Thyroidal autoregulation: Iodide-induced suppression of thyrotropin-stimulated cyclic AMP production and iodinating activity in thyroid cells

In continuing our study of the thyroidal autoregulation phenomenon, we have investigated the effects of iodide on several thyroidal responses to thyrotropin. Thus, we have found that the 2–4-fold thyrotropin stimulation of protein iodination in beef thyroid cells was reduced about 30% by 4 h of preincubation with 10 μM iodide, and virtually abolished with 50 μM iodide. Similarly the 8-fold thyrotropin stimulation of cyclic AMP accumulation in the cells was reduced about 30% by 3 h of preincubation with 50 μM iodide. It appears therefore that the so-called autoregulation of the thyroid gland does include influences of iodide on the thyrotropin stimulation of cyclic AMP production, iodide transport, and protein iodination which can be demonstrated in vitro in the dispersed thyroid cell system. Two other effects of thyrotropin, namely, the stimulation of [¹⁴C]leucine incorporation into protein and of iodide efflux were not at all affected by treatment with excess iodide, and hence may not be subject to the autoregulatory influence of iodide.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 2. Positive regulation.

Two different independent processes are operating in cultured thyroid cells to regulate adenylate cyclase/cyclic AMP responsiveness to thyroid stimulators (thyrotropin and prostaglandin E2): firstly, refractoriness or negative regulation [preceding paper], which is specific for each thyroid stimulator, is not mediated by cyclic AMP and is not accompanied by alteration of adenylate cyclase activity; secondly, positive regulation which is characterized by an augmentation of the cyclic AMP response stimulated by thyrotropin and prostaglandin E2. This process is not specific for each thyroid stimulator and is a state of increased susceptibility of cyclic AMP synthesis to stimulation, accompanied by increased activity of the catalytic subunit of adenylate cyclase. Positive regulation is apparently mediated by increased intracellular cyclic AMP levels. It is a time-dependent and dose-dependent process. Very low concentrations (5-50 micronU/ml) of thyrotropin augmented cyclic AMP synthesis stimulated by thyrotropin and prostaglandin E2 whereas higher concentrations (above 0.1 mU/ml) augmented prostaglandin E2 stimulation but induced refractoriness to thyrotropin. Prostaglandin E2 (0.1 to 10 micronM) augmented thyrotropin stimulation and dibutyryl adenosine 3':5'-monophosphate (0.3 to 2 mM) augmented thyrotropin and prostaglandin E2 stimulation. Positive regulation is a slow process which develops within days and increases up to day 5 in culture. Experiments using inhibitors suggested that protein synthesis is required for the full expression of the increase in adenylate cyclase activity induced by the studied thyroid stimulators.     

Inhibition of thyroid secretion by sodium fluoride in vitro

NaF mimicked the activation by thyrotropin of iodide binding to proteins and of glucose C-I oxidation but not the accumulation of intracellular colloid droplets or the stimulation of secretion in dog thyroid slices in vitro. On the contrary, NaF inhibited the two latter thyrotropin effects. The inhibitory action of F⁻ was partially relieved by the addition of glucose to the medium; it was mimicked by sodium oxamate. These data suggest that NaF depresses the endocytosis of colloid and thyroid secretion by inhibiting aerobic glycolysis in the follicular cell. NaF inhibited the activation of colloid droplet accumulation and secretion by N⁶,O^2′-dibutyryl-adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) and the accumulation of cyclic AMP in thyrotropin-stimulated slices. This suggests an inhibition at the level of both cyclic AMP accumulation and cyclic AMP action. The inhibition by NaF and sodium oxamate of colloid droplet formation and thyroid secretion but not of glucose C-I oxidation in stimulated slices further confirms our conclusion that the latter effect is not merely a consequence of the activation by thyrotropin of colloid endocytosis.     

Negative control of TSH action by iodide and acetylcholine: mechanism of action in intact thyroid cells.

