Sheep Thyroid Research Articles

Iodide induces transforming growth factor beta 1 (TGF-beta 1) mRNA in sheep thyroid cells.

We examined TGF-beta mRNA levels in primary sheep thyroid cell cultures to determine whether the inhibitory effects of iodide on thyroid cells could be explained by an induction of TGF-beta mRNA and if this induction was mediated by iodine organification. Thyroid cells were incubated with TSH and five additives (insulin, somatostatin, growth hormone, transferrin, and glycyl-L-histidyl-L-lysin) for 2-3 weeks and then were exposed to sodium iodide (NaI) or 1-methylimidazole-2-thiol (methimazole, MMI), or both for 72 h. Iodide at 10(-6) M and 10(-4) M significantly increased the amount of TGF-beta mRNA as determined by Northern blot analysis with a rat TGF-beta 1 cDNA probe. This increase in TGF-beta 1 mRNA was abolished by the addition of methimazole, an inhibitor of organification. These data indicate that the effects of iodide on thyroid growth and function may be mediated by a process that involves organification of iodide and increases in TGF-beta 1 mRNA levels.

Hormonal regulation of insulin-like growth factor (IGF)-binding proteins secreted by isolated sheep thyroid epithelial cells: relationship with iodine organification

Isolated sheep thyroid follicles release specific insulin-like growth factor-binding proteins (IGFBPs). Since IGFBPs can modulate IGF bioactivity, at least in vitro, their presence in thyroid tissue may influence synergistic interactions between TSH and endogenous IGF-I or -II which are known to control both thyroid growth and function. We have examined the hormonal control of IGFBP release in relation to iodine organification. Sheep thyroid follicles were isolated by incubation with collagenase and differential centrifugation, grown in Coon's modified Ham's F12M medium with the addition of transferrin, glycylhistidyl-lysine, somatostatin (3H), TSH, cortisol and insulin (6H), and maintained in OH (hormone-free) or 3H medium with or without further supplements for 48 h. Conditioned culture medium was separated by 8% sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis, transferred to nitrocellulose and incubated with 125I-labelled IGF-II followed by autoradiography (ligand blot). Additionally, the radioactive bands were cut from the filters and quantified by gamma-spectrometry. Iodine organification was assessed by incubation of follicles with 10(6) c.p.m. Na125I for 3 h before washing, solubilization in 0.1 mol NaOH/l and the precipitation of organified radioisotope with 10% (v/v) trichloroacetic acid. Cells conditioned in OH or 3H medium released specific IGFBPs of 46, 34, 28 and 19 kDa on ligand blot analysis. The proteins of 34 and 19 kDa were immunopositive on Western blot analysis using anti-bovine IGFBP-2 antiserum. The 46-kDa IGFBP was retained by Concanavalin A-Sepharose chromatography and demonstrated to be glycoprotein. This is probably ovine IGFBP-3. The addition of TSH, or TSH plus cortisol to OH or 3H medium significantly decreased the 125I-labelled IGF-II associated with the 34- and 28-kDa IGFBP species. All IGFBP species were substantially reduced in 6H medium, which was predominantly due to the effects of TSH and cortisol. When total 125I-labelled IGF-II associated with IGFBPs was considered, a significant (P less than 0.01) inverse correlation existed between IGFBP activity and iodine organification in the same cultures; the latter being greatest in OH or 3H medium supplemented with TSH and cortisol. None of these hormone additions altered the endogenous release of IGF-II by the cells. These results suggest that endogenous IGFs, under hormonal control, may modulate the action of endogenous IGF in the regulation of thyroid function.

