Effects of interleukin 1 alpha on porcine thyroid follicles in suspension culture

Interleukin 1, a product primarily of activated macrophages, was found to exert both a stimulatory and an inhibitory effect on porcine thyroid follicles in suspension cultures. A stimulatory effect on [125I] iodide incorporation into protein (iodination) was seen in cultures exposed to human recombinant interleukin 1 alpha (20 micrograms/l) for 1 h and (0.1 or 10 micrograms/l) for 18 h, whereas an inhibitory effect on iodination of interleukin 1 (10 micrograms/l) was registered after pre-incubation for 42 h and significant upon stimulation with TSH (200 mU/l). Cyclic AMP levels were stimulated in cells exposed to interleukin 1, however, significantly only after 42 h of pre-incubation, whereas TSH-stimulated cAMP response was inhibited by interleukin 1 already after 18 h of pre-incubation. The lumen of interleukin 1 incubated follicles was highly dilated and surrounded by a flattened epithelium. There were, however, no morphological signs of follicle disintegration or cellular cytotoxicity in the cultures exposed to interleukin 1. The data suggest that interleukin 1 might have a modulating role on thyroid function in autoimmune thyroid diseases associated with activation of intrathyroidal macrophages.     

Thyroid-stimulating antibody bioassay using porcine thyroid cells cultured in follicles

Isolated porcine thyroid cells were cultured in agarose-coated dishes and allowed to reform follicles with normal polarity. The thyroid cells reaggregated into follicles were compared with cells cultured in monolayer for cAMP responsiveness to TSH and thyroid-stimulating antibody (TSAb). The cells in follicles were sensitive to TSH stimulation and responded to the hormone at concentrations as low as 3.3-10 microU/ml with an increase in cAMP production. In contrast, 10-50 microU/ml TSH were required to elicit a cAMP response in cells cultured in monolayer using identical conditions. cAMP responsiveness to TSAb also was greater in the cells organized into follicles. TSAb was detected in serum from 89.4% of 66 untreated patients with hyperthyroid Graves' disease using thyroid follicles, but TSAb was detected in serum from only 60% of the patients when assayed using cells in monolayer. The assay using thyroid follicles was used to measure TSAb in 27 euthyroid patients who were euthyroid while receiving thionamide therapy and compared with 20-min thyroid 131I uptake after T3 suppression. TSAb was present in 11 of 12 nonsuppressible patients and in 5 of 15 suppressible patients. TSAb was positively correlated with 20-min 131I thyroid uptake. We conclude that thyroid cells cultured in follicles are suitable for measuring TSAb.     

Sodium dependence of the thyrotrophin-induced depolarization in cultured porcine thyroid cells

Cultured porcine thyroid cells exhibit a resting membrane potential of about -73 mV and depolarize to about -54 mV on exposure to TSH. The depolarizing response to TSH was preserved in a medium consisting only of inorganic salts and buffers, but was abolished in sodium-free medium, demonstrating dependence on an inward sodium current. Increasing the potassium concentration of the medium resulted in a reduction in the resting membrane potential of 60 mV per tenfold change in potassium concentration, and a diminished TSH response. A hyperpolarizing TSH response was observed in a sodium- and bicarbonate-free medium, indicating that a hyperpolarizing ion current (probably carried by potassium) was also enhanced in the presence of TSH. Tetrodotoxin blocked the TSH response. We conclude that the response of the thyroid cell membrane to TSH involves increases in permeability to sodium and potassium, and that the thyroid membrane ion channels bear some similarity to the voltage-dependent sodium channels of excitable tissues, despite the absence of action potentials in the thyroid.     

Decrease in cAMP-dependent protein kinase activity in suspension cultures of porcine thyroid cells exposed to TSH or forskolin

Suspension cultures of porcine thyroid cells were used to study the action of TSH and forskolin (Fk) on cAMP-dependent (PKa) and Ca2+-phospholipid-dependent (PKc) protein kinase--enzymes which represent the key step in the transduction of extracellular signals. The PKa activity in cells cultured for 2 days in the presence of TSH was decreased to about 50% of control level with a TSH dose of 0.1 mU/ml. This decrease is dose dependent; only traces of PKa activity remained at very high doses of TSH (50 mU/ml). Similar results were obtained with Fk (10(-5) M), the adenylate cyclase activator. It decreased the PKa activity to the level obtained with 0.1-1.0 mU/ml TSH. The loss of the PKa activity was parallel in cytosol and particulate fractions, suggesting that there is no translocation of enzymes under the action of either TSH or Fk. Neither TSH nor Fk had any effect on PKc, which became the predominant activity in cells exposed to either of the regulators. The cAMP-dependent phosphorylation of endogenous proteins was lower in TSH- or Fk-treated cells than in controls, and was dependent, like the PKa activity, on the dose of TSH. Polyacrylamide gel electrophoresis (PAGE) revealed the specific substrates of PKa in cultured thyroid cells. Proteins of 28, 30 and 33 kDa were regularly found, while 58 kDa protein was not present in all experiments. PAGE patterns showed that the decrease in endogenous phosphorylation in TSH- and Fk-treated cells was due to decreased labelling of PKa-specific substrates. The observed down-regulation of PKa activity could have an influence on the expression of thyroid cell differentiation.     

Electrical responses of cultured porcine thyroid cells to adrenergic agents

The membrane potential of cultured porcine thyroid follicular cells depolarized by up to 20 mV from the resting value of about -73 mV on exposure to beta-adrenoceptor agonists. A similar response was induced by TSH or dibutyryl cyclic AMP. alpha-Adrenoceptor agonists were without effect. The receptor subtype was shown to be (at least predominantly) beta 2 by the order of potency for beta-agonists (isoprenaline approximately equal to fenoterol much greater than adrenaline greater than noradrenaline) and by the relative potency of selective beta-antagonists (ICI 118,551 much greater than atenolol). The alpha-agonist phenylephrine had no effect on the TSH response but weakly inhibited the beta-agonist response. Rather than a physiological antagonism between alpha- and beta-adrenoceptor-mediated responses, this effect was shown to be due to the weak beta-antagonist effect of phenylephrine since the alpha-antagonist phentolamine failed to potentiate the depolarizing response to the mixed agonist noradrenaline, and also failed to block the inhibitory action of phenylephrine on the beta-agonist effect. Sensitivity to beta-agonist was enhanced by omission of serum from the culture medium and reduced by exposure to beta-agonists or a high concentration of TSH or dibutyryl cyclic AMP.     

Thyrotropin-stimulated electrical excitation and its refractoriness in cultured porcine thyroid cells

This report demonstrates TSH-stimulated electrical excitation in the thyroid and its refractoriness after exposure to TSH. TSH depolarizes the membrane potentials and causes action potentials. TSH-induced electrical activity is characterized by a latent period, rapid depolarization, action potentials (usually two spikes were observed), and then repolarization to the potential level of the silent phase. This TSH-induced electrical excitation is associated with iodide discharge. Previous exposure to TSH induces refractoriness of electrophysiological excitation and iodide discharge to further TSH stimulation.     

Effect of thyroid hormone on steroidogenic enzyme induction in porcine granulosa cells cultured in vitro

In order to elucidate the mechanism of thyroid hormone action on the ovary, direct effects of L-thyroxine (T4) or L-triiodothyronine (T3) on steroidogenic enzyme induction were investigated in vitro using a monolayer culture system of porcine granulosa cells obtained from small follicles. The cells were cultured in the absence or presence of porcine FSH (20ng/ml) for 6 days, with or without T4 or T3, under sparsely (4%) serum supplemented condition. The mechanism of thyroid hormone action on the granulosa cells was studied by testing the capability of thyroid hormone to enhance the steroidogenesis in response to exogenously provided substrates. Concomitant treatment with FSH (20ng/ml) and T4 (10(-7) M) caused a further increased production of progesterone in response to the addition of pregnenolone compared to that in the absence of pregnenolone. The same treatment with FSH and T4 also caused a further increased production of estrone in response to the addition of androstenedione. Concomitant treatment with 10(-9) MT3 demonstrated similar stimulatory effects on the steroidogenesis in cultured granulosa cells. T4 or T3 alone without FSH was incapable of exhibiting these stimulatory effects. Furthermore, aromatase activity in cultured granulosa cells assessed by the release of tritiated water from [1 beta-3H, 4-14C] androstenedione was significantly higher in the cells treated concomitantly with FSH (20ng/ml) and T4 (10(-7) M) than that in the cells treated with FSH alone. These results suggest that thyroid hormone synergizes with FSH and increases FSH-mediated induction of 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) and aromatase activity in immature granulosa cells. Since the effective dose of T4 and T3 observed in our studies is in the physiological range of circulating total levels of T4 and T3, it can be concluded that the synergism between FSH and thyroid hormone is of physiological importance to the full expression of FSH actions in the functional differentiation of immature granulosa cells.     