Iodide, a substrate of thyroid metabolism, and acetylcholine depress cyclic AMP intracellular content and secretion in dog thyroid slices under TSH stimulation. A direct or indirect pseudocompetitive effect at the level of TSH receptor interaction has been rejected. Iodide and carbachol, both inhibited cyclic AMP accumulation in TSH stimulated dog thyroid slices but only the effect of carbachol was suppressed in the presence of isobutylmethylanthine. Ro 20-1724 did not relieve either inhibitory effect. Carbachol greatly enhanced cyclic AMP disposal in TSH prestimulated slices after the cut off of hormone action by a trypsin treatment. This effect was also suppressed by isobutylmethylxanthine but not by Ro 20-1724. No action of iodide could be evidenced on cyclic AMP disposal in similar slices, although a clear effect after the same time of iodide action was observed on cyclic AMP accumulation. Neither carbachol, nor iodide depresses ATP levels in these slices. The data suggest that carbachol exerts its action through an activation of cyclic AMP disappearance probably by an activation of cyclic AMP phosphodiesterase and that iodide, through an oxidized intermediate, experts its inhibitory effect at the level of cyclic AMP synthesis.     

Adenosine 3':5'-monophosphate metabolism and turnover in dog thyroid slices.

The metabolism and turnover of adenosine 3':5'-monophosphate (cyclic AMP) have been studied in intact thyroid cells incubated in vitro. Thyroid slices have been stimulated by 1 mU thyrotropin/ml, then washed with buffer, or with buffer containing thyrotropin antibody, or trypsin so as to cut off the stimulation. The decline of cyclic AMP levels has been followed and the time required to decrease this level to half of the initial value estimated. Computer simulation taking into account the penetration of trypsin in the slices, the kinetics of thyrotropin inactivation and the relation between thyrotropin concentration and cyclic AMP concentration at the steady state has made it possible to estimate the true cellular half-life of cyclic AMP in the stimulated cell to 1 min 50 s. The method provides an experimental approach to the demonstration in intact cells of effective on cyclic AMP disappearance. The methodology of the calculation of half-life and turnover from such data is discussed.     

Effects of calcium ionophore (A-23187) on glucose oxidation and iodide transport in dog thyroid slices.

A calcium ionophore (A-23187, 20 mug/ml) stimulted 14C-1-glucose oxidation in dog thyroid slices to an extent equivalent to that obtained by the optimal concentration of dibutyryl cyclic AMP (1mM). Furthermore, the ionophore augmented the stimulation by dibutyryl cyclic AMP much more than the simple additive effect. The ionophore also enhanced the effect of TSH, but to a lesser extent. Under conditions where organic binding was blocked, T/M ratio of radioiodine concentration was lowered in slices by the ionophore; the findings similar to those obtained with TSH and dibutyryl cyclic AMP. The ionophore exhibited a slightly depressive effect on the basal cyclic AMP level. The elevation by TSH of cyclic AMP levels was also slightly depressed by the ionophore, but statistically insignificant in most cases. These results indicate that calcium ion may play an important role in the TSH regulation of iodide transport and glucose metabolism in the thyroid, in some cases by augmenting the effects of cyclic AMP.     

Thyroid cell responses to thyrotropin and 12-O-tetradecanoyl-phorbol-13-acetate: translocation of protein kinase C and phosphorylation of thyroid cell polypeptide substrates

Not all of the effects of thyroid-stimulating hormone (TSH) on the thyroid are mediated by activation of the adenylate cyclase-cyclic AMP system, indicating that other control systems must also exist. Although a calcium-phospholipid-dependent protein kinase (protein kinase C) and specific substrates had been identified in thyroid tissue, their responsiveness to TSH and other stimulators has not been determined. In thyroid cells which had been preloaded with [32P]orthophosphate, TSH and 12-O-tetradecanoyl-phorbol-13-acetate (TPA) increased the phosphorylation of a 33K polypeptide substrate within 5 min in a dose-dependent fashion. The effect was observed with 1 mU/ml TSH and 3 nM TPA and was maximal with 100 mU/ml TSH and 100 nM TPA. The biologically inactive analog of TPA, 4 alpha-phorbol, had no effect. Isobutylmethylxanthine (IBMX) decreased the phosphorylation of the 33K polypeptide and inhibited the effect of TSH and TPA, indicating that the phosphorylation is not mediated by cyclic AMP. TSH and IBMX, but not TPA, augmented phosphorylation of a 38K polypeptide, suggesting involvement of cyclic AMP. In contrast TPA, but not TSH, increased the phosphorylation of 58K and 28K polypeptides. TSH, but not TPA or 4 alpha-phorbol, elevated the cyclic AMP level of thyroid slices. Incubation of thyroid slices with TSH or TPA significantly decreased protein kinase C activity in the 100,000g cytosol fraction and increased it in an extract of plasma membranes. The effect was present within 5 min and was maximal by 30 min. The effect was observed with 100 mU/ml TSH or 1 nM TPA. The stimulation by TSH or TPA of protein kinase C and its translocation from the cytosol to the plasma membranes of thyroid tissue may provide another mechanism for control of thyroid cell metabolism.     