Regulation of Insulin-Like Growth Factor-Binding Protein Messenger Ribonucleic Acid Levels in Sheep Thyroid Cells*

The insulin-like growth factors (IGFs) exist primarily bound to cell surface receptors or complexed to specific binding proteins (IGFBPs). The IGFBPs modulate the bioavailability of the IGFs and may enhance or inhibit IGF actions. Several distinct forms of IGFBPs have been described on the basis of size, immunological determinants, and distribution in biological fluids; the IGFBPs may differ as well in their biological function. Sheep thyroid cells produce IGFBPs under hormonal regulation. Cells grown in basal medium or with six-hormone (6H) medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) release nonglycosylated BPs that migrate at 24, 27, 29, and 32 kDa on Western ligand blot. Cells cultured with the thyroid mitogens epidermal growth factor and phorbol ester release additional glycosylated IGFBPs of 40-44 kDa. Immunoprecipitation experiments indicate that 29- and 32-kDa IGFBPs are antigenically related to IGFBP-2, and the 40- to 44-kDa proteins are related to IGFBP-3. Using specific cDNA probes IGFBP-1, -2, and -3, we examined the regulation of IGFBP mRNA levels in sheep thyroid cultures. The rat IGFBP-2 cDNA probe hybridized to an approximately 1.6-kilobase mRNA species in cells under all culture conditions. However, IGFBP-3 mRNA was detectable only in epidermal growth factor- or phorbol ester-treated cells and appeared within 4 h, preceding the release of IGFBP-3 protein into the medium. The 6H additives, which stimulate differentiated function in thyroid cells, inhibited the mRNA levels of both IGFBP-2 and IGFBP-3. IGFBP-1 mRNA was not detectable. The distinct regulation of these IGFBPs suggest that they may play different biological roles in modulating thyroid physiology.

Thyrotropin Inhibits while Insulin, Epidermal Growth Factor and Tetradecanoyl Phorbol Acetate Stimulate Insulinlike Growth Factor Binding Protein Secretion from Sheep Thyroid Cells

Six insulin-like growth factor binding proteins (IGFBP) have been identified in the conditioned medium from sheep thyroid cells cultured under serum-free conditions. IGFBPs of 32, 28, 23 and 19 kDa were secreted by cells cultured for 14 days in serum-free and hormone-free medium. The constitutive secretion of IGFBP was inhibited by thyrotropin (TSH, 0.3 mU per mL). The effect was most marked on the secretion of the 28 kDa BP. High insulin concentrations stimulated the secretion of this IGFBP. The stimulatory effects of insulin were inhibited by TSH. Growth hormone treatment decreased the secretion of the 28 kDa protein. Tetradecanoylphorbol-13 acetate (TPA) and epidermal growth factor (EGF) both of which stimulate thyroid cell growth but inhibit differentiated function, markedly stimulated IGFBP secretion and induced the appearance of a 46 and a 150 kDa IGFBP. The effects of EGF and TPA were not identical. A rat IGFBP-2 cDNA reacted with sheep thyroid RNA of approximate size 1.6 kb. TPA treatment increased IGFBP-2 mRNA. Other hormones used to enhance differentiation and growth in thyroid cells in culture i.e. transferrin, somatostatin, cortisol and glycyl-histidyl-lysine acetate had no marked effects on IGFBP secretion nor on TSH-dependent, insulin-mediated iodide uptake and organification and cell growth. We show a correlation between secretion of high molecular weight IGFBP with enhanced growth but decreased function. Conversely, we find a correlation between decreased secretion of the 28 kDa BP and increased growth and function.

Immunohistochemical demonstration of calcitonin gene-related peptide and chromogranin A in C cells of sheep thyroid.

[in Japanese]

Is Long-term Levothyroxine Therapy Safe?

Our understanding of thyroid hormone action has increased tremendously since 1891, when George Murray¹first successfully treated a hypothyroid patient with sheep thyroid extract injections. As time and experience progressed, we have learned that the thyroid gland synthesizes and secretes two major active components, thyroxine and triiodothyronine. In fact, each of these products has been synthesized and is used now for the treatment of various clinical conditions. Presently, thyroid preparations are prescribed frequently for a variety of common clinical conditions (eg, hypothyroidism, suppression of solitary nodules, or multinodular goiter) See also pp 2077-2081. and, in 1988, accounted for about 1.3% of prescriptions filled in the United States (S. C. Kaufman, MD, T. P. Gross, MD, and D. L. Kennedy, MPH, unpublished data, 1990).²Endocrinologists are virtually unanimous in recommending synthetic levothyroxine for long-term use, and about 70% of prescriptions written for thyroid preparations in the United States by

