体外培养的人甲状腺细胞对TSH反应的实验研究

目的 研究促甲状腺激素（TSH）对甲状腺细胞分泌细胞的影响，探讨TSH的细胞内信号传导通路。方法 用含10%小牛血清的RPMI-1640培养液体外培养人甲状腺细胞，对照组48h后加入TSH，实验组24h内加入TSH（2U/L)，观察细胞培养1，3，5，7d的T3，T4分泌量及培养细胞内的cAMP含量和TPO活性。结果 对照组甲状腺细胞呈上皮样贴壁生长，实验组细胞则呈假滤泡样立体生长。实验组细胞T3、T4分泌量及细胞内cAMP含量均较同期对照组明显增加，但实验组TPO活性仅于3d之内较对照组增加，3d之后显著下降，至培养第5d2组细胞TPO活性均消失。结论 TSH作为一种生长刺激因子至少有一部分是以cAMP为第二信使起作用的；TSH对细胞增殖及分化的作用与细胞周期相关，可通过同一信号分子调节细胞生长，是细胞增值与分化的关键因素。     

On the mechanism of 'escape' from desensitization of the cyclic AMP response to TSH in cultured human thyroid cells

Studies were conducted to examine the mechanism by which 'escape' from TSH desensitization of the cyclic AMP response to TSH (Endocrinology 109, 1156, 1981) occurs in confluent cultured thyroid cells. At confluence, cell replication and DNA synthesis are suppressed. An attempt was therefore made to reproduce escape in sparse thyroid cell monolayers using inhibitors of DNA synthesis. The concurrent presence of TSH and mitomycin C (5 micrograms/ml) did not influence the induction of desensitization to TSH after 6 h of stimulation, but cAMP levels then rebounded by 24 h; that is, mitomycin C reproduced escape in sparse cells. Hydroxyurea (10 mM) did not reproduce escape in sparse cells. Adenylate cyclase activity was unaltered in plasma membranes prepared from sparse thyroid cells treated with mitomycin C for 24 h. These data suggest that 'escape' from TSH desensitization is related to events occurring during the cell cycle associated with DNA synthesis, and is caused by an alteration in adenylate cyclase substrate or co-factor availability rather than in enzyme activity itself.     

Age-related changes of TSH receptors in thyroid tissues obtained from euthyroid patients

The results obtained in the comparative study of age-related changes in TSH binding by thyroid tissues of euthyroid patients indicated that the content of high affinity binding sites of TSH receptors is not significantly decreased during aging, while the level of low affinity binding sites is increased in thyroid tissues of aged people. However, the dissociation constant of low affinity binding sites for TSH is significantly increased in thyroid tissues of old patients, compared with middle-aged people. Further investigation of functional characteristics of observed changes in TSH receptors and their binding parameters will provide a better understanding of the mechanism involved in the regulation of thyroid gland function during the aging.     

Tonic effect of endogenous TSH on the in vitro thyroid cAMP response to TSH

The present study was undertaken to compare the effects of 3,5,3'-triiodothyronine (T3) alone and T3 plus bovine thyrotrophin (bTSH) given chronically in vivo on the TSH-stimulated cyclic adenosine 3',5'-monophosphate (cAMP) production in a mouse thyroid in vitro. Mice were given T3 (5 micrograms/ml) in drinking water for 4 days. The thyroid cAMP concentrations after an incubation with 10 mU/ml of TSH for 10 min were decreased by 50% in T3-treated mice as compared to the control. In the second experiment, mice were given T3 alone or T3 plus 0.5 mU of bTSH ip daily for 4 days. The combined treatment with T3 and TSH partially restored the reduction of cAMP response to TSH that was induced by T3 alone. In the third experiment, mice were given T3 alone for 7 days, or T3 for 7 days plus TSH for the last 3 days. The reduced cAMP response to TSH induced by T3 alone was again partially restored by the concomitant treatment with TSH. These results indicate 1) that the capacity of the thyroid cAMP to respond to TSH is regulated, at least in part, by a trophic effect of endogenous TSH and 2) that the impaired capacity caused by a loss of tonic effect of endogenous TSH is reversible.     