Modulation of adenylate cyclase/cyclic AMP response by thyrotropin and prostaglandin E2 in cultured thyroid cells. 1. Negative regulation.

Isolated porcine thyroid cells, cultured in the presence of thyrotropin (greater than or equal to 0.25 mU/ml) or prostaglandin E2 (greater than or equal to 0.1 micron), showed decreased adenosine 3':5'-monophosphate (cyclic AMP) response to further thyrotropin or prostaglandin E2 stimulation, respectively. Kinetics of the refractory process to thyrotropin and prostaglandin E2 are different: (a) maximal refractoriness to prostaglandin E2 was attained after 2--6 h exposure to prostaglandin E2 while refractoriness to thyrotropin was maximal only after 12--24 h; (b) the degree of refractoriness to prostaglandin E2 was much greater than that to thyrotropin. Refractoriness to thyrotropin or prostaglandin E2 is characterized: by specificity for each thyroid stimulator; by dependence upon the dose of thyrotropin or prostaglandin E2 in culture, e.g. induction of high degree of refractoriness with 0.5 mU/ml thyrotropin (or 1 micron prostaglandin E2), which elicits only a small cyclic AMP increase; by time requirement for induction; by partial effect; by changes of maximum activation of cyclic AMP response; by reversibility. This refractoriness of the cyclic AMP response was not induced by dibutyryl adenosine 3':5'-monophosphate. It was not attributed to increased cyclic AMP-phosphodiesterase activity, but to alterations in the receptor-adenylate cyclase system. Prevention of refractoriness to thyrotropin or prostaglandin E2 by incubation of cells in the presence of actinomycin D, puromycin and cycloheximide suggests that new RNA and protein syntheses are required for the development of the refractory state.     

Thyrotropin controls cyclic nucleotide metabolism of thyroid follicular cells without affecting membrane potential or input resistance

The action of thyrotropin on the rat thyroid follicular cell has been investigated using continuous transmembrane potential and input resistance recording from individual cells in in vitro preparations. The membrane potential in this study was -68.0 mV +/- 0.6 (mean+/-S.E.). Over a wide range of concentrations (1.5-50 mU/ml), thyrotropin failed to affect membrane potential or input resistance while 20 mU/ml thyrotropin was shown to elicit complex time-dependent changes in tissue levels of both cyclic AMP and cyclic GMP. The present results reveal that thyrotropin-receptor interaction does not affect plasma membrane permeability, but is characterized by complex changes in endogenous cyclic nucleotide metabolism.     

Cyclic AMP in the thyroid of the rat fed prophylthiouracil: in vitro unresponsiveness to thyrotropin.

Fragments of thyroid from rats fed Purina +0.1% propylthiouracil were incubated in vitro and the concentration of cyclic AMP measured. The normal gland showed a 3-fold increase in cyclic AMP with 50 mU thyrotropin per ml or 10(-4) M prostaglandin E1; tissue from rats fed propylthiouracil for 10 days to 6 months responded to prostaglandin E1 but not to thyrotropin. Feeding Purina without propylthiouracil for 1 month after 5 months of goitrogen restored thyrotropin-responsiveness, as did, to a lesser extent, injection of triiodothyronine, 50 mug twice daily for 5 days.     

Inhibition by iodide of iodide binding to proteins: the "Wolff-Chaikoff" effect is caused by inhibition of H2O2 generation

H2O2 generation is limiting the oxidation and binding to proteins of iodide. In dog thyroid slices thyrotropin and carbamylcholine greatly enhance protein iodination and H2O2 generation. The action of thyrotropin is mimicked by dibutyryl cyclic AMP and forskolin which suggests that it is mediated by cyclic AMP. The action of carbamylcholine was mimicked by ionomycin and by phorbol myristate ester. This suggests that the effect of carbamylcholine is mediated by the two intracellular signals generated by the Ca++ phosphatidylinositol cascade: Ca++ and diacylglycerol. The Wolff-Chaikoff effect is the inhibition by iodide of its own organification. In dog thyroid slices, iodide greatly inhibited H2O2 generation stimulated by thyrotropin and by carbamylcholine. Iodide decreased the production of intracellular signals induced by TSH and carbamylcholine but it also inhibited the action of probes of these intracellular signals (dibutyryl cAMP, forskolin, ionomycin, phorbol-myristate ester) on the H2O2 generating system itself. These effects were suppressed by methimazole an inhibitor of iodide oxidation.     

