RESUMEN
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.
Asunto(s)
Yoduros/farmacología , Sulfatos/metabolismo , Glándula Tiroides/metabolismo , Tirotropina/farmacología , Ácido 4,4'-Diisotiocianostilbeno-2,2'-Disulfónico/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Epitelio/metabolismo , Cinética , Biosíntesis de Proteínas , Radioisótopos de Azufre , Porcinos , Tiosulfatos/farmacología , Glándula Tiroides/efectos de los fármacosRESUMEN
Cell volume regulation by thyrotropin (TSH) and iodide, the main effectors involved in thyroid function, was studied in cultured thyroid cells. The mean cell volume, determined by performing 3-D reconstitution on confocal microscopy optical slices from living octadecylrhodamine-labeled cells cultured with both TSH and iodide (control cells), was 3.73 +/- 0.06 pl. The absence of iodide resulted in cell hypertrophy (136% of control value) and the absence of TSH in cell shrinkage (81%). These changes mainly affected the cell heights. The effect of TSH on cell volume was mediated by cAMP. The proportion of cytosolic volume (3-O-methyl-D-glucose space vs. total volume) decreased in the absence of iodide (85% of control value) and increased in the absence of TSH (139%), whereas protein content showed the opposite changes (121 and 58%, respectively). The net apical-to-basal fluid transport was also inversely controlled by the two effectors. Iodide thus antagonizes TSH effects on cell volumes and fluid transport, probably via adenylylcyclase downregulation mechanisms. The absence of either iodide or TSH may mimic the imbalance occurring in pathological thyroids.
Asunto(s)
Espacio Extracelular/metabolismo , Yoduros/farmacología , Glándula Tiroides/citología , Tirotropina/farmacología , 3-O-Metilglucosa/metabolismo , Animales , Tamaño de la Célula , Células Cultivadas , Citosol/metabolismo , ADN/biosíntesis , Yoduros/metabolismo , Microscopía Confocal , Proteínas/metabolismo , Porcinos , Glándula Tiroides/ultraestructuraRESUMEN
Thyroid hormone synthesis is under the control of thyrotropin (TSH), which also regulates the sulfation of tyrosines in thyroglobulin (Tg). We hypothesized that sulfated tyrosine (Tyr[S]) might be involved in the hormonogenic process, since the consensus sequence required for tyrosine sulfation to occur was observed at the hormonogenic sites. Porcine thyrocytes, cultured with TSH but without iodide in the presence of [(35)S]sulfate, secreted Tg which was subjected to in vitro hormonosynthesis with increasing concentrations of iodide. A 63% consumption of Tyr[S] (1 residue) was observed at 40 atoms of iodine incorporated into Tg, corresponding to a 40% hormonosynthesis efficiency. In addition, hyposulfated Tg secreted by cells incubated with sodium chlorate was subjected to in vitro hormonosynthesis. With 0.5 Tyr[S] residue (31% of the initial content), the efficiency of the hormonosynthesis was 29%. In comparison, when hormonosynthesis was performed by cells, with only 0.25 Tyr[S] residue (16% of the initial content), the hormonosynthesis efficiency fell to 18%. These results show that there exists a close correlation between the sulfated tyrosine content of Tg and the production of thyroid hormones.
Asunto(s)
Tiroglobulina/química , Hormonas Tiroideas/biosíntesis , Tirosina/análogos & derivados , Animales , Células Cultivadas , Cloratos/farmacología , Yodo/metabolismo , Yodo/farmacología , Cinética , Oligosacáridos/química , Oligosacáridos/metabolismo , Sulfatos/metabolismo , Porcinos , Tiroglