Free diiodotyrosine effects on protein iodination and thyroid hormone synthesis catalyzed by thyroid peroxidase.

Free diiosotyrosine exerts two opposite effects on the reactions catalyzed by thyroid peroxidase, thyroglobulin iodination and thyroid hormone formation. 1. Inhibition of thyroglobulin iodination catalyzed by thyroid peroxidase was observed when free diiodotyrosine concentration was higher than 5 muM. This inhibition was competitive, suggesting that free diiodotyrosine interacts with the substrate site(s) of thyroid peroxidase. Free diiodotyrosine also competively inhibited iodide peroxidation to I2. 2. Free diiodotyrosine, when incubated with thyroid peroxidase in the absence of iodide was recovered unmodified; in the presence of iodide an exchange reaction was observed between the iodine atoms present in the diiodotyrosine molecule and iodide present in the medium. Using 14C-labelled diiodotyrosine, 14C-labelled non-iodinated products were also observed, showing that deiodination occurred as a minor degradation pathway. However, no monoiodo[14C]tyrosine or E114C]tyrosine were observed. Exchange reaction between free diiototyrosine and iodide is therefore direct and does not imply deiodination-iodination intermediary steps. Thyroglobulin inhibits diiodotyrosine-iodide exchange and vice versa, again suggesting competition for both reactions. These results support, by a different experimental approach, the two-site model for peroxidase previously described by us in this journal. 3. Free diiodotyrosine when present at a very low concentration, 0.05 muM, exerts a stimulatory effect on throid hormones synthesis. The relationship between diiodotyrosine concentration and thyroid hormone synthesis give an S-shaped curve, suggesting that free diiodotyrosine acts as a regulatory ligand for thyroid peroxidase. Evidence is also presented that free diiodotyrosine is not incorporated into thyroid hormones. Therefore, thyroid peroxidase catalyzes only intra-molecular coupling between iodotyrosine hormonogenic residues. 4. Finally, although no direct proof exists that these free diiodotyrosine effects upon thyroglobulin iodination and thyroid hormone synthesis are physiologically significant, such a possibility deserves further investigation.     

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.     

Chemical determination of iodinated compounds in human thyroid

As a tool with which to detect iodinated compounds in human thyroid specimens, we have reevaluated a nonincineration technique which has so far been employed in the determination of thyroxine-iodine in peripheral blood. The catalytic action of iodoamino acids in the Ce-As reaction was enhanced by a small amount of Cl2. On the contrary, a large amount of Cl2 inhibited the reaction unexpectedly. Among iodide, iodotyrosine and iodothyronine, iodide was the most effective catalyst in the Ce-As reaction and iodothyronine was the least effective one. Protein seemed to inhibit this reaction of thyroglobulin. But the result of iodine content in thyroglobulin by this technique agreed well with that by incineration when measured 127I was corrected by percent activity of dializable part of the total activity of 131I-thyroglobulin with the same protein concentration, after the NaClO treatment. The results of human thyroid specimens were as follows: the thyroglobulin content of five normal subjects was 8.0 +/- 1.5% of wet thyroid weight. That of Hashimoto's disease was significantly decreased which seemed compatible with the decrease in iodine content of thyroglobulin, whereas thyroglobulin content of Graves disease treated with 1-methyl, 2-mercaptoimidazole followed by a large dose of iodide was well preserved in spite of a lower degree of iodination of thyroglobulin. As for the distribution of iodoamino acids-iodine in normal thyroid, T4 was 20.5 +/- 0.7%. This technique ultimately looks promising as a tool with which to study intrathyroidal iodine metabolism in human.     

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.     

