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.     

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.     

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.     

Stimulation by iodide of H(2)O(2) generation in thyroid slices from several species

The regulation of thyroid metabolism by iodide involves numerous inhibitory effects. However, in unstimulated dog thyroid slices, a small inconstant stimulatory effect of iodide on H(2)O(2) generation is observed. The only other stimulatory effect reported with iodide is on [1-(14)C]glucose oxidation, i.e., on the pentose phosphate pathway. Because we have recently demonstrated that the pentose phosphate pathway is controlled by H(2)O(2) generation, we study here the effect of iodide on basal H(2)O(2) generation in thyroid slices from several species. Our data show that in sheep, pig, bovine, and to a lesser extent dog thyroid, iodide had a stimulatory effect on H(2)O(2) generation. In horse and human thyroid, an inconstant effect was observed. We demonstrate in dogs that the stimulatory effect of iodide is greater in thyroids deprived of iodide, raising the possibility that differences in thyroid iodide pool may account, at least in part, for the differences between the different species studied. This represents the first demonstration of an activation by iodide of a specialized thyroid function. In comparison with conditions in which an inhibitory effect of iodide on H(2)O(2) generation is observed, the stimulating effect was observed for lower concentrations and for a shorter incubation time with iodide. Such a dual control of H(2)O(2) generation by iodide has the physiological interest of promoting an efficient oxidation of iodide when the substrate is provided to a deficient gland and of avoiding excessive oxidation of iodide and thus synthesis of thyroid hormones when it is in excess. The activation of H(2)O(2) generation may also explain the well described toxic effect of acute administration of iodide on iodine-depleted thyroids.     

Forskolin stimulates adenylate cyclase and iodine metabolism in thyroid

Forskolin is a potent activator of the cyclic AMP-generating system in many tissues. In dog thyroid slices, the enhancement of cyclic AMP level was rapid, sustained in the presence of forskolin, but easily reversible after its withdrawal. Contrary to TSH, forskolin induced little apparent desensitization. Forskolin potentiated the effects of TSH, PGE1 and cholera toxin. However, the forskolin-induced cyclic AMP accumulation was still sensitive to inhibitors of dog thyroid adenylate cyclase such as iodide, norepinephrine and adenosine. As fluoride, but contrary to TSH and PGE1, forskolin stimulated adenylate cyclase in a medium where Mg2+ was replaced by Mn2+. This suggests that in thyroid, as in other tissues, forskolin acts beyond the receptor level but, as it potentiates hormone action and does not impair modulation by inhibitors, it may interact with the nucleotide-binding regulatory proteins. Forskolin mimicked the effect of TSH on iodide organification and secretion.     

Carbamylcholine and thyrotropin activate distinctive pathways of protein phosphorylation in dog thyroid

The role of protein phosphorylation in the regulation of thyroid function by carbamylcholine was investigated using dog thyroid slices incubated in the presence of [³²P]phosphate and two-dimensional electrophoresis. In these intact cells, carbachol increased the phosphorylation of three polypeptides with M_r values of 21 500, 24 000 and 29 000. Maximal [³²P]phosphate incorporation occurred within 5 min of addition of carbamylcholine for 10 min increased the phosphorylation of 11 polypeptides whcih were identical to those observed previously after 2 h of hormone action (Lecocq, R., Lamy, F. and Dumont, J.E. (1979) Eur. J. Biochem. 102, 147–152). All three polypeptides whose phosphorylation is increased by carbamylcholine were different from those whose phosphorylation is increased by thyrotropin. Under our experimental conditions, the calcium ionophore A23187 did not stimulate significantly [³²P]phosphate incorporation in these three polypeptides. In conclusion, our results show that carbamylcholine and thyrotropin, which have some antagonist and some similar effects on dog thyroid, do not act through the phosphorylation of the same proteins. Although we have, in our previous chapter, established that in a rise in intracellular cyclic AMP could accout for the effect of thyrotropin on protein phosphorylation, the nature of the intracellular mediator of carbamylcholilne action on this parameter is still uncertain.     