Expression of G(alpha)(s) proteins and TSH receptor signalling in hyperfunctioning thyroid nodules with TSH receptor mutations

OBJECTIVE: Constitutively activating mutations of the thyrotrophin receptor (TSHR) are the main molecular cause of hyperfunctioning thyroid nodules (HTNs). The G protein coupling is an important and critical step in the TSHR signalling which mainly includes G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins. DESIGN: We investigated the in vitro consequences of overexpressing G(alpha) proteins on signalling of the wild-type (WT) or mutated TSHR. Moreover, we investigated whether changes in G(alpha) protein expression are pathophysiologically relevant in HTNs or cold thyroid nodules (CTNs). METHODS: Wild-type TSH receptor and mutated TSH receptors were coexpressed with G(alpha)(s), G(alpha)(i) or G(alpha)(q)/11, and cAMP and inositol phosphate (IP) production was measured after stimulation with TSH. The expression of G(alpha)(s), G(alpha)(i) and G(alpha)(q)/11 proteins was examined by Western blotting in 28 HTNs and 14 CTNs. RESULTS: Coexpression of G(alpha)(s) with the WT TSH receptor in COS 7 cells significantly increased the basal and TSH-stimulated cAMP accumulation while coexpression of the G(alpha)(q) or G(alpha)11 protein significantly increased the production of cAMP and inositol triphosphate (IP(3)). The coexpression of the TSH receptor mutants (I486F, DEL613-621), known to couple constitutively to G(alpha)(s) and G(alpha)(q) with G(alpha)(s) and G(alpha)(q)/11, significantly increased the basal and stimulated cAMP and IP(3) accumulation. Coexpression of the TSH receptor mutant V556F with G(alpha)(s) only increased the basal and stimulated cAMP production while its coexpression with G(alpha)(q)/11 increased the basal and stimulated IP(3) signalling. The expression of G(alpha)(s) protein subunits determined by Western blotting was significantly decreased in 14 HTNs with a constitutively activating TSH receptor mutation in comparison with the corresponding surrounding tissue, while in 14 HTNs without TSH receptor or G(alpha)(s) protein mutation and in 14 CTNs the expression of G(alpha)(s) protein was not different compared with the surrounding tissue. The expression of G(alpha)(i) and G(alpha)(q)/11 proteins in HTNs or CTNs was not significantly different compared with the surrounding tissue. CONCLUSIONS: The reduced expression of G(alpha)(s) protein subunits in HTNs with TSHR mutations could act as a feedback mechanism to desensitise the chronically stimulated cAMP cascade. As G(alpha) protein expression was not significantly increased in the majority of CTNs and HTNs an influence of G(alpha) overexpression on TSH signalling could be excluded in these nodules.     

Effect of dexamethasone on thyroid hormone response to TSH.

The effect of short-term dexamethasone administration (8 mg daily for 3 days) on thyroid hormone response to exogenous TSH (bovine TSH, 5 IU i.m.) was studied in 16 euthyroid volunteers. Serum T3 and T4 concentrations were measured by radio-immunoassay prior to and 2, 6, 12, 24, and 49 hr after bTSH injection, both under basal conditions and during dexamethasone treatment. In all subjects bTSH administration raised both T3 and T4 concentrations significantly. Dexamethasone treatment induced a slight depression of endogenous TSH (m +/- SEM = 2.0 +/- 0.4 versus 1.6 +/- 0.3 muU/ml) and T4 (6.8 +/- 0.4 versus 6.1 +/- 0.2 mug/100 ml) basal values and a significant decrease in T3 value (1.16 +/- 0.09 versus 0.64 +/- 0.06 ng/ml, p = 0.005). The mean increment of both T3 and T4 after bTSH injection was percentually unchanged during dexamethasone treatment but, due to lowered basal value, T3 levels at each time interval after TSH + dexamethasone were significantly lower than the corresponding values observed after TSH alone. The present data show that high dexamethasone doses decrease T3 serum levels significantly without inhibiting T3 response to TSH stimulation. Only a slight lowering was observed in T4 levels.     