Thyrotrophin and prostaglandin E2 increase calmodulin levels and cyclic AMP phosphodiesterase activity in cultured porcine thyroid cells

Thyrotrophin (TSH) and prostaglandin E2 (PGE2) increased cellular cyclic AMP (cAMP), calmodulin levels and cAMP phosphodiesterase activity in cultured porcine thyroid cells. Dibutyryl cAMP (dbcAMP), a stable analogue of cAMP, increased calmodulin levels and cAMP phosphodiesterase activity. These results indicate that TSH- and PGE2-stimulated increases in calmodulin are mediated by cAMP. This increased concentration of calmodulin in turn stimulates cAMP phosphodiesterase. Double reciprocal plots of cAMP hydrolysis yielded two apparent Michaelis constants (Km); the lower in the 1 mumol/l and the higher in the 10 mumol/l range. Thyrotrophin, PGE2 and dbcAMP increased the values of maximal velocity without changing the Km values.     

Interaction of insulin-like growth factor I with porcine thyroid cells cultured in monolayer

The interaction of insulin-like growth factor I (IGF-I) with porcine thyroid cells cultured in monolayer was studied. Specific binding of [125I]iodo-IGF-I to thyroid cells was a reversible process dependent on the time and temperature of incubation. A steady state was achieved in 18 h at 4 C and averaged 14.2 +/- 2% (mean +/- SD)/10(6) cells. Binding of [125I]iodo-IGF-I was inhibited by unlabeled IGF-I; half-maximal inhibition occurred at concentrations of 2-5 ng/ml. Multiplication-stimulating activity (rat IGF-II) and pork insulin had relative potencies of 1:20 and 1:300 compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with a Ka of 4.3 X 10(10) M-1, 49,000 binding sites were estimated per cell. Affinity cross-linking and autoradiography demonstrated the presence of type I IGF receptors. Thyroid cells also had specific receptors for insulin, but specific binding of [125I]iodoinsulin (2.03 +/- 0.03%/10(6) cells) was much lower than that of [125I]iodo-IGF-I. Preincubation of thyroid cells with IGF-I or insulin caused a concentration-dependent decrease in [125I]iodo-IGF-I binding due to an apparent loss of receptors. Preincubation with epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, or TSH did not alter subsequent binding of [125I]iodo-IGF-I. Low concentrations of IGF-I stimulated DNA synthesis and proliferation of thyroid cells and acted synergistically with epidermal growth factor. Multiplication-stimulating activity and insulin had relative potencies in stimulating DNA synthesis comparable to their abilities to inhibit the binding of [125I]iodo-IGF-I to thyroid cells, suggesting that their effects are mediated primarily by IGF-I receptors. Preincubation with IGF-I did not alter cAMP responsiveness to TSH. We, thus, demonstrated the presence of functional and regulated IGF-I receptors on porcine thyroid cells.     

Effect of epidermal growth factor on the membrane potential of cultured porcine thyroid cells

Cultured porcine thyroid cells maintained in media containing TSH exhibited a membrane potential of -50 mV, and hyperpolarized by about 10 mV within 1 h of the addition of epidermal growth factor (EGF; 10 ng/ml). Follicle cells had depolarized to -45 mV after 4 h of exposure to EGF. Cells maintained in dibutyryl cyclic AMP (dbcAMP) did not alter their membrane potential when exposed to EGF for up to 4 h. Cultures washed to remove the TSH or dbcAMP hyperpolarized to -75 mV within 30 min, and a reversible depolarization to -60 mV was observed on addition of EGF. It was concluded that EGF acts as a physiological antagonist of TSH and also exerts a separate depolarizing influence on cultured thyroid cells.     