Involvement of three intracellular messenger systems, protein kinase C, calcium ion and cyclic AMP, in the regulation of c-fos gene expression in Swiss 3T3 cells

In quiescent cultures of Swiss 3T3 cells, platelet-derived growth factor or fibroblast growth factor known to induce both protein kinase C activation and Ca2+ mobilization raised c-fos mRNA. This action of the growth factors was mimicked by the specific activators for protein kinase C, such as phorbol esters and a membrane-permeable synthetic diacylglycerol, and also by the Ca2+ ionophores, such as A23187 and ionomycin. Prostaglandin E1 known to elevate cyclic AMP also raised c-fos mRNA, and this action was mimicked by 8-bromo-cyclic AMP, dibutyryl cyclic AMP and forskolin. These results suggest that expression of the c-fos gene is regulated by three different intracellular messenger systems, protein kinase C, Ca2+ and cyclic AMP, in Swiss 3T3 cells.     

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.     

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.     

Effects of acetylcholine, TSH and other stimulators on intracellular calcium concentration in dog thyroid cells

The intracellular free calcium concentration, [Ca2+]i, has been measured in dog thyroid cells using the fluorescent Ca2+-indicator, quin2. Acetylcholine or its non-hydrolyzable analog, carbamylcholine rapidly increased [Ca2+]i by 40 +/- 4% (mean +/- SE) over the basal level of 81 +/- 2 nM. This increase was totally abolished by atropine, a muscarinic cholinergic receptor blocker, but was not influenced by verapamil, a voltage dependent-calcium channel blocker. Depletion of extracellular Ca2+ by the addition of EGTA, diminished but did not abolish the response to carbamylcholine. These data suggest that cholinergic effectors increase [Ca2+]i by mobilization of Ca2+ from intracellular stores rather than from an influx of Ca2+. Addition of TSH, isoproterenol, phorbol ester, dibutyryl cyclic GMP or cyclic AMP did not elicit any change in [Ca2+]i suggesting that their action may not involve any mobilization of intracellular Ca2+. These data provide direct evidence that in the thyroid cell, cholinergic agents act via their receptors to cause a rapid increase in [Ca2+]i, which may mediate their metabolic effects.     

Effects of prostaglandins F alpha on dog thyroid cyclic AMP level and function

Prostaglandins F1 alpha and F2 alpha, at high concentrations (greater than or equal to 28 microM) enhanced cyclic AMP accumulation in dog thyroid slices. At lower concentrations, they inhibited the cyclic AMP accumulation induced by thyrotropin (TSH), prostaglandin E1, and cholera toxin. This effect was rapid in onset and of short duration, calcium-dependent and suppressed by methylxanthines. Prostaglandin F alpha also inhibited TSH-induced secretion and activated iodide binding to proteins. These characteristics are similar to those of carbamylcholine action, except that prostaglandins F did not enhance cyclic GMP accumulation. The effect of prostaglandin F alpha was not inhibited by atropine, phentolamine and adenosine deaminase and can therefore not be ascribed to an induced secretion of acetylcholine, norepinephrine or adenosine. It is suggested that prostaglandins F act by increasing influx of extracellular Ca2+. Arachidonic acid also inhibited the TSH-induced cyclic AMP accumulation. However this effect was specific for TSH, it was enhanced in the absence of calcium and was not inhibited by methylxanthines or by indomethacin at concentrations which completely block its conversion to prostaglandin F alpha. Arachidonic acid action is sustained. This suggests that arachidonic acid inhibits thyroid adenylate cyclase at the level of its TSH receptor and that this effect is not mediated by prostaglandin F alpha or any other cyclooxygenase product.     

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.     

Thyrotropin-stimulated recruitment of free monoribosomes on to membrane-bound thyroglobulin-synthesizing polyribosomes.

Treatment of ox and dog thyroid slices in vitro with either thyrotropin or dibutyryl cyclic AMP elicited a variety of changes in polyribosome distribution. The most marked and consistent responses were decreases in both free and membrane-bound monoribosomes with a concomitant increase in the specific peak of thyroglobulin-synthesizing polyribosomes. On some occasions there was a shift towards heavier aggregates in the free polyribosomes. The increase in the amount of thyroglobulin-synthesizing polyribosomes was not accompanied by a shift in its location on the gradients. These changes were apparent within 30 min of thyrotropin addition and within 60 min of the addition of dibutyryl cyclic AMP. It is suggested that the major initial effect on translation of both thyrotropin and dibutyryl cyclic AMP is to stimulate the recruitment of pre-existing free monoribosomes on to pre-existing unloaded or under-loaded thyroglobulin mRNA molecules.     