Characterization of triton-solubilized TSH receptors from human thyroid plasma membranes

The TSH receptor from human thyroid plasma membranes has been solubilized in 10 mM Tris/HCl, 50 mM NaCl, pH 7.4 containing 0.5% triton X-100. Binding of [125I]TSH to the soluble receptor showed rapid and reversible kinetics and reached a maximum within 30 min at 37 degrees C, by 1 h at 25 degrees C and by 24 h 4 degrees C. Optimal pH was 7.4. The soluble receptor retained specificity with cross-reactivity only to crude hCG (0.03%). Scatchard plots were curvilinear indicating the presence of at least two binding sites. The high affinity site showed an affinity content of 1.1 X 10(9) M-1 with binding capacity of 1.3 X 10(-10) M/mg protein. TSH-binding inhibitor immunoglobulins from patients with Graves' disease inhibited [125I]TSH binding to the soluble receptor in a dose-dependent manner. NaCl inhibited the TSh binding and this was ascribed to the decrease in the receptor capacity. Trypsin, neuraminidase and phospholipase C treatment of the solubilized receptor had no effect on TSH binding. The apparent molecular weight of the receptor, determined by gel filtration of Sepharose 6B, was approximately 300 000.     

Age modifies the pituitary TSH response to thyroid failure.

OBJECTIVE: To investigate the association between serum TSH, total T4 and various patient characteristics when hypothyroidism is diagnosed in a population, and to study how age, sex and serum T4 levels influenced pituitary TSH response. DESIGN: A computer-based register linked to laboratory databases prospectively identified all patients with new, biochemically overt hypothyroidism (n = 685) in an open cohort in Denmark. The diagnosis was verified in each patient, and disease was classified into nosological type. Serum TSH and total T4 were recorded at the time of diagnosis in untreated patients with spontaneous autoimmune hypothyroidism (n = 578). MAIN OUTCOME: In untreated primary, spontaneous autoimmune hypothyroidism, we observed a four fold difference in average serum TSH levels between the youngest (0-20 years: TSH = 100 mU/l) and the oldest (80+ years: TSH = 24.4 mU/l) group of patients. No age dependent variation was observed in serum total T4. Log TSH showed an inverse linear correlation with age. An inverse linear correlation was present between log TSH and total T4 in both young and old patients, but for all total T4 values we observed lower median serum TSH values in elderly patients. CONCLUSIONS: For the same degree of thyroid failure, the serum TSH is lower among the elderly. This is most likely caused by a decrease in the hypothalamic/pituitary response to low serum T4. A certain increase in serum TSH may indicate more severe hypothyroidism in an old than in a young patient, and longer time may be needed after thyroid hormone withdrawal before elderly patients with thyroid cancer reach sufficiently high TSH values to allow for an effective radio-iodine treatment.     

Inhibition of TSH activation of human cultured thyroid cells by tumor necrosis factor: an explanation for decreased thyroid function in systemic illness?

Thyroidal economy in systemic nonthyroidal illness (the sick euthyroid syndrome) is marked by reductions in both thyroid function and peripheral T4 to T3 conversion, presumed to reflect a homeostatic mechanism to conserve energy. TSH levels tend to be normal in these patients, and the mechanism underlying reduced thyroidal secretion is unknown. Since increased blood levels of tumor necrosis factor (TNF) are found in many of the conditions associated with the sick euthyroid syndrome, we hypothesized that TNF might affect the function of the thyroid gland. We, therefore, explored the effects of TNF on TSH stimulation of the thyroid, employing a human thyrocyte cell culture model. Cells were incubated with various concentrations (0-1000 pg/mL) of recombinant human TNF-alpha and bTSH (1 mU/mL), with measurement of secreted thyroglobulin (Tg) and cyclic AMP (cAMP) as the end points of stimulation. TNF had no effect on either basal or TSH-stimulated cAMP generation, but significantly blunted TSH-stimulated Tg secretion. No loss of cell viability and growth was observed based on trypan blue exclusion and thymidine incorporation by cells. These studies demonstrated an inhibitory effect on TNF on human thyrocytes in concentrations comparable to blood levels seen in humans during systemic illness. We conclude that increases in serum TNF may be responsible for reduced thyroid function in patients with the sick euthyroid syndrome.     

