Polarized secretion of tissue-plasminogen activator in cultured thyroid cells.

We studied the polarized secretion of tissue-type plasminogen activator in porcine thyroid cells cultured as a monolayer on porous bottom chambers. The presence of tissue-type plasminogen activator was detected by zymographic analysis on two independent media that were in contact either with the apical surface or with the basolateral membrane. The amount of tissue-type plasminogen activator was determined in both media by ELISA and enzyme assay. Measurable tissue-type plasminogen activator activity was found in the basal but not in the apical medium. However, on zymogram, a lytic zone corresponding to tissue-type plasminogen activator was visible in both media. In addition, a lytic band at 130 kDa suggested presence of a complex formed by tissue-type plasminogen activator and an inhibitor. Preferential basolateral tissue-type plasminogen activator antigen secretion (70%) has been observed, showing the possible relation between tissue-type plasminogen activator and extracellular matrix components. Neither tissue-type plasminogen activator level nor polarized secretion seemed to be regulated by thyrotropin (0.1 mU/ml).

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 microM 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.

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.

Long-term iodination of thyroglobulin by porcine thyroid cells cultured in porous-bottomed culture chambers: regulation by thyrotrophin.

Thyroid cells cultured as monolayers on the porous bottom of culture chambers have been shown to express some specific functions of thyroid follicles. This system, which allows independent access to apical and basal media, is suitable for the long-term study of polarized processes, as the cells maintain their polarized organization. Iodination of thyroglobulin has been investigated under different culture conditions in the presence or absence of TSH. Apical thyroglobulin accumulation, apical iodide concentration and thyroglobulin iodination have been followed simultaneously. Iodide (0.5 mumol/l) was added to basal medium at various stages: only once for 4-day incubations and at each medium change or daily for longer experiments. TSH increased the amount of thyroglobulin secreted into the apical medium by five- to sixfold, whereas high basal iodide concentrations (greater than 5 mumol/l) inhibited thyroglobulin secretion by TSH-stimulated cells. TSH increased iodide uptake giving an iodide concentration ratio between apical and basal media of about 5. Thyroglobulin iodination was dependent upon TSH. Thyroglobulin was iodinated only in the apical compartment. Secretion and iodination of thyroglobulin were polarized phenomena, but the polarity of iodination was total whereas the polarity of secretion was only partial (10% basal secretion). This functional asymmetry was maintained for up to 29 days. The maximal incorporation of iodine into thyroglobulin obtained was never higher than 3.5 atoms/mol. Apical iodide concentrations from 1 to 15 mumol/l, depending on culture conditions, did not increase this value. These results suggest that cells cultured in this culture system are able to reproduce several steps of thyroidal iodide metabolism although there may be unknown factors which could interfere and reduce the efficiency of thyroglobulin iodination.

Thyrotrophin regulation of apical and basal exocytosis of thyroglobulin by porcine thyroid monolayers.

Exocytosis, the ultimate step in thyroglobulin secretion, has been studied in porcine thyroid cells cultured in monolayers on the permeable bottom of culture chambers. We have previously demonstrated, using this culture system, that apical secretion accounts for 85-95% of total secretion of newly synthesized thyroglobulin. When cells were cultured for several days with bovine TSH (25 microU/ml) in the basal medium, the rate of glycoprotein accumulation in the upper compartment was three times higher than that in the absence of TSH. In contrast, the rate of thyroglobulin released into the basal medium (5-15% of total secreted thyroglobulin) appeared unmodified by chronic TSH stimulation. To investigate the effect of acute TSH stimulation on thyroglobulin exocytosis in the apical and basal compartments, pulse-chase experiments were carried out with the same culture system. The release of radiolabelled thyroglobulin (1.5-h pulse) into the apical medium was increased threefold during the 2-h chase period under TSH stimulation. The radiolabelled thyroglobulin released into the basal medium was increased only 1.5- to 2-fold, and stimulation disappeared after 1 h. The effect of TSH was maximal when the chase medium contained 50 microU TSH/ml. However, cells cultured for several days in the presence of 25 microU TSH/ml before the pulse-chase experiment, appeared desensitized to acute TSH stimulation. Similar responses were observed when the chase medium contained 8-chloro-cyclic AMP or cholera toxin. This study provides another example of the pleiotropic effect of TSH, mediated by cyclic AMP, on the sequential steps of thyroglobulin gene expression in cultured thyroid cells in which the polar character of the epithelial cells is well preserved.