stimulatory activity in Graves' disease and Hashimoto's thyroiditis remained after heat inactivation of serum and is thought to reflect surface binding of thyroid autoantibodies.     

Kinetics of dog thyroid secretion in vitro.

The time sequence of radioiodine sequestration and secretion (BE131I) have been compared in dog thyroid slices prelabeled with 131I in vivo and incubated in vitro with or without TSH. Sequestration has been taken to be the amount of radioiodine present in phagocytic vacuoles or colloid droplets; the TSH or (Bu)2cAMP stimulation of the basal values was suppressed by endocytosis blocking drugs. TSH induced a sequestrated radioactivity (S) after 5 min and a stimulated secretion after 20 min. The secretion rate was constant: 1%/h (mean +/- SD = 1.0 +/- 0.4; n = 7) of the total radioactivity of the slices. At equilibrium, S was constant and equal to less than 1% of the total radioactivity. The half-life of S, assuming a disappearance rate proportional to S, was 26 min (26 +/- 4; n = 5); assuming a disappearance rate independent of S, the lifetime was 44 min (44 +/- 7; n = 6). At the steady state, the limiting step of maximally stimulated secretion was the hydrolysis of the sequestrated radioactivity and endocytosis rate was equal to secretion rate. Without TSH, a constant BEI release (0.23% +/- 0.07%/h; n = 7), insensitive to cytochalasin B, was observed, which corresponded to basal secretion.     

Thyrotropin-dependent proliferation of in vitro rat thyroid cell systems

This review is focused on the most recent knowledge on growth control of rat thyroid cell lines. We analyzed the effect of mitogenic as well as inhibitory agents, but mainly the proliferative effect elicited by thyrotropin (TSH). The classic cAMP-dependent protein kinase (PKA) signal transduction pathway involved in TSH-mediated cell growth is analyzed exhaustively. We have also reviewed new concepts about the participation of other effectors such as small GTPases and phosphatidyl inositol-3-kinase (PI3-K) and the new data about the existence of a cAMP-dependent but PKA-independent pathway. Finally, we give information about TSH induction of cell cycle-related genes, such as G1 cyclins, cyclin-dependent kinases (CDKs) and CDK inhibitors.     

Inhibition by monensin of dog thyroid secretion in vitro

Monensin, a carboxylic ionophore, raises the pH of prelysosomal and lysosomal compartments and inhibits lysosomal protein degradation. We tested this drug in dog thyroid slices to ascertain the role of lysosomal pH in thyroglobulin hydrolysis and hormone secretion. Monensin (10(-7)-10(-5) M) and another carboxylic ionophore, nigericin (10(-7)-10(-5) M), inhibited in a concentration-dependent manner TSH-stimulated secretion of T4, T3, and iodide. This inhibition was not toxic since: 1) 10(-5) M monensin did not affect TSH stimulation of protein iodination and cAMP accumulation; and 2) the inhibition was reversible. Secretion was blocked at a postphagocytotic, presumably lysosomal step because the time lag for the fall in secretion rate after 10(-5) M monensin addition was 19 min +/- (SD) 3 min (six experiments), i.e. the same as after lysosomotropic amine addition and significantly shorter than after addition of cytochalasin B (time lag, 43 min +/- 7 min, nine experiments), an inhibitor of phagocytosis. In addition, 10(-5) M monensin blocked the TSH-induced formation of apical pseudopods and colloid droplets and induced a swelling of the Golgi structures. In conclusion, monensin interfered with phagocytosis and with a postphagocytotic, presumably lysosomal, step in secretion by dog thyroid in vitro. Our data provide the first biochemical evidence, in the intact cell, that an acidic pH in the prelysosomal and/or lysosomal compartment is necessary for thyroid hormone secretion.     

Pharmacological interference with in vitro tests of thyroid function

Numerous drugs may cause changes in the serum concentrations of T4 and of T3. If such alterations are not recognized an incorrect diagnosis may result. In moderate degrees of hypo- and hyperthyroidism thyroid hormone levels may be spuriously normal, or the influence of pharmacological substances may lead to false diagnosis of thyroid disease in euthyroid patients. Since prediction of such alterations remains uncertain, it may be necessary to perform additional investigations when a potential artefact is recognized. On the other hand many pharmacological agents, especially those which interact with neurotransmitters, may influence TSH secretion, too. The TRH-test may show an increase or decreased TSH response, although complete suppression is only rarely seen during high-dose glucocorticoid treatment when low TRH doses are applied. Because of TRH-test gives such wide separation between different clinical states false interpretations are generally less likely than with drug-induced changes in T4 and T3 values.     

Effects of amiodarone on thyroid iodine metabolism in vitro

Since alterations of thyroid function have been reported in patients treated with amiodarone, 2-butyl,3-(4-diethylaminoethoxy-3,4-diiodo, benzoyl) benzufuran, the effects of this drug on the active iodide transport, organic iodine formation, thyroid peroxidase and the enzymatic iodotyrosine deiodination, were studied. In pig thyroid slices the iodide transport was affected by amiodarone at concentrations of 10(-4) M and 10(-5) M, showing a decrease of T/M (tissue/medium) ratios of 20% and 23%, respectively. Lower concentrations produced no significant differences from the controls. Iodotyrosine synthesis was only, but poorly, affected by 10(-4) M and 10(-5) M amiodarone. Inhibition of the DIT formation was greater than that produced for MIT. Thyroid peroxidase activity, as measured by the tyrosine-iodinase assay, showed a 20% decrease at 10(-3) M amiodarone. None of the other concentrations have affected the activity of the enzyme, except for 7% at a concentrations of 10(-4) M. The iodotyrosine deiodination was affected by amiodarone only at a concentration of 10(-3) M and 10(-4) M. The inhibitions were of 22.5% and 16.8%, respectively. We have concluded that, under the conditions of our study, amiodarone per se does not affect the intrathyroidal iodine metabolism in concentrations which are usually present in the sera of patients treated with this drug. However, it is not possible to rule out an in vivo direct action, if amiodarone is substantially concentrated in the human thyroid gland.     