Characterization of insulin-like growth factor-binding proteins secreted by isolated sheep thyroid epithelial cells

We have characterized the insulin-like growth factor-binding proteins (IGF-BPs) released by isolated sheep thyroid epithelial cells. Thyroid follicles were isolated with collagenase and cultured in Coon's modified F-12 M (0H medium) supplemented with insulin, cortisol, transferrin, glycyl-histidyl-lysine and somatostatin (5H medium) and TSH (6H medium). Conditioned 0H medium specifically bound both 125I-labelled IGF-I and -II, although binding capacity was reduced following acid-gel filtration to separate endogenous IGF-BP complexes, suggesting some destruction of BPs. The binding of 125I-labelled IGF-I or -II to conditioned (0H) medium was progressively displaced by increasing amounts of unlabelled homologous peptides, while fractionation on concanavalin A-Sepharose showed that the IGF-BPs consisted of both glycoprotein and non-glycoprotein components. The molecular sizes of the IGF-BPs were resolved by separation of 0H medium on SDS-PAGE and ligand blot analysis with 125I-labelled IGF-I or -II. Conditioned medium contained four specific binding species for IGF-II of 19, 30, 38 and 46 kDa; all but the smallest also binding radiolabelled IGF-I. Prior fractionation on concanavalin A-Sepharose showed that the 46 kDa binding species was a glycoprotein. Competition studies with increasing concentrations of unlabelled IGF-I or -II during ligand blotting suggested that the 46 and 30 kDa binding species had a greater affinity for IGF-II than IGF-I, while the 38 kDa had a greater relative affinity for IGF-I. Incubation of cells in 5H medium reduced the abundance of the 46 kDa binding protein, while incubation in 6H medium decreased the release of all binding protein species. Results show that isolated thyroid follicles released several forms of IGF-BP with differing relative affinities for IGF-I and -II. Gross changes seen in the presence of BPs between 0H, 5H and 6H media suggest acute hormonal control of release.

Effect of Hyperthermia on Thyroid Structure in the Late Gestation Fetal Lamb

This study examined the effect of hyperthermia in late gestation on fetal thyroid structure. Mild heat stress (increase of ewe's rectal temperature to 40.4 degrees C) caused significant changes of fetal thyroid structure. Intra-follicular colloid area was significantly larger in the heat-stressed group (2,532 +/- 181 microns2) compared to a control group where rectal temperature of the ewe was within the normal range (1,660 +/- 188 microns2; p less than 0.01). Epithelial cell height was significantly reduced (2.5 +/- 0.19 vs. 4.8 +/- 0.26 microns; p less than 0.01). Fetal plasma glucose concentrations were significantly lower in the heat-stressed group (0.7 +/- 0.07 mmol/l) compared to the non-heated group (1.0 +/- 0.11 mmol/l; p less than 0.05). We suggest that the fetal thyroid gland in sheep is responsive to environmental temperature changes and is important in the neuroendocrine regulation of fetal body temperature. Fetal hypoglycaemia may also contribute to the changes in fetal thyroid gland by altering the peripheral utilization of thyroid hormone. Such changes, if prolonged, may contribute to the decreased rate of fetal growth and maturation associated with chronic heat stress in sheep.

Immunohistochemical demonstration of parafollicular (C) cells in sheep thyroid and parathyroid glands.

Immunohistochemical demonstration of parafollicular (C) cells in sheep thyroid and parathyroid glands.

Interaction of TSH, Insulin and Insulin-like Growth Factors in Regulating Thyroid Growth and Function

Primary cultures of sheep thyroid cells have been used to study regulation of thyroid growth and function by growth factors and TSH. Cells were plated at low density to minimize contributions from the endogenously produced insulin-like growth factors and their binding proteins and other proteins or hormones secreted by thyroid cells in culture. Growth of the cells was followed for 7-11 days in medium without serum. We found that TSH by itself was unable to stimulate thyroid growth. However, the ability of insulin and IGF-I to stimulate thyroid cell growth was markedly potentiated by TSH. Thyroid function was assayed by measurement of uptake of pertechnetate and organification of iodide and also by synthesis of thyroglobulin mRNA. TSH alone was unable to stimulate thyroid function appreciably. Insulin and IGF-I were ineffective by themselves at stimulating thyroid differentiated function, but in the presence of TSH, all indices were stimulated markedly. We conclude that TSH by itself is not a growth factor for thyroid cells. However, in the presence of insulin or IGF-I, TSH potentiates the growth-stimulating properties of this hormone. Similarly, TSH by itself does not stimulate thyroid function but requires the presence of insulin or IGF-I. These data show the cooperative interactions between growth factors and TSH in regulating both thyroid growth and function.

Characterization of Insulin-Like Growth Factor-Binding Proteins from Sheep Thyroid Cells*

The insulin-like growth factors (IGFs) are bound by specific, high affinity binding proteins. Distinct classes of IGF-binding proteins have been described in human serum, amniotic fluid, cerebrospinal fluid, and conditioned medium from cultured cells. Sheep thyroid cells produce IGF-binding proteins under hormonal regulation. Cells grown without or with standard medium supplements (transferrin, glycyl-histidyl-lysine, hydrocortisone, somatostatin, insulin, and TSH) released binding proteins with apparent mol wt of 23, 29, and 32 kDa on Western ligand blot (nonreduced). Binding proteins from these cells appeared as 21, 26, 34, 36, and 41 kDa bands when cross-linked to [125I]IGF-I under reducing conditions. The addition of epidermal growth factor (EGF) or phorbol esters, thyroid cell mitogens stimulated the production of larger binding proteins with mol wt of 40-44 and 48-52 by ligand blot and cross-linking methods, respectively. Deglycosylation of conditioned medium cross-linked to [125I]IGF-I with endoglycosidase-F did not alter the size of the smaller binding proteins, but reduced EGF-stimulated binding proteins to 36-40 kDa. Similarly, tunicamycin treatment, which inhibits glycosylation, reduced only the size of this larger binding protein species. Polyclonal antisera directed against the human amniotic fluid binding protein (BP-28) immunoprecipitated the 32 kDa sheep thyroid binding protein seen on ligand blot and the cross-linked binding protein at 36-38 kDa. Antibody against the major human serum binding protein (BP-53) recognized only the larger EGF-stimulated binding proteins. In contrast to sheep thyroid cells, rat FRTL5 thyroid cells produced no detectable IGF-binding proteins. We conclude that the predominant binding proteins produced by sheep thyroid cells under standard culture conditions are non-glycosylated and immunoreact with antiserum directed against BP-28. EGF and phorbol esters stimulate production of larger glycosylated binding proteins antigenically related to BP-53.

Regulation of thyroperoxidase, thyroglobulin and iodide levels in sheep thyroid cells by TSH, tumor promoters and epidermal growth factor

Using sheep thyroid cells in culture, we have studied the effects of thyroid stimulating hormone (TSH), epidermal growth factor (EGF) and the tumor promoter, 12- O-tetradecanoylphorbol-13-acetate (TPA) on the activity and expression of both thyroglobulin (Tg) and thyroid peroxidase (TPO) and on the ability of cells to trap and organify iodide. Using Western blotting techniques, we found that TSH increased the absolute cellular levels of Tg. The optimum TSH concentration for Tg mRNA production was between 0.1–1.0 mU/ml. Thyroglobulin mRNA levels were stimulated by TSH but detectable levels were also present in cultures grown in its absence containing cortisol, insulin, transferrin, somatostatin and glycyl-lysyl-histidyl acetate. Unlike Tg, TPO protein levels were found to be completely dependent upon TSH. A time course of TSH stimulation of TPO mRNA showed increases after 8 h of TSH stimulation, whereas induction of Tg mRNA by TSH was seen at 24 h. Iodide trapping and organification were also TSH-dependent processes, showing maximum activities at 300–500 μU/ml of TSH. The addition of 10 nM TPA caused a biphasic decrease in radiolabeled pertechnetate uptake, with complete inhibition being seen at 14 h. Inhibition of iodide organification occurred more rapidly. TPA and EGF (1 nM) reduced the amount of newly synthesized Tg in TSH-stimulated cells by 50% but the absolute amount of Tg within the cells was not markedly inhibited at these early times. The amount of TPO protein, however, was markedly decreased by TPA and EGF. The results show that 2 agents which inhibit differentiated function do so, at least in part, by reduction of the protein levels of both TPO and Tg. Regulation of the amount of TPO protein appears to be more sensitively regulated than that of Tg by TSH, TPA and EGF.

Integrated regulation of growth and of function.

In vivo studies have shown that thyrotropin regulates thyroid growth and function. How TSH is able to exert these dramatic effects is not clear. The last decade has seen such refinements in cell culture systems that this question can finally be addressed. The major advance in cell culture technology has been the removal of serum from the culture medium of primary cultures of thyroid cells released from tissues. Serum can be replaced by a cocktail of hormones and other factors which have been determined empirically to be essential for the maintenance of most cells in culture.1 This chemically-defined medium permits researchers to add hormones both individually and in combination to examine direct, cooperative, and indirect interactions between them. Another unanticipated benefit from serum-free culture of thyroid cells has been that thyroid cell conditioned medium has been found to contain several proteins of potential biological importance.2–5 This finding has opened up a new area examining potential endocrine functions other than thyroid hormone production for the thyroid. Alternatively, these newly discovered products could be for autocrine or paracrine use. In this brief review we shall discuss the direct ways in which TSH is thought to regulate thyroid growth and function and how TSH interacts with other hormones and growth factors to influence these parameters. We shall examine the intracellular pathways thought to be activated by TSH, growth factors and by agents known to be capable of discretely activating second messengers of some partially defined intracellular pathways. We shall use as a model system data we have obtained with the sheep thyroid cell culture system. This culture system differs from FRTL5, dog and human thyroid cells in culture because cells reorganize to form 3D follicles6 and synthesize and secrete physiologic quantities of thyroid hormones.7 KeywordsThyroid CellInositol TrisphosphateFRTL5 CellHuman Thyroid CellThyroid GrowthThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Thyrotropin induces the acidification of the secretory granules of parafollicular cells by increasing the chloride conductance of the granular membrane.

Secretory granules of sheep thyroid parafollicular cells contain serotonin, a serotonin-binding protein, and calcitonin. Parafollicular cells, isolated by affinity chromatography, were found to secrete serotonin when activated by thyrotropin (TSH) or elevated [Ca2+]e. TSH also induced a rise in [Ca2+]i. We studied the effect of these secretogogues on the pH difference (delta pH) across the membranes of the secretory granules of isolated parafollicular cells. The trapping of the weak bases, acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine (DAMP), within the granules was used to evaluate delta pH. In contrast to lysosomes, which served as an internal control, the secretory granules of resting parafollicular cells displayed a limited and variable ability to trap either acridine orange or 3-(2,4 dinitro anilino)-3'-amino-N-methyl dipropylamine; however, when parafollicular cells were stimulated with TSH or elevated [Ca2+]e, the granules acidified. Weak base trapping was also used to evaluate the ATP-driven H+ translocation into isolated parafollicular granules. The isolated parafollicular granules did not acidify in response to addition of ATP unless their transmembrane potential was collapsed by the K+ ionophore, valinomycin. Secretory granules isolated from TSH-treated parafollicular cells had a high chloride conductance than did granules isolated similarly from untreated cells. Furthermore, ATP-driven H+ translocation into parafollicular granules isolated from TSH-stimulated parafollicular cells occurred even in the absence of valinomycin. These results demonstrate that secretogogues can regulate the internal pH of the serotonin-storing secretory granules of parafollicular cells by opening a chloride channel associated with the granule membrane. This is the first demonstration that the pH of secretory vesicles may be modified by altering the conductance of a counterion for the H+ translocating ATPase.

Insulin-like growth factors in sheep thyroid cells: Action, receptors and production

Sheep thyroid cells cultured in serum-free medium were used to study the biologic activity, binding, and production of the insulin-like growth factors (IGFs). IGF-I, IGF-II, and insulin stimulated thyroid cell division. Abundant, specific IGF receptors on sheep thyroid cell membranes were identified by binding displacement studies. Maximal specific binding of [125I]-labeled IGF-I and IGF-II to 25 ug of membrane protein averaged 21% and 27% respectively. The presence of type I and type II IGF receptors was confirmed by polyacrylamide gel electrophoresis of [125I]IGFs covalently cross-linked to cell membranes. Under reducing conditions. [125I]IGF-I bound to a moiety of approximate Mr =135,000 and [125I]IGF-II to a moiety of approximate Mr=26,000. Cross-linking of [125I]IGF-I to medium conditioned by thyroid cells indicated the presence of four IGF binding proteins with apparent Mr=34,000, 26,000, 19,000 and 14,000. Thyroid cells also secreted IGF-I and II into the medium. IGF synthesis was enhanced consistently by recombinant growth hormone. These data indicate that sheep thyroid cells are a site for IGF action, binding, and production and provide further evidence that IGFs may modulate thyroid gland growth in an autocrine or paracrine manner.

131I in the thyroid glands of sheep from Wales and south-west England after the Chernobyl accident.

Radioactive material from the Chernobyl reactor was deposited over the United Kingdom between May 2 and May 4, 1986. In this report, the 131I content of ovine thyroid glands, collected from seven abattoirs in Wales and south-west England is used to monitor the geographical distribution of the contamination. The results indicate that there was considerable local variation in the degree of contamination and suggest that, in addition to the major areas of contamination, deposition of high levels of radioactive material also occurred in isolated areas in the south-west of England. These observations confirm the value of monitoring 131I in the thyroid glands of grazing ruminants as a sensitive index of environmental contamination with the products of nuclear fission.

Serotonin-storing secretory granules from thyroid parafollicular cells

The subcellular storage of 5-HT was studied in sheep thyroid parafollicular cells. These cells are neural crest derivatives and were investigated as a serotonergic model system. Light and electron microscopic immunocytochemistry was used to examine the distributions of 5-HT, 45 and 56 kDa forms of 5-HT binding protein (SBP), and calcitonin. A single type of parafollicular cell was found that contained calcitonin, 5-HT, and 45 kDa SBP but not 56 kDa SBP. The secretory granules of parafollicular cells all displayed calcitonin immunoreactivity, and many were also immunoreactive for 5-HT and 45 kDa SBP. Granules were isolated, first by size and then by density, on successive metrizamide gradients. These provided a granular fraction that was enriched in calcitonin, endogenous 5-HT, and 45 kDa SBP. Immunoblots confirmed the presence of 45 kDa SBP in the isolated granules and in suspensions of parafollicular cells that were purified by an affinity chromatographic technique. Parafollicular cell granules did not appear to contain substantial stores of ATP. Granules isolated on Percoll gradients were morphologically homogeneous and took up 3H-5-HT. The specificity of this uptake was confirmed by quantitative electron microscopic radioautography. The granular uptake of 3H-5-HT was inhibited by reserpine (10 microM). It is concluded that parafollicular cell granules are different from other amine-storing vesicles that do contain ATP; nevertheless, since parafollicular cell granules store 5-HT and have the same 45 kDa SBP as is found in serotonergic axon terminals, parafollicular cell granules may be analogous to the synaptic vesicles of serotonergic neurons.

Induction of a neural phenotype in a serotonergic endocrine cell derived from the neural crest