Direct regulating effects of transforming growth factor-beta 1 on lactate production in cultured porcine Sertoli cells.

In the present study we have tested the direct effects of transforming growth factor-beta 1 (TGF beta 1) on lactate production by Sertoli cells isolated from immature porcine testes. In Sertoli cells cultured in a defined medium, TGF beta 1 was shown to stimulate lactate production in a time- and dose-dependent manner. The maximal and half-maximal effects of TGF beta 1 on lactate production were obtained in the picomolar concentration range, respectively 24 and 8 pM TGF beta 1. TGF beta 1 action was found closely related to that of insulin since 1) both TGF beta 1 (40 pM) and insulin (1 microgram/ml) induced the secretion of similar and nonadditive amounts of lactate; and 2) TGF beta 1 and insulin induced comparable increases in lactate production in FSH (1 microgram/ml)-treated Sertoli cells. Because lactate is derived from glucose, 2-deoxy-D-glucose (2-DOG) was used to investigate the hexose transport system of Sertoli cells after insulin, FSH, and TGF beta 1 treatments. Insulin (1 microgram/ml) and FSH (1 microgram/ml) were found to stimulate 2-DOG transport with a similar time course, with an effect detected up to 30 min and maximal at 150 min. In contrast, although TGF beta 1 also enhanced 2-DOG uptake by Sertoli cells, the increase in glucose transport was delayed, since the TGF beta 1 effect was first detected at 150 min and was maximal at 360 min. These effects of TGF beta 1 action on Sertoli cell activity are exerted through specific membrane TGF beta 1 receptors. Scatchard analysis of the binding of TGF beta 1 to cultured Sertoli cells revealed the presence of both a high affinity (Kd, approximately 180 pM) and a low affinity binding site systems for TGF beta 1. Affinity labeling of these receptors by covalent attachment to [125I] TGF beta 1 with disuccinimidyl suberate and subsequent electrophoretic analysis of the labeled complexes revealed the specific binding of [125I] TGF beta 1 to three predominant molecules of 260, 130, and 70 kDa. In conclusion, the present study demonstrates that testicular Sertoli cells are targets for TGF beta 1 action. In view of the importance of lactate as a substrate for germ cells, it is suggested that TGF beta 1 might also be involved in the development of normal germinal epithelium.     

Anti-apoptogenic function of TGFbeta1 for human synovial cells: TGFbeta1 protects cultured synovial cells from mitochondrial perturbation induced by several apoptogenic stimuli

OBJECTIVE: To investigate anti-apoptogenic mechanism of transforming growth factor beta1 (TGFbeta1) towards synovial cells. METHODS: Isolated synovial cells, treated or not with TGFbeta1, were cultured in the presence or absence of anti-Fas IgM, proteasome inhibitor Z-Leu-Leu-Leu-aldehyde (LLL-CHO), etoposide, or C2-ceramide. After cultivation, apoptosis of synovial cells was examined by the presence of hypodiploid DNA(+) cells, the presence of terminal deoxy (d)-UTP nick end labelling(+) cells (TUNEL(+) cells), activation of caspases, and disruption of mitochondrial transmembrane potential (DeltaPsim). RESULTS: Activation of caspase-9 and DeltaPsim was found in anti-Fas IgM treated synovial cells. The increment of both hypodiploid DNA(+) cells and TUNEL(+) cells accompanied by the activation of caspase-8 and caspase-3 was also determined in anti-Fas IgM treated synovial cells. These hallmarks for apoptosis induced by anti-Fas IgM were significantly suppressed in TGFbeta1 treated synovial cells. LLL-CHO, etoposide, and C2-ceramide also caused DeltaPsim, the increment of both hypodiploid DNA(+) cells and TUNEL(+) cells, and the activation of both Leu-Glu-His-Asp ase (LEHDase; caspase-9 like activity) and Asp-Glu-Val-Asp ase (DEVDase; caspase-3 like activity) in synovial cells. As determined in anti-Fas IgM treatment, TGFbeta1 significantly reduced apoptotic cell death of synovial cells induced by the above chemicals. CONCLUSIONS: The protective effect of TGFbeta1 for mitochondrial homoeostasis may be important in the anti-apoptogenic function of TGFbeta1 for synovial cells.     