"In vitro" study on release of cyclic AMP and thyroid hormone in autonomously functioning thyroid nodules

The TSH effect on slice and the incubation medium cyclic AMP levels and T3 and T4 released from 8 autonomously functioning thyroid nodules (AFTN) and their respective perinodular (PN) tissues were examined. The thyroid slices were incubated in Eagle's Medium containing TSH (5 to 100 mU/ml) for 60 min and 300 min for tissue cyclic AMP generation and for cyclic AMP, T3 and T4 release, respectively. Basal cyclic AMP levels were not different either in AFTN and in PN slices or into the incubation medium. In both tissues TSH produced a similar cyclic AMP generation. In contrast, cyclic AMP released into the incubation medium was significantly higher in AFTN than in PN tissues, after TSH stimulation. Basal T3 values and TSH-stimulated T3 release in AFTN were not different from PN tissue. However, basal T4 levels were significantly higher in AFTN than in PN tissue as well as T4 released in response to TSH. In addition, T3/T4 ratio was lower in AFTN than in PN tissues. The cyclic AMP released into the incubation medium correlated with both T3 and T4 release in PN tissue but in the AFTN tissue no correlations were found. These findings suggest that the adenylate cyclase-cyclic AMP system is more sensitive to TSH-stimulation in AFTN when compared with PN tissue and that AFTN tissue has a preferential T4 secretion.     

Effects of microtubule inhibitors and cytochalasin B on thyroid metabolism in vitro.

Mitotic spindle inhibitors (colchicine, vinblastine, vincristine, 020, ethanol) and cytochalasin B inhibit the phagocytosis of colloid by thyroid cells and the secretion of thyroid hormones. This inhibition has been linked to interferences with the microtubular microfilament system of the follicular cell. In order to test the possibility of using such inhibitors to selectively block secretion, the action of suppressing or highly inhibitory concentrations on other metabolic parameters has been studied on dog thyroid slices in vitro: glucose oxidation, lactate formation, iodide binding to protein, cyclic 3'5' AMP accumulation. It is shown that at a concentration of 10 mM colchicine is entirely non specific as it greatly inhibits all facets of metabolism and all the stimulatory effects of cyclic 3'5' AMP and thyrotropin. The other mictrotubule inhibitors, although affecting thyroid metabolism in various ways were more specified. The enhancement by vineblastine of glucose oxidation ald iodine binding to proteins suggests an activation of they thyroid H2O2 generating system. D2O on the other hand selectively inhibits secretion and the binding of iodide to proteins. Cytochalasin B, presumably by inhibiting hexose transport, decreased glycolysis and the uptake of iodide. However this effect cannot account for the complete inhibition of thyroid secretion.     

Pattern of protein phosphorylation in intact stimulated cells: thyrotropin and dog thyroid

Two-dimensional, high-resolution electrophoretic technique of O'Farrell has been adapted to the analysis of thyroid phosphorylated proteins. Proteins were extracted from dog thyroid slices which had been incubated in the presence of [32P]phosphate with thyrotropin or with different agents which enhance the intracellular accumulation of cyclic AMP. About 350 phosphorylated polypeptides have been separated. Thyrotropin stimulates the phosphorylation of at least eight of these polypeptides. An increase in the phosphorylation of the same polypeptides was observed was observed when dog thyroid slices were incubated with dibutyryl adenosine 3':5'-monophosphate, cholera toxin or prostaglandin E1 instead of thyrotropin. Our results confirm that most of dog thyroid protein phosphorylation is independent of cyclic AMP. They offer a first link between the action of cyclic AMP on protein kinase and the physiological effects of thyrotropin. They strongly substantiate the hypothesis that most thyrotropin effects are mediated by cyclic AMP.     

Bovine thyrotropin inhibits DNA synthesis inversely with stimulation of cyclic AMP production in cultured porcine thyroid follicles

The effects of bovine thyrotropin (TSH) on DNA synthesis and cyclic AMP production were studied in porcine thyroid follicles using suspension culture. During the early 72 hours incubation, the time-dependent uptake of [3H]thymidine by the follicles was observed. In the presence of 10 mU/ml TSH, the uptake of [3H] thymidine was significantly depressed at 72 hours incubation. TSH inhibition of [3H] thymidine incorporation was related to its concentration and the 50% inhibition was observed by using 1.0 mU/ml TSH. Under the same conditions, cyclic AMP production was stimulated by TSH and the stimulation was observed to be related to TSH concentration. In these experiments, the incubation time was 30 min. Dibutyryl cyclic AMP, an analogue of cyclic AMP, inhibited the [3H] thymidine uptake at 72 hours incubation. From these results, it is suggested that TSH inhibits DNA synthesis, and that the inhibition may be mediated by cyclic AMP that is produced by TSH stimulation.     