The thyroid parafollicular cell is an endocrine cell derived from the neural crest that stores 5-hydroxytryptamine (5-HT). In common with serotonergic neurons, but in contrast to 5-HT-storing cells that are not neurectodermal derivatives, parafollicular cells also contain a specific 5-HT binding protein. Despite this similarity to serotonergic neurons, parafollicular cells in situ were found to express an endocrine phenotype with few neural characteristics. Thus, the cells costore 5-HT with calcitonin, not calcitonin gene-related peptide (CGRP), which is the product of the calcitonin gene expressed in neurons, and they do not contain neurofilaments. The ability of adult parafollicular cells to respond to microenvironmental perturbations by expressing neuronal characteristics was examined. Sheep thyroid glands were dissociated, and parafollicular cells were purified by affinity chromatography. The purified parafollicular cells were grown in culture on a variety of substrates in the presence or absence of the beta subunit of nerve growth factor (beta-NGF). Parafollicular cells survived in culture for at least a week but retained a roughly spherical shape. Nevertheless, a subset of the cultured parafollicular cells began to display CGRP immunoreactivity. The addition of beta-NGF to the cultured parafollicular cells induced a number of them to extend neurites and increased the proportion of cells in which CGRP immunoreactivity could be found. Neurite-bearing parafollicular cells appeared not to survive for more than 2 d. While their survival was not enhanced when they were grown on collagen, polylysine, laminin, or reconstituted basal lamina, parafollicular cells that had extended neurites in response to beta-NGF survived for at least a week when cocultured with an explant of aneuronal chick hindgut. The effect of the gut was local and only those neurite-bearing parafollicular cells that were growing in direct contact with the explant survived. The thyroid parafollicular cell therefore resembles another crest-derived endocrine cell, the adrenal chromaffin cell, in being able to manifest neural properties in culture. For the parafollicular cell these neural properties include the processing of RNA encoded by the calcitonin gene to express CGRP and neurite outgrowth in response to beta-NGF.

Role of non-TSH factors in thyroid cell growth.

The effects of insulin, the tumour promotor tetradecanoyl phorbol acetate (TPA), TSH and combinations of these factors on growth and DNA synthesis have been examined in the FRTL-5 cell strain and in sheep thyroid cells. In addition the regulation of the production by sheep thyroid cells of the insulin-like growth factors (IGF) by TSH and their possible autocrine roles have been investigated. We found that insulin and the IGF's stimulated DNA synthesis in both rat FRTL-5 cells and sheep cells. TPA also stimulated growth in both cell types, and its effects were additive to those of insulin. In the FRTL-5 cells, TPA was a less potent stimulator of growth than TSH, but the effects of TPA and TSH were not additive which may imply growth stimulation through a common pathway. In sheep cells TSH was not mitogenic and did not appear to activate protein kinase C, the receptor for TPA. Sheep cells, unlike FRTL-5 cells, were found to produce IGF-I and IGF-II, and their syntheses were regulated by TSH. Sheep cells were also found to produce IGF-binding proteins which may modulate the biologic effects of the IGF's. Sheep thyroid IGF binding proteins were found to copurify with urokinase-like plasminogen activator on immunoaffinity chromatography. The production of this serine protease has also been shown to be regulated by TSH.

Regulation of differentiated thyroid function by iodide: preferential inhibitory effect of excess iodide on thyroid hormone secretion in sheep thyroid cell cultures.

Primary cultures of sheep thyroid cells have been used to study the inhibitory effects of iodide on thyroid function. Under the influence of TSH, iodide was concentrated with a cell to medium ratio of 20. When thyroid hormone secretion was measured from cells cultured without addition of exogenous iodide, preferential T3 secretion was evident. The optimum iodide concentration for T4 and T3 synthesis and secretion was 10(-6) M. Prior exposure to 10(-5) M or more iodide decreased subsequent iodide transport in a concentration-dependent manner compared to that in cells acutely exposed to iodide. Although cell to medium ratios were decreased, intracellular iodide concentrations continued to rise with increasing external iodide concentrations, and iodide available for thyroid hormone synthesis was not in limited supply. Iodide concentrations of 10(-4) M or greater inhibited iodothyronine synthesis and thyroid hormone secretion, assessed by both assay of trichloroacetic acid-insoluble Na125I activity in cells and RIA of T4 and T3 in the medium and cell layer. An intermediate concentration of 10(-5) M iodide had a marked inhibitory effect on T4 and T3 secretion, but iodothyronine formation on thyroglobulin was only slightly affected. Our results suggest a preferential inhibitory effect of elevated iodide concentrations on thyroid hormone secretion. The adaptive advantages of this selective inhibition would allow storage of iodothyronines in times of iodide sufficiency while maintaining euthyroidism.