The differential effect of thyrotropin on the electrical responses of thyroid cells in monolayer cultures of varying duration

The acute effects of thyrotropin on the membrane potential of thyroid cells maintained in the presence or absence of thyrotropin (0.2 U/ml) in the culture medium was determined. Monolayer cultures were prepared from porcine thyroid glands and cultured for 4–17 days after which the culture medium was exchanged for a buffered salt solution for intracellular measurements of the membrane potential. Cells were serially impaled with a microelectrode, first in the absence and then in the presence of 10 mU/ml thyrotropin. Cells cultured for 4–9 days depolarized from ?29.6 ± 1.7 (mean ± S.E.) to ?19.3 ± 1.3 mV within 10 min after acute addition of 10 mU/ml thyrotropin. From 11 to 17 days of culture, basal membrane potentials were lower and, in most instances, cell hyperpolarization occurred within 30 min in response to thyrotropin. There was no difference in electrical response of cells maintained in culture with or without thyrotropin. However, cells cultured with thyrotropin formed follicle-like structures in contrast to the monolayer formation of cells cultured without thyrotropin. The changes in the basal and stimulated electrical responses occur within a time frame similar to that reported for changes in the biosynthetic capacity of thyroid cells in culture. The data further emphasize the possible regulatory role of the cell membrane in stimulus-secretion coupling in the thyroid.     

The effect of indomethacin on the response of thyroid tissue to thyrotropin

Indomethacin, in concentrations up to 400 μM, fails to alter the stimulation by thyrotropin in various thyroid preparations of [1-¹⁴C]glucose oxidation, hormone and iodine secretion, and adenylate cyclase activity. It is concluded that prostaglandin synthesis is not an obligatory step in the activation of these thyroid functions by thyrotropin.     

Noninactivation of thyrotropin by cultured porcine thyroid cells.

Freshly isolated porcine thyroid cells were cultured in the presence of highly purified porcine thyrotropin. Cells associate into follicles between the second and tenth day of culture and later form a monolayer. The biological and immunological activity of thyrotropin was measured daily in the media. Thyrotropin concentration and biological activity remained unchanged from the onset of the culture up to day 14. Limiting factors influencing thyroglobulin biosynthesis do not appear before day 13. The loss of follicular organization at day 10 cannot be explained by thyrotropin degradation in the medium. Considering the number of receptors per cell and the half life of the thyrotropin . receptor complex in the two dissociation compartments previously demonstrated, it appears in terms of both biological activity and affinity for the receptors that the thyrotropin molecules released from the first compartment do not differ from native molecules. It can be calculated that at least 31% of the molecules released from the second compartment are not inactivated. Thus, it is probable that the catabolism of thyrotropin on the receptor, or near the receptor site, does not play an important role in the regulation of thyroid cell function in vitro.     

Effects of thyrotropin and thyroid hormones in vivo on thyroid responsiveness to thyrotropin in vitro.

The thyroid gland of rats fed propylthiouracil is known to be unresponsive in vitro to thyrotropin; to investigate further the underlying mechanism groups of rats were variously treated with propylthiouracil and thyroid hormone or subjected to hypophysectomy. In vitro responsiveness of the thyroids was tested by measuring an increase in the concentration of c AMP when thyrotropin or prostaglandin E1 was added to the medium. Results showed that responsiveness to thyrotropin partially returned with rats fed prophylthiouracil and hypophysectomized 5, but not 2, days before death; hypophysectomy of normal rats led to increased in vitro responsiveness to thyrotropin and this was partially reversed by injections of thyrotropin for a week before death. Administration of thyroid hormone had little effect in these investigations and in vitro responsiveness to prostaglanding E1 was not consistently influenced by any of the in vivo regimens. From this experience we conclude that, at least as studied in vitro, circulating thyrotropin has a significant role in modulating responsiveness of the thyroid to thyrotropin.     

Kinetic studies of the localization of aminopeptidase N in monolayer and in follicle-associated cultures of porcine thyroid cells

Summary The fate of aminopeptidase N (AP_N) was investigated by means of immunofluorescence in porcine thyroid-cell cultures. At day 1, control cells cultured without TSH formed flat, cylinder-like aggregates in which AP_N was mainly located on the lateral surface. At day 2, the fluorescence completely disappeared. At days 3 and 4, AP_N appeared in the apical pole of monolayer cells directed toward the medium and in intracellular organelles. The cilium in each cell was intensely labelled and located in the center of the apical pole. In thyrotropin-stimulated cell cultures, the fluorescence at days 1 and 2 was located over the entire plasma membrane. At days 3 and 4, morphological polarization of cells, reorganized into follicles, occurs, AP_N being located either in the apical or basolateral regions and in intracellular vesicles. Between day 4 and 6, segregation of the enzyme to the apical membrane occurred. Between day 6 and 14, fluorescence was generally retained in the apical region and in an intracellular pool. In growing cell cultures, after 2 to 5 subcultures, the cells exposed AP_N in a pattern similar to primary monolayers. After conversion of monolayer cells into follicles, the antigen was recovered in the apical pole of cells facing the follicular lumen.     

The role of proteases in fibronectin matrix remodeling in thyroid epithelial cell monolayer cultures

Fischer rat thyroid (FRT) cells organize a matrix of extracellular fibronectin (FN) fibrils, which undergoes extensive remodeling according to cell culture confluence. In non-confluent cells FN forms a fibrillar array associated with the ventral cell surface. However, basal FN is progressively removed in confluent cultures and substituted by non-fibrillar FN deposits at lateral cell domains in regions of cell-cell contacts. FRT cells secrete and expose on the plasma membrane the tissue-type plasminogen activator and, in serum-free cultures, plasminogen induces a rapid loss of FN fibrils. Incubation with plasmin inhibitors greatly reduces this effect. FRT cells also express annexin II, a plasminogen receptor, suggesting that plasmin activity is associated with the pericellular enviroment. This is in agreement with the observation that a great reduction in FN degradation is observed if the cells are pre-incubated with carboxypeptidase B, which prevents plasminogen binding to the cells. A gelatinolytic activity with a molecular weigth equivalent to MMP-2 has been demonstrated by zymography of culture media, and the presence of MMP-2 and MT1-MMP on the cell plasma membrane has been detected by immunofluorescence. These results indicate that in the FN remodeling process, occurring during FRT epithelium maturation, both plasmin-dependent (tPA activated) and plasmin-independent proteolytic activities are involved.     

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.     

In vivo effect of thyrotropin on intracellular translocation of thyroid peroxidase in rat thyroid cells by an indirect immunofluorescence method

The in vivo effect of thyrotropin (TSH) on the intracellular localization of thyroid peroxidase (TPO) in rat thyroid epithelial cells was examined by an indirect immunofluorescence method. The staining for TPO in the epithelial cells of normal rats appeared all over the cytoplasm, especially in the apical region. The injection of propylthiouracil for 3-10 days increased the staining in the apical region. The administration of L-thyroxine for 7-10 days to normal rats abolished the relatively high localization of TPO in the apical region, and resulted in TPO staining all over the cytoplasm. Six hours after TSH was injected into the thyroxine-treated rats, localization of TPO staining in the apical region was observed. These results suggest that TSH may play a role in the translocation of preexisting TPO to the apical region before TSH-induced biosynthesis becomes evident.     

Differential effects of thyrotropin on various glycosyltransferases in porcine thyroid cells

Porcine thyroid cells were cultured with or without thyrotropin for 9 days. It is known that the hormone enhances the synthesis of thyroglobulin, with an increase in the content and stability of its m-RNA. We show in the present work that thyrotropin also stimulates a number of glycosyltransferases diversely situated along the process of N-glycosylation. The most increased was oligosaccharyltransferase, responsible for attachment to nascent peptides of preformed core carbohydrate sequences. The relatively low activity level of oligosaccharyltransferase and its preferential responsiveness to thyrotropin supports the possibility of a regulation point at this enzyme.     

Effect of thyrotropin on glycosaminoglycans synthesized by primocultured thyroid cells

The synthesis of glycosaminoglycans (GAGs) was investigated in porcine thyroid cells under the influence or not of thyrotropin. After labelling with [³H] glucosamine and [³⁵S] $\text{SO}{}_4{}^{\mathrm{2?}}$, enriched GAG-fractions prepared from culture media, cells, and eventually substrate adhering materials, were analyzed by cellulose acetate electrophoresis combined with specific degradations. They comprised heparan sulfate and hyaluronic acid together with an unknown sulfated component labile to endo-β-galactosidase. Whereas global labellings of newly made GAGs were not significantly modified by thyrotropin, we reproducibly observed with the hormone a substantial increase in the proportion of hyaluronic acid [³H] label and, when cells organized into follicles, of the proportion of cell-associated [³H] GAGs. This system thus offers an interesting model to study how the responsiveness to an hormone and the reorganization that follows might implicate specific glycoconjugates.