Differential effects of interleukin 1 alpha and 1 beta on cultured human and rat thyroid epithelial cells

The effects of human recombinant interleukin 1 alpha (20 pg/1-2 micrograms/l) and 1 beta (200 pg/1-20 micrograms/l) on two systems of thyroid cells have been compared. The thyroglobulin and cAMP secretion and the DNA content of human thyroid cells cultured in monolayer and of continuously grown rat thyroid cells, Fischer rat thyroid cell line have been studied. The growth of the rat thyroid cell line was inhibited by interleukin 1 beta (20 ng/1-20 micrograms/l), but not by interleukin 1 alpha. None of the cytokines changed the cAMP production of the rat thyroid cells. In contrast, both cAMP production and thyroglobulin secretion were inhibited dose-dependently by the cytokines in human thyroid cells in secondary cultures. These results caution the interpretation and extrapolation of changes induced by interleukin 1 from one cell system to the other.     

Cytoplasmic pH in cultured porcine thyroid cells: alkalinization by Ca2+

The role of calcium in cytoplasmic pH (pHi) changes was studied using 2',7'-bis(2-carboxyethyl)-5-(and 6-)carboxyfluorescein, an internalized fluorescent pH indicator, in cultured porcine thyroid cells. The Ca2+ ionophores A23187 and ionomycin stimulated thyroid cell alkalinization. An increase in cytoplasmic free calcium resulted in activation of Na+/H+ exchange which alkalinized the cells.     

Cytoplasmic pH in the action of insulin-like growth factor-I in cultured porcine thyroid cells

The effects of insulin-like growth factor-I (IGF-I) on cytoplasmic pH (pHi) and [3H]thymidine incorporation were studied in primary cultures of porcine thyroid cells. IGF-I alkalinized thyroid cells and stimulated thymidine incorporation in a dose-dependent manner; the effects of IGF-I on alkalinization (the maximal rates of change of cytoplasmic pHi/min ((dpHi/dt)max)) and thymidine incorporation were observed at 2 ng/ml and were maximal at 100 ng/ml, with half-maximal stimulation at approximately 10 ng/ml. The results indicate that Na+/H+ exchange or cell alkalinization may function as a transmembrane signal transducer in the action of IGF-I on thyroid cell proliferation. Several mitogens and comitogens which activate sodium hydrogen exchange, including epidermal growth factor, platelet-derived growth factor and nerve growth factor, have been listed. Activation with IGF-I has not, however, been presented before. Thus the present study constitutes the first demonstration of IGF-I-stimulated activation of Na+/H+ exchange or cell alkalinization.     

Inhibition of cyclic AMP formation by iodide in suspension cultures of porcine thyroid follicle cells

In the present study porcine thyroid cells in suspension cultures were employed to investigate the suppressive effect of iodide on adenylate cyclase under basal conditions and following incubation with TSH, PGE1, cholera toxin and forskolin. Within 30 min of incubation with iodide (half-maximal effect 10(-5) M), inhibition was established and remained unchanged up to 40 h of culture. The inhibitory action was abolished by methimazole. TSH, PGE1, cholera toxin and forskolin stimulated cAMP accumulation 10-, 3-, 24- and 22-fold, respectively. Iodide pretreatment reduced basal cAMP levels and also made the cells less sensitive to stimulation by the various agents. High concentrations of TSH or PGE1 could not overcome the suppressive influence of iodide, whereas with high concentrations of cholera toxin and forskolin the reduction in cAMP levels in iodide-treated cultures was less pronounced. Membranes isolated from iodide-treated cultures produced significantly lower amounts of cAMP compared to control membranes. Furthermore, iodide did not inhibit basal or forskolin-stimulated cAMP production in human fibroblasts. The results demonstrate that iodide via an iodination-dependent mechanism influences cAMP generation in thyroid cells. It is suggested that the inhibitory activity, which has a long half-life, involves stable modification of the membrane-localized catalytic unit of adenylate cyclase such that its activation by the regulatory unit is rendered less efficient.     