Iodide induced suppression of thyrotropin-stimulated adenosine 3',5'-monophosphate production in cat thyroid slices.

Cat thyroid slices were studied to investigate their responsiveness to thyrotropin stimulation of cyclic AMP accumulation. Ovine and bovine thyrotropin, in the presence of 2.5 mM aminophylline, induced a dose-dependent increase in the cyclic AMP content of cat thyroid tissue. Half-maximal stimulation of cyclic AMP accumulation was obtained at a thyrotropin concentration of 1-2 mU/ml. The maximal effect of thyrotropin was observed at 10 mU/ml, and was associated with a mean 77 +/- 19-fold increase in thyroidal cyclic AMP. Preincubation of cat thyroid tissue for 2 h with 50 micron NaI resulted in an impairment in the subsequent ability of thyrotropin to enhance cyclic AMP accumulation, without altering the level of cyclic AMP in tissues not exposed to the hormone. Preincubation alone was without effect on thyrotropin stimulation of cyclic AMP, and the inhibitory effect of iodide was prevented by addition of 3 mM methimazole to the preincubation medium. In addition, the time course of thytrotropin stimulation of cyclic AMP accumulation in cat thyroid slices was not significantly altered by the preincubation with excess iodide. These studies provide additional evidence that excess iodide inhibits the adenylate cyclase-cyclic AMP system in thyroid tissue.     

Effect of thyrotropin-releasing hormone on dog thyroid in vitro

The in vitro action of thyrotropin-releasing hormone (TRH) on the cyclic AMP level and iodine metabolism in dog thyroid, has been studied. TRH inhibited cyclic AMP accumulation and subsequent secretion in slices stimulated by thyrotropic hormone (TSH), prostaglandin E1, cholera toxin and to a lesser extent forskolin. The effect of TRH was suppressed in a medium deprived of calcium or in the presence of isobutylmethylxanthine. TRH also stimulated iodide binding to proteins, but not cyclic GMP accumulation. Although all these characteristics of TRH action on dog thyroid fit those of prostaglandin F1 alpha in this tissue, TRH effects were not relieved by indomethacine. The possibility of a TRH action through other known inhibitors of the cyclic AMP system in dog thyroid such as: acetylcholine, alpha-adrenergic agents, adenosine, iodide were checked and ruled out. The possible involvement of other neurotransmitters, such as ATP or vasoactive intestinal peptide were studied but could not be substantiated. Our data suggest the existence of a direct negative action of TRH on the thyroid itself besides its stimulatory role at the pituitary level. The great variability of the TRH effect was overcome by pretreatment of the dog by pyridostigmine, an acetylcholinesterase inhibitor.     

Effect of thyrotropin-releasing hormone on dog thyroid in vitro

The in vitro action of thyrotropin-releasing hormone (TRH) on the cyclic AMP level and iodine metabolism in dog thyroid, has been studied. TRH inhibited cyclic AMP accumulation and subsequent secretion in slices stimulated by thyrotropic hormone (TSH), prostaglandin E₁, cholera toxin and to a lesser extent forskolin. The effect of TRH was suppressed in a medium deprived of calcium or in the presence of isobutylmethylxanthine. TRH also stimulated iodide binding to proteins, but not cyclic GMP accumulation. Although all these characteristics of TRH action on dog thyroid fit those of prostaglandin F_1α in this tissue, TRH effects were not relieved by indomethacine. The possibility of a TRH action through other known inhibitors of the cyclic AMP system in dog thyroid such as: acetylcholine, α-adrenergic agents, adenosine, iodide were checked and ruled out. The possible involvement of other neurotransmitters, such as ATP or vasoactive intestinal peptide were studied but could not be substantiated. Our data suggest the existence of a direct negative action of TRH on the thyroid itself besides its stimulatory role at the pituitary level. The great variability of the TRH effect was overcome by pretreatment of the dog by pyridostigmine, an acetylcholinesterase inhibitor.     