Thyroid-stimulating immunoglobulin bioassay using cultured human thyroid cells

Modifications are described in the cultured thyroid cell cAMP assay for TSH which make it suitable for the measurement of thyroid-stimulating immunoglobulins. Comparison was made between this assay and two others measuring cAMP responsiveness in human thyroid tissue, namely the thyroid slice and thyroid plasma membrane adenylate cyclase assays, all performed with the same tissue sample. Of immunoglobulin G (IgG) samples from 7 unselected patients with untreated hyperthyroidism associated with Graves' disease, 5 produced significant stimulation of cAMP content in cultured thyroid cells when compared to pooled normal IgG. None of these 7 produced a statistically significant increase in thyroid slice cAMP content when assayed in triplicate, the same replicate number used in the cultured thyroid cell assay. Similarly, none of the same Graves' IgG samples produced significant stimulation (vs. control IgG) in the membrane adenylate cyclase assay, in which sensitivity to TSH stimulation was very poor. With a scaled-down modification of the assay using microtiter wells and acetylation to enhance detection of cAMP in the RIA, significant TSI activity was observed in 15 of 18 (83%) IgG samples from patients with untreated Graves' disease. The data indicate the excellent sensitivity and precision of the thyroid cell cAMP assay, as well as its convenience.     

Desensitization of the thyroid cyclic AMP response to thyroid stimulating immunoglobulin: comparison with TSH

Studies were conducted to examine the characteristics of thyroid cell cAMP stimulation by thyroid stimulating immunoglobulins (TSI) and to compare the cAMP response to TSI and TSH in desensitized human thyroid cells. In terms of cAMP production, preexposure (eight hours) of the cells to TSI induced a desensitization very similar to TSH-induced desensitization: both TSH- and TSI-desensitized cells showed a normal response to cholera toxin and forskolin stimulation; TSH and TSI desensitization was interchangeable in that desensitization by either stimulator affected the action of the other; the time of recovery from either TSH and TSH desensitization was identical; the cycloheximide (10(-4) mol/L) prevented both TSI- and TSH-induced desensitization; preexposure of the cells to iodine, which affects mainly the adenylate cyclase catalytic unit, or to epinephrine, which activate the inhibitory regulatory protein Ni by the alpha 2-adrenergic stimulation, induced a similar inhibition of the subsequent stimulation by both TSH or TSI. The remarkable similarities between TSH and TSI in stimulating and desensitizing thyroid cells strongly support the concept that TSI activates thyroid adenylate cyclase by interacting with the TSH receptor and not through an allosteric mechanism.     

The effects of TSH, cholera toxin and Graves' IgG on cAMP production in cultured human thyroid adenoma cells in monolayer

Monolayer cultures of human thyroid cells derived from thyroid adenoma were utilized for the assay of thyroid stimulating substances such as thyrotropin (TSH), cholera toxin and thyroid stimulating immunoglobulin (TSI) in patients with Graves' disease. Adenoma cells were treated with 0.1% collagenase or 2000 unit/ml dispase to thyrocytes. The cells were cultured in MEM containing 10% fetal calf serum under an atmosphere of 5% CO2 in air. Within 24 hours, the cells attached themselves to the plastic surface and formed a monolayer. Cyclic AMP responses to TSH, cholera toxin or Graves' IgG were tested in a medium (PBS) containing 0.5 mM IBMX. The cyclic AMP responses to TSH were generally maximal on the 3rd day of culture and declined thereafter. The response was dose-dependent, and 10 microU/ml of TSH produced a significant increase of cellular cyclic AMP. The response by 1 microU/ml of TSH was 28 approximately 57 fold above the basal. The response was also a function of the incubation period. The maximal response was attained after 1 h incubation. When the cultures were washed after exposure to TSH, the cellular cyclic AMP levels rapidly declined, suggesting that removal of receptor-bound TSH results in a prompt cessation of cyclic AMP production. The thyroid cells in monolayer also responded to cholera toxin. The response was dose-dependent, and cholera toxin as low as 1 ng/ml was able to increase cyclic AMP production. In contrast to the observations in TSH, the cyclic AMP responses induced by cholera were hardly affected by washing the cultures after exposure to cholera toxin. Treatment of the cells with cholera toxin for only 3 min resulted in a continuous stimulation of cyclic AMP production for more than 4 hours. Confirming recent observations by others, most of Graves' IgG stimulated cyclic AMP production in a dose-dependent manner, but some of them inhibited the response at high concentrations. IgG derived from normal subjects did not increase cellular cyclic AMP. The time course in the cyclic AMP responses induced by Graves' IgG was variable among the IgG preparations from different patients. In some patients, the maximal responses were attained after 4 hours of incubation. A significant difference was noted between TSH and Graves' IgG in the stimulation of cyclic AMP production after washing the cultures. When the cultures were treated with Graves' IgG for 30 min, washed and then incubated without Graves' IgG, cellular cyclic AMP levels remained at the levels which were almost equivalent to those observed in the continuous presence of the IgGs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blocking anti-thyrotropin receptor antibodies desensitize cultured human thyroid cells

Stimulating anti-TSH receptor antibodies (TSAb) mimic TSH in the induction of refractoriness in cultured thyroid cells; TSAb and TSH desensitize one another. We investigated whether blocking anti-TSH receptor antibodies (TBkAb) have the same desensitizing effects in cultured human thyroid cells. Prolonged exposure of cells (20 h) to TBkAb followed by antigen-antibody dissociation by an acid wash step was required to induce refractoriness to subsequent stimulation of cAMP accumulation with TSH and TSAb. Cycloheximide prevented this desensitization effect. The cAMP response to forskolin was not reduced in cells pretreated by TBkAb and was increased in cells desensitized by TSH or TSAb. The pattern of the TSH dose-response curves suggested that desensitization by TSH or TSAb involved only a postreceptor mechanism but both receptor and postreceptor phenomena in the case of TBkAb. In conclusion, like TSH or TSAb, TBkAb may induce a homologous desensitization in human thyroid cells which is not mediated by cAMP.     

Effect of gonadotrophins on progesterone secretion by cultured granulosa cells obtained from human preovulatory follicles

Granulosa cells were obtained from human preovulatory follicles in 31 women undergoing in vitro fertilization and embryo transfer due to tubal infertility. Follicular maturation was stimulated and synchronized by treatment with Clomiphene or human menopausal gonadotrophin (hMG), or both, plus human chorionic gonadotrophin (hCG). Follicles were aspirated by ultrasound guided puncture approximately 34-36 h after the hCG injection. The granulosa cells were washed and suspended in modified medium 199 containing 10% foetal bovine serum and cultured as monolayers for 6-8 days in the absence and presence of hormones and reactants. Progesterone formation was analyzed by RIA. In general, the cells underwent morphological luteinization and secreted high amount of progesterone. Under basal conditions the secretion of progesterone was highest during the first 2 days in culture and then gradually declined. Progesterone secretion was stimulated by human LH, hCG and the adenylate cyclase stimulator forskolin, with a maximal effect between days 2-6. The beta-adrenergic agonist isoproterenol in preliminary experiments potentiated the stimulatory effect of hCG but had no own stimulatory effect. No clear differences in progesterone secretion or responsiveness to in vitro stimulation relating to the various in vivo stimulation protocols were found.