Aminoglutethimide augments follicle-stimulating hormone-induced aromatase activity in cultured porcine granulosa cells

The role of endogenous progestin synthesis in the modulation of FSH-induced aromatase activity was examined. Granulosa cells isolated from nonatretic medium-sized (3-5 mm) follicles of prepubertal pigs were cultured for an initial 48-h period, during which time aromatase activity was induced by FSH in the absence or presence of aminoglutethimide (AG). After induction, the cell monolayers were washed before being cultured for a further 6-h period in the presence of the substrate testosterone (0.5 microM). The aromatase activity was assessed by measuring the accumulation of estradiol during the test period. Basal aromatase activity was negligible and was unaffected by the presence of AG (0.1-100 microM) during the induction period. But when cells were cultured with FSH and AG (0.1-1000 microM) during the induction period, there was a dose-dependent, biphasic increase in the FSH-induced estradiol synthesis during the test period. Maximal enhancement was obtained with 10 microM AG (3.5-fold). Thereafter the aromatase activity declined and, at 1000 microM AG, was significantly (P less than 0.05) inhibited. At the same time, the FSH-stimulated progestin production during the induction period was inhibited in a dose-related fashion by AG. This AG-enhanced aromatase activity was dose and time dependent but was independent of the FSH concentration used. The apparent median effective dose of AG was 2.4 microM and a minimal time of 24 h or less was needed to potentiate the induction of aromatase activity by FSH. If AG was, however, added to the cell cultures during the test period, the FSH-induced aromatase activity was inhibited, showing that AG is an inhibitor of FSH-induced aromatase activity. This action of AG during the test period could be alleviated by the addition of testosterone during the induction period. The viability of the granulosa cells and the total cellular protein were not significantly (P greater than 0.05) altered by AG. These results show that the induction of aromatase activity by FSH could be enhanced by AG, which probably acts by inhibiting progestin production during the induction period, leading us to conclude that endogenous progestins might play an important role in modulating the induction of aromatase activity by FSH.     

Phenol red: an inhibitor of thyroglobulin iodination in cultured porcine thyroid cells.

Phenol red, commonly used as a pH indicator in tissue culture media, is known to possess estrogenic properties. We investigated the effect of phenol red on the process of thyroglobulin iodination which occurs only at the apical surface of porcine thyroid cells when cultured in porous bottom chambers. When phenol red was added simultaneously to both compartments (apical and basolateral), separated by the polarized monolayer, thyroglobulin iodination was inhibited by about 86% without any effect on thyroglobulin secretion and apical iodine concentration. When phenol red was added separately to either the apical or basal media, inhibition was 68% and 43%, respectively. A large amount of phenol red which was introduced into the basal medium crossed through the monolayer. Thus, inhibition was dependent upon the concentration of phenol red present in the apical compartment. A maximal inhibition was observed from 30 microM apical concentration. Phenol red acts as a substrate for thyroperoxidase in the iodination reaction.     

The biocellular effect of thyroid hormone on functional differentiation of porcine granulosa cells in culture

To elucidate if the thyroid hormone acts directly on the ovary, the biocellular effect of L-thyroxine (T4) on porcine granulosa cells cultured in vitro was investigated. Monolayer cultures of porcine granulosa cells obtained from small (1 approximately 2 mm), medium (3 approximately 5 mm) or large (6 approximately 11 mm) follicles were carried out in the presence of porcine FSH (100 ng/ml). Concomitant treatment with T4 promoted FSH-dependent morphological luteinization, i.e. alteration of immature granulosa cells obtained from small follicles to epithelioid form. T4 also increased FSH-stimulated induction of hCG/LH receptor on immature granulosa cells. Furthermore, T4 augmented FSH-mediated production of progesterone and estradiol by immature granulosa cells cultured in vitro. The concentration of T4 to produce the maximal stimulatory effect was 10-7 M, demonstrating that optimal concentration of thyroid hormone is required for the expression of this stimulatory action. Since T4 alone demonstrated no effect on the differentiation of porcine granulosa cells and all the stimulatory effect of T4 seems to have a permissive action on FSH-induced granulosa cell luteinization. Although insulin showed a similar effect on porcine granulosa cells, no stimulation of estradiol production by porcine granulosa cells was observed with insulin in the culture system used in this study. These results suggest that the thyroid hormone acts directly on the ovary and plays an important role in modifying the FSH-dependent cellular differentiation of immature granulosa cells.