Pairs of cyclic AMP analogs, that are specifically synergistic for type I and type II cAMP-dependent protein kinases, mimic thyrotropin effects on the function, differentiation expression and mitogenesis of dog thyroid cells

The role of the two different isozymes of the cAMP-dependent protein kinase is still unclear. We have investigated the potential roles for each isozyme in dog thyroid cells, a model in which the function, expression of differentiation and proliferation are positively regulated by thyrotropin acting through cyclic AMP. The dog thyroid contains both type I and type II cAMP-dependent protein kinases. These isozymes were selectively activated in vitro by type-I-directed and type-II-directed analog pairs. In thyroid slices, both type-I directed and type II-directed analog pairs synergistically activated thyroid hormone synthesis, as measured by incorporation of 131I into proteins and thyroid hormone secretion as determined by the release of butanol-extractable 131I. In primary cultures of dog thyroid cells both isozyme-directed analog pairs synergistically enhanced iodide trapping, a marker of differentiation, and DNA synthesis, as measured by the percentage of cells incorporating [3H]thymidine into their nuclei. However, DNA synthesis was more sensitive to type-I-directed pairs. The results demonstrate that both cAMP-dependent protein kinase isozymes can mediate the action of cAMP on function, differentiation expression and cell proliferation in dog thyroid cells.     

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.     

Iodide-induced inhibition of adenylate cyclase activity in horse and dog thyroid

The characteristics of the iodide-induced inhibition of cyclic AMP accumulation in dog thyroid slices have been previously described [Van Sande, J., Cochaux, P. and Dumont, J. E. (1985) Mol. Cell. Endocrinol. 40, 181-192]. In the present study we investigated the characteristics of the iodide-induced inhibition of adenylate cyclase activity in dog and horse thyroid. The inhibition of cyclic AMP accumulation by iodide in stimulated horse thyroid slices was similar to that observed in dog thyroid slices. The inhibition was observed in slices stimulated by thyroid-stimulating hormone, cholera toxin and forskolin. Increasing the concentration of the stimulators did not overcome the iodide-induced inhibition. Adenylate cyclase activity, assayed in crude homogenates or in plasma-membrane-containing particulates (100,000 x g pellets), was lower in homogenates or in particulates prepared from iodide-treated slices than from control slices. This inhibition was observed on the cyclase activity stimulated by forskolin, fluoride or guanosine 5'-[beta, gamma-imino]triphosphate, but also on the basal activity. It was relieved when the homogenate was prepared from slices incubated with iodide and methimazole. Similar results were obtained with dog thyroid. The inhibition persisted when the particulate fraction was washed three times during 1 h at 100,000 x g, in the presence of bovine serum albumin or increasing concentration of KCl. It was similar whatever the duration of the cyclase assay, in a large range of protein concentration. These results indicate that a stable modification of adenylate cyclase activity, closely related to the plasma membrane, was induced when slices were incubated with iodide. Iodide inhibition did not modify the affinity of adenylate cyclase for its substrate (MgATP), but induced a decrease of the maximal velocity of the enzyme. The percentage inhibition was slightly decreased when Mg2+ concentration increased, and markedly decreased when Mn2+ concentration increased. A detectable adenylate cyclase activity was demonstrated when intact slices were incubated in the presence of [alpha-32P]ATP, probably because of the presence of broken cells produced during the slicing. Iodide had no direct effect on this cyclase system, which confirms that iodide needs the integrity of the cell to induce the inhibition and suggests that the inhibition is not transmitted between cells.     

1,25-Dihydroxycholecalciferol attenuates thyrotropin stimulated iodide accumulation in rat thyroid follicular FRTL-5 cells by reducing iodide porter number.

We have recently shown that rat thyroid follicular FRTL-5 cells have functional receptors for 1,25-dihydroxycholecalciferol (1,25- (OH)2D3) and that 1,25-(OH)2D3 attenuates the thyrotropin (TSH) induced iodide uptake. Here we show that the dibutyrylcyclic AMP induced iodide uptake was significantly reduced by 1,25-(OH)2D3, indicating that 1,25-(OH)2D3 affects the cAMP signal pathway beyond cAMP generation. The Vmax of the iodide porter was significantly reduced in 1,25-(OH)2D3 treated cells as compared to cells treated with TSH alone, indicating that 1,25-(OH)2D3 reduces the effective number of iodide porters in FRTL-5 cells.