Effects of cholera toxin on thyroid cyclic AMP-dependent protein kinase and ornithine decarboxylase activities.

Cholera toxin activated beef thyroid cyclic AMP-dependent protein kinase in a dose (0.2 to 8 microgram/ml)-related fashion. Thus, when beef thyroid slices were incubated with toxin (8 microgram/ml) for 90 minutes and then assayed for protein kinase, the activity ratio (i.e. -cyclic AMP/+cyclic AMP) increased from 0.32 +/- 0.02 to 0.77 +/- 0.06. The toxin (5 microgram/ml)-induced increase was abolished by inclusion of ganglioside GM1 in the incubation medium (I50, 0.7 microgram/ml), whereas, gangliosides GD1a and GT1 were without effect. In contrast, TSH-activated protein kinase was unaffected by ganglioside addition. Cholera toxin increased rat thyroid ornithine decarboxylase (ODC) activity in-vitro in a dose (0.1 to 10 microgram/ml)-related fashion [basal, 100 cf cholera toxin (10 microgram/ml), 1500 pmol 14CO2/g tissue/30 min]. The toxin (1 microgram/ml)- (but not TSH-) induced increase in ODC was abolished by inclusion of ganglioside Ga and GT1 were without effect. Cholera toxin stimulation of ODC was inhibited by indomethacin or iodide as are the stimulatory effects of TSH or dibutyryl cyclic AMP. These results demonstrate that although there are differences in the TSH and cholera toxin responses with respect to receptor (ganglioside) interaction, they nevertheless elicit similar intracellular responses in thyroid.     

Iodide modulation of Ca2+ efflux from mouse thyroid

In a preceding report, we showed evidence that thyrotropin (TSH) stimulates Ca2+ efflux from mouse thyroid gland and that TSH stimulation of Ca2+ efflux is inhibited by acute administration of excess iodide to mice fed a low iodine diet (Hashizume et al., 1984). The observations suggested that iodide inhibits Ca2+ efflux through an inhibition of TSH-sensitive adenylate cyclase activity. We found further, that iodide inhibits dibutyryl cyclic AMP (DBC)-stimulated Ca2+ efflux. The results suggested that iodide influences the step subsequent to the generation of cyclic AMP. In this report, we studied whether iodide can inhibit Ca2+ efflux by a mechanism which is distinct from adenylate cyclase inhibition. The acute administration of excess iodide to mice fed a regular diet did not decrease the basal Ca2+ efflux rate in the thyroid. TSH-induced stimulation of Ca2+ efflux in thyroids obtained from regular diet-treated mice was not modified by iodide administration. Iodide injection to mice fed a low iodide diet, however, decreased the basal Ca2+ efflux rate though the content of cyclic AMP in the thyroids was not altered by this treatment. The decreased-Ca2+ efflux rate induced by iodide in the low iodine diet-treated thyroids was not modified by TSH in vitro. The results indicate that an acute administration of excess iodide in thyroid inhibits Ca2+ efflux not only by an inhibition of adenylate cyclase but also by an inhibitory action which is distinct from the adenylate cyclase inhibiting action of iodide.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation.

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into folicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 microU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 microU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.     

Effect of gelatin on the cyclic AMP response of primocultured hog thyroid cells to acute thyrotropin stimulation

The cyclic AMP response of cultured hog thyroid cells to acute thyrotropin stimulation was shown to be under a dual regulatory control by thyrotropin: both positive and negative regulation have been described. When added to the culture medium, gelatin (0.25%) promoted the reorganization of the cells into follicle-like structures, as does thyrotropin. Unlike thyrotropin, gelatin did not induce an increase in intracellular cyclic AMP but enhanced the acute cyclic AMP response to thyrotropin in cells cultured in gelatin-containing medium. When both gelatin and thyrotropin were present, the positive effect of low concentrations of hormone (less than 50 μU/ml) was increased whereas the refractory process observed in the presence of higher concentrations of hormone (greater than 50 μU/ml) was unchanged. These effects of gelatin might be mediated by interaction of the denatured collagen molecules with external proteins of the plasma membrane of thyroid cells.