Identification of an intracellular pathway of thyroxine synthesis by dispersed thyroid cells

This study deals with the identification of the biochemical events involved in the metabolic sequence leading from the synthesis to the release of thyroxine in the dispersed thyroid cell system. (1) Using an experimental model allowing the differentiation between intracellular and extracellular sites of iodination, it is shown that thyroxine is synthesized inside the cells by an iodinating system sensitive to thyrotropin stimulation. (2) The secretion of thyroxine synthesized inside the cells is not mediated by an exocytotic-endocytotic phenomenon. Colchicine, vinblastine, fluoride, propanolol and chlorpromazine, at concentrations equal to or 10–100-times higher than those required to inhibit hormone release in follicular-organized thyroid tissue have no effect on thyrotropin-stimulated thyroxine secretion. (3) The secretion involves the intracellular proteolysis of hormone-containing iodoprotein(s) which, in addition to free thyroxine, generates free mono- and diiodotyrosines. Free thyroxine is released into the incubation medium and iodotyrosines are deiodinated under normal conditions and accumulate in the presence of an inhibitor of iodotyrosine deiodinase: 3,5-dinitrotyrosine. This proteolysis is inhibited by 5 mM chlorpromazine. These data indicate that the complete metabolic sequence leading from the uptake of iodide to the release of free thyroxine into the incubation medium can be described as an ‘intracellular metabolic sequence for thyroxine synthesis’.     

Hormonogenesis in thyroid cells cultured on porous bottom chambers

Different processes implied in thyroid hormonogenesis (thyroglobulin, thyroperoxidase and hydrogen peroxide generating system expressions) and their regulation by TSH and iodide have been studied using porcine thyroid cells cultured in porous bottomed chambers. This system allowed to reproduce the functional bipolarity. Cells form a tight and polarized monolayer. Both apical and basolateral poles of epithelial cells were independently accessible and the cell layer separated two compartments which can contain different media. A major polarized secretion of thyroglobulin into the apical compartment was observed; it was increased in the presence of TSH as well as the thyroglobulin synthesis and mRNA level. These TSH effects were the consequence of adenylcyclase stimulation. Active transport of iodide, iodination of thyroglobulin and hormonosynthesis took place only in the presence of TSH. These steps occurred at the apical pole of cells. In the culture chamber system, thyroglobulin was weakly iodinated (6 atoms of iodide per mole of thyroglobulin; in vivo up to 40 atoms per mole) but hormonogenesis efficiency was close to this one observed in vivo (40%). Iodide concentrations higher than 0.5 µM daily added to the basal medium inhibited iodination of thyroglobulin and hormonosynthesis. Some components contained in culture media were inhibitors for iodination when they were present in the apical medium such as vitamins, amino acids and phenol red. The culture system appears to be interesting for pharmacological and toxicological studies.     

Differential effects of methylmercuric chloride and mercuric chloride on oxidation and iodination reactions catalyzed by thyroid peroxidase

Thyroid peroxidase (TPO), the major enzyme in the thyroid hormone synthesis, multifunctionally catalyzes (1) iodide oxidation, (2) iodination of the precursor protein, and (3) a coupling reaction of iodotyrosyl residues. The present study was carried out to examine the mercurial effects on the iodination, the second step of TPO. Purified porcine thyroglobulin or bovine serum albumin as acceptor protein was iodinated with [125I]NaI and H2O2 by purified porcine TPO. Iodinated protein was separated by acid precipitation on membrane filter or paper chromatography. Both CH3HgCl and HgCl2 dose-dependently inhibited the iodination, but HgCl2 was more potent to inhibit the iodination than CH3HgCl. These mercurial effects on the second step resemble the effects on the third step which were already reported; but are in marked contrast to the effects on the first step, where TPO was inhibited by HgCl2 but never by CH3HgCl.     