Localization of iodine binding in the thyroid gland in vitro

Protein-bound radioiodine was localized by electron microscopic autoradiography in follicles and cells isolated from pig and rat thyroid tissue after incubation with 125I- for 1-10 min. Labeled proteins were analyzed by gel electrophoresis and immunoprecipitation. In closed follicles, with colloid-filled follicle lumina, the autoradiographic labeling was concentrated over the lumina and no labeling occurred over the cells. In open follicles, without colloid content, autoradiographic grains were invariably found along the apical cell surface and in some cells over intracellular lumina. Isolated cells with preserved polarity showed some labeling associated with remaining microvilli whereas isolated cells with lost polarity showed no grains at the cell surface. In isolated cells, particularly those with lost polarity, most grains were located over intracellular lumina (which were common in such cells) and some grains over vesicular elements associated with these lumina. About 80% of the labeled soluble proteins and 40% of the labeled proteins solubilized by sodium dodecyl sulfate were thyroglobulin. It is concluded that the site of iodination in follicles is the same in vitro as in vivo, viz. the apical surface of the follicle cells. When thyroid cells are removed from the follicle and lose their polarity, intracellular lumina become the major site of iodination. This shift in iodination sites is thought to be due to retention of Golgi-derived secretory vesicles in nonpolarized cells, leading to coalescence of vesicles with the formation of intracellular lumina and activation of membrane-bound enzymes catalyzing iodination.     

Mechanism of cholinergic inhibition of dog thyroid secretion in vitro

It has been previously shown that carbamylcholine (10(-5) M) decreases TSH-induced cAMP accumulation and hormone secretion in dog thyroid slices. The mechanism of the latter effect has been investigated in this work. The role of a decrease of cAMP level as the sole mediator of the inhibition of secretion was excluded: the inhibition persisted in the presence of 1-methyl-3-isobutylxanthine at 10(-4) M, which completely abolished the carbamylcholine-induced decrease in cAMP. Moreover, carbamylcholine also inhibited secretion when the slices were incubated with 0.4 mM (Bu)2cAMP. Scanning electron microscopic studies showed that carbamylcholine added at the same time as TSH blocked the formation of pseudopods in response to TSH within 2 min. The kinetic and morphological effects of carbamylcholine added at the same time as, or 90 min after, TSH were similar to those of cytochalasin B (3 micrograms/ml). After carbamylcholine addition at time 90 min, the stimulated secretion rate persisted unchanged for 46 +/- 10 min (mean +/- SD) (n = 6). During this period the colloid droplets disappeared from the cells. Carbamylcholine, like cytochalasin B, did not affect the basal secretion, which is independent of phagocytosis. It is concluded that carbamylcholine (10(-5) M) inhibits stimulated thyroid secretion at a step beyond cAMP accumulation by blocking pseudopod formation and not by inhibiting thyroglobulin hydrolysis or hormone diffusion.     

Inhibition by lysosomotropic amines of dog thyroid secretion in vitro

The lysosomotropic amines are well-known inhibitors of lysosomal protein degradation. These drugs were used in dog thyroid slices to ascertain the role of the lysosome in thyroglobulin hydrolysis and in hormone secretion. NH4Cl (1-20 mM) and chloroquine (5-500 microM) inhibited, in a concentration-dependent manner, the TSH-stimulated secretion. The high concentrations of these compounds also inhibited the basal secretion. The inhibition was not toxic since 1) the nonbutanol extractable iodine, an index of follicle disruption, was not increased, except slightly by 20 mM NH4Cl; 2) the compounds did not inhibit the TSH-induced stimulation of protein iodination; 3) the inhibition of secretion caused by these compounds was reversible; 4) the ultrastructural changes induced by NH4Cl were reversed after its withdrawal. The inhibition of secretion was presumably related to the lysosomal trapping of the drugs because: 1) the time lag for the fall in secretion rate after drug addition was shorter than for the inhibition of secretion at the level of pseudopod formation by carbamylcholine and cytochalasin B; 2) for NH4Cl this delay and the degree of inhibition were modulated by the [H+] gradient between the medium and the lysosome; 3) 20 min after NH4Cl addition, 92% of the lysosomes were vacuolated and swollen (median section area, 126 mu2 vs. 50 mu2 for the controls) and 8% of the lysosomes were swollen and still dense (median, 206 mu2); 20 min after chloroquine addition, 90% of the lysosomes remained dense and had a significantly higher section area (median, 79 mu2) than the controls (P less than 0.001), whereas 10% of the lysosomes were vacuolated and large (median area, 438 mu2). The number of pseudopods measured by scanning electron microscopy significantly decreased only after 1 h (P less than 0.001). This late decrease could not account for the early block of secretion and suggested a lack of membrane recycling. In conclusion, lysosomotropic amines interfere with a post phagocytotic, presumably lysosomal, step in secretion by dog thyroid. These data constitute the first biochemical evidence in the intact cell of the role of lysosomes in TSH-induced thyroglobulin hydrolysis and thyroid hormone secretion.