Reorganization of porcine thyroid cells into functional follicles in a chemically defined, serum- and thyrotropin-free medium

In the serum-free, chemically defined medium NCTC 109, freshly isolated porcine thyroid cells aggregate and form functional follicles in culture even in the absence of thyrotropin. The follicular pattern observed under light and electron microscopy express the main morphological characteristics of in vivo thyroid cells. Follicles are large, replete with dense colloid, and the apical pole of cells is characterized by well-developed microvilli and the presence of aminopeptidase N. The index of iodide transport activity (125I-C/M ratio) decreases vs. days of culture to a resting value of about 1 or 2 at day 2. Addition of thyrotropin (200 microU/ml final concentration) at day 4 is followed by a 10-fold increase in iodide transport activity within 24 h and a 40-fold increase 4 d later. Incorporation and organification of iodide are dose dependent between 0 and 250 microU/ml thyrotropin; highest concentrations (4,000--16,000 muU/ml) are significantly inhibitory. In the absence of thyrotropin each cell synthesizes 8.2 pg thyroglobulin/d. Acute stimulation by thyrotropin at day 4 resulted in a slight decrease in the quantity of thyroglobulin present in the cell layer but in an increase in the total amount of thyroglobulin recovered in both cells and medium, reaching 34.3 pg/cell/d. The protein exported into the medium is thyroglobulin, as shown by SDS PAGE and immunological properties. Here we demonstrate that porcine thyroid cells can be maintained in culture as resting, highly differentiated, follicular-associated cells, sensitive to acute stimulation by thyrotropin.     

Resistance to Pediococcus cerevisiae to amethopterin as a consequence of changes in enzymatic activity and cell permeability. II. Permeability changes to amethopterin and other folates in the drug-resistant mutant.

the accumulation of amethopterin in a Pediococcus cerevisiae strain resistant to this analogue was about 30% of that in P. cerevisiae/PteGlu, the sensitive parent. The uptake in the resistant strain was strictly glucose dependent, whereas in the sensitive parent about 16% accumulation occurred in absence of glucose. The transport in both strains was inhibited by iodoacetate and KF. Amethopterin uptake exhibited saturation kinetics with an apparent Km of 5 muM in P. cerevisiae/AMr and 0.5 muM in P. cerevisiae/PteGlu. The apparent V was 0.2 nmol per min per mg cells (dry weight); the same for both strains. The optimum pH for the uptake of amethopterin by P. cerevisiae/AMr and P. cerevisiae/PteGlu was pH 6.0. Folate and methyltetrahydrofolate competitivity inhibited amethopterin uptake with apparent Ki values of 8 and 0.7 muM, respectively. The uptake of folate exhibited a slightly increased Km value as compared to that of the sensitive strain, whereas the uptake activity velocity was in the same range. Methyltetrahydrofolate accumulated up to about 60-fold higher intracellular concentration than that of the medium, which is a markedly lower accumulation from that in the sensitive strain. The uptake was glucose dependent and inhibited by iodoacetate and KF. The pH optimum for methyltetrahydrofolate uptake in the resistant strain was the same as that in the sensitive parent (pH 5.7--6). In contrast to the increase in the apparent Km value for amethopterin in the resistant strain, the affinity of the carrier for methyltetrahydrofolate was apparently unchanged, whereas the V value was about 16 times lower than that in the sensitive strain. The Ki for amethopterin when added to increasing concentrations of methyltetrahydrofolate was 5.2 muM, a value about the same as that of the Km.     

Effect of long-term injection of high doses of potassium iodide on iodine metabolism in rat thyroid gland

Concentration of thyroid hormones in the serum of the rats after 14-day injections of potassium iodide (1, 3, 10, 100, and 500 physiological daily doses) did not differ from the control values. Excessive administration of potassium iodide increased the total iodide content in the rat thyroid tissue by 60–121% (35–108% and 94–128% for the protein-bound and free iodide, respectively), indicating the activation of the uptake and organification of iodide. The long-term injection of both low and high doses of potassium iodide increased the activity of catalase by 8–18% and SOD by 33–50% and enhanced the level of toxic LPO products reacting with thiobarbituric acid by 15–38%. It is suggested that reactive oxygen species and the excessive iodination of proteins (particularly thyroglobulin) induced by the long-term administration of high doses of potassium iodide can play an important role in the development of thyroid dysfunctions and autoimmune diseases.     

Formation of intracellular lumina in dispersed pig thyroid cells

Intracellular cavities characterized by the presence of microvilli have been identified in dispersed thyroid cells. These structures resembling follicular lumina were called intracellular lumina or ICL. Freshly dispersed cells did not contain ICL. At 37 degrees C, ICL formation was a rapid process. After 60 min of incubation, ICL were present in 15 to 20% of the cells; the number of ICL remained rather constant during 3 to 4 h of incubation. In the presence of thyrotropin, the number of ICL increased with time to reach a value ranging from 40 to 60 ICL per 100 cells after 4 h of incubation. ICL formation was also increased in the presence of dibutyryl cyclic AMP (2 mM). Vinblastine (30 microM), a microtubule-disrupting agent and monensin (30 microM), an ionophore inhibiting Golgi functions blocked the formation of ICL in control and thyrotropin-stimulated cells. Cycloheximide (0.5 mM) and puromycin (0.5 mM) did not inhibit ICL formation in either control or thyrotropin stimulated cells. The iodination capacity of ICL was studied by quantitative electron microscopic autoradiography after incubation of thyroid cells with 125 I-iodide for 2 to 60 min. Radioiodinated products appeared first in ICL. After 1 h of labeling autoradiographic grains were found mainly in ICL (60-70%) and over the cytoplasm. The labeling of ICL was heterogeneous; ICL contained either few or numerous overlapping grains. Whatever the labeling time, a high proportion of ICL (70-80%) were labeled. The labeling of ICL as well as the labeling over the cytoplasm was increased in the presence of thyrotropin and almost completely inhibited in the presence of an iodide trapping inhibitor: sodium perchlorate. Pulse-chase experiments revealed that thyrotropin stimulated the discharge of 125 I-labeled material from ICL.     

Association of the changes in cytosolic Ca2+ and iodide efflux induced by thyrotropin and by the stimulation of alpha 1-adrenergic receptors in cultured rat thyroid cells

Thyrotropin causes a time- and concentration-dependent increase in cytosolic Ca2+ in FRTL-5 rat thyroid cells as measured by Quin2 fluorescence; the half-maximal response occurs in response to 1 X 10(-7) M thyrotropin. The effect of added thyrotropin is the same whether cells have been previously and chronically exposed to thyrotropin or whether they have been thyrotropin "starved" for several days. The thyrotropin effect on cytosolic Ca2+ has no relationship to intracellular cAMP levels with respect to dose and time course. Norepinephrine (1 X 10(-7) M) also causes increases in cytosolic Ca2+ in FRTL-5 thyroid cells. With the use of a variety of adrenergic inhibitors, norepinephrine was found to exert its effect via an alpha 1-adrenergic receptor. The exposure of FRTL-5 cells to physiological thyrotropin concentrations enhances the effect on cytosolic Ca2+ level induced by norepinephrine in vitro; the shape of the dose-response curve indicates a cooperative effect of the thyrotropin and norepinephrine. The increase in cytosolic Ca2+ seems to be derived from an intracellular pool rather than from the extracellular space. It is not prevented by nifedipine, a blocker of Ca2+ channels; it is present in cells exposed to ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid; and it is not associated with increased Ca2+ uptake into the cell. the thyrotropin- and norepinephrine-induced increase in cytosolic Ca2+ parallels the efflux of iodide and the organification of thyroglobulin in a dose-dependent manner.     

Monoiodotyrosine formation during thyroglobulin processing in Golgi vesicles

A golgi-enriched subfraction was obtained from porcine thyroid glands by differential centrifugation. When incubated in a suitable medium, these vesicles were able to concentrate iodide from the medium and bind it to protein. The iodination process was inhibited by methylmercapto-imidazole and was increased by the addition of an H2O2 generating system to the medium. Analysis of the protein content of the vesicles revealed the presence of 18 and 12-13 S thyroglobulin molecules, lacking mannose residues, and containing only monoiodotyrosine. It is concluded that in vitro, iodination can begin before exocytosis, in the smooth-surfaced vesicles derived from the golgi apparatus, as soon as N-acetylglucosamine is incorporated onto the pre-thyroglobulin molecule.     

Sulfate transport in porcine thyroid cells. Effects of thyrotropin and iodide

In porcine thyroid cells, thyroglobulin sulfation is controlled by thyrotropin (TSH) and iodide, which contribute to regulating the intracellular sulfate concentration, as we previously established. Here, we studied the transport of sulfate and its regulation by these two effectors. Kinetic studies were performed after [(35)S]sulfate was added to either the basal or apical medium of cell monolayers cultured without any effectors, or with TSH with or without iodide. The basolateral uptake rates were about tenfold higher than the apical uptake rates. TSH increased the basolateral and apical uptake values (by 24 and 9%, respectively, compared with unstimulated cells), and iodide inhibited these effects of TSH. On the basis of results of the pulse-chase experiments, the basolateral and apical effluxes appeared to be well balanced in unstimulated cells and in cells stimulated by both TSH and iodide: approximately 40-50% of the intracellular radioactivity was released into each medium, whereas in the absence of iodide, 70% of the intracellular radioactivity was released on the basolateral side. The rates of transepithelial sulfate transport were increased by TSH compared with unstimulated cells, and these effects decreased in response to iodide. These results suggest that TSH and iodide may each control the sulfate transport process on two sides of the polarized cells, and that the absence of iodide in the TSH-stimulated cells probably results in an unbalanced state of sulfate transport.     

The role of calcium and guanosine 3':5'-monophosphate in the action of acetylcholine on thyroid metabolism

The role of calcium and guanosine 3':5'-monophosphate (cyclic GMP) in the regulation of thyroid metabolism has been investigated in dog thyroid slices. Carbamoylcholine enhanced glucose carbon-1 oxidation, protein iodination, cyclic GMP accumulation and decreased thyrotropin-induced adenosine 3':5'-monophosphate (cyclic AMP) accumulation and iodine secretion; it did not affect protein synthesis. The effects of carbamoylcholine were reproduced under various experimental conditions by supplementary calcium in the medium, ouabain, and in media in which Na+ had been replaced by choline chloride. They were inhibited by lanthanum. These results further support the hypothesis that free intracellular Ca2+ is the intracellular signal for carbamoylcholine effects and suggest that a Na+ -gradient-driven Ca2+ extrusion mechanism operates in the thyroid cell. Mn2+ reproduced the effect of Ca2+ on glucose oxidation, protein iodination and cyclic GMP accumulation in Ca2+ -depleted slices and medium, and thus mimicked some intracellular effects of Ca2+. On the other hand Mn2+ inhibited the carbamoylcholine effect on thyrotropin-induced thyroid secretion and cyclic AMP accumulation, and Ca2+ inhibited the Mn2+-induced cyclic GMP accumulation. This suggests that the two ions compete for the same channel. Similarly Mn2+ inhibited calcium effects in the presence of ionophore A23187. Procaine inhibited protein iodination under all conditions suggesting a primary effect; it also inhibited all carbamoylcholine and ouabain actions. However the drug did not inhibit the effects of choline chloride and its action was reversed by raising carbamoylcholine but not Ca2+ concentration; it is therefore doubtful that procaine acts by blocking Ca2+ channels. In media without added Ca2+, Mn2+ increased cyclic GMP accumulation but did not decrease thyrotropin-induced cyclic AMP accumulation or iodine secretion, which suggests that cyclic GMP cannot be the sole mediator of the latter two effects of carbamoylcholine.     

Investigations on the sodium dependence of bile acid fluxes in the isolated perfused rat liver.

At [Na+]o = 118 mM the concentrative transfer of cholic and taurocholic acid from the perfusate into the isolated rat liver displays saturation kinetics (taurocholate: V = 299 nmol-min-1-g-1, Km = 61 muM; Cholate: V=327 nmol-min-1-g-1, Km = 436 muM). Perfusion with an isotonic sodium-free medium did not change the feature of a carrier-mediated transport but did markedly reduce V without affecting Km (taurocholate: V = 65 nmol-min-1-g-1, Km = 78 muM; cholate: V = 104 nmol-min-1-g-1, Km = 354 muM). It was experimentally assured that the observed reduction of bile salt uptake was not a consequence of regurgitation of bile salts or due to an excessive intracellular accumulation during cholestasis in the sodium-free state. The rate of taurocholate efflux is very low when compared with the rapid rate of the uptake. A stimulatory action of extracellular sodium on this pathway was also observed. Inhibition of the (Na+ + K+)-ATPase by 1 mM ouabain resulted in a decrease of bile salt uptake. Activation of the enzyme by potassium readmission to a K+-deprived liver enhanced bile salt uptake. The immediate response to alteration of the enzyme activity suggests a close association of a fraction of bile acid active transport with the sodium pump.     

The effect of ouabain on the guinea pig ileum longitudinal smooth muscle: 2. Intracellular levels of Ca, Na, K, and Mg during the ouabain response and the dependence of the response on extracellular Ca.

Isotonic Tris-HCl containing 10 mM LaCl3 at 4 degrees C effectively removed extracellular ions in 30 min while preventing loss of intracellular ions. Intracellular Ca and Na increased during the contraction in the presence of 10 mM ouabain and then decreased during relaxation. Intracellular Na increased again during the latter part of the relaxation phase when K loss became apparent. Mg levels remained essentially constant. Ouabain responses were rapidly lost in Ca-free medium indicating that they were dependent on extracellular Ca. A 5.5-fold increase in the normal levels of extracellular K did not reduce the contraction to a submaximal dose of ouabain. A full phasic response to high K (60 mM) was observed after a 10-min exposure of the tissue to ouabain, at which time the ouabain response had returned to basal tension. The contraction to ouabain appears to be dissociated from inhibition of the Na,K-ATPase at the K site. The changes in intracellular ions indicated that ouabain contracted the muscle by increasing the plasma membrane permeability to Ca and Na and later decreased the K and Na concentration gradients, probably by inhibition of the Na,K-ATPase.     

Antagonistic effects of thyrotropin and epidermal growth factor on thyroglobulin mRNA level in cultured thyroid cells

Both thyrotropin (TSH) and epidermal growth factor (EGF) are potent mitogenic agents when added to dog thyroid cells in primary culture [Roger, P. P. and Dumont, J. E. (1984) Mol. Cell. Endocrinol. 36, 79-93]. The concomitant effect of these agents on the differentiation state of the cells was appreciated using cell morphology, iodide trapping, thyroglobulin synthesis and cytoplasmic thyroglobulin mRNA content as markers. Together with previous results [Mol. Cell. Endocrinol. 36, 79-93 (1984)] it is shown that cells cultured in the continuous presence of TSH maintain all the parameters at a near normal level. In the absence of TSH, thyroglobulin mRNA decreased to very low, though still detectable levels. Addition of TSH restored subnormal mRNA levels. Culture of cells in the presence of EGF for 4-6 days affected profoundly their morphology, abolished iodide trapping and decreased thyroglobulin synthesis and cytoplasmic mRNA content to undetectable levels. Addition of TSH to cells previously exposed to EGF reversed the growth factor effect on all four indexes. The redifferentiating effect of TSH was well observed within 3-4 days and was mimicked by the adenylate cyclase activators, forskolin and cholera toxin. When administered simultaneously, TSH and EGF achieved an intermediate situation, EGF antagonizing partially the effect of TSH on the expression of thyroglobulin gene. Another growth factor, fibroblast growth factor, while promoting thyroid cell proliferation also, did not interfere at all with TSH effects on cytoplasmic thyroglobulin mRNA content. Our results make the dog thyroid cell in primary culture an appropriate model to study the mechanisms involved in gene regulation by cyclic AMP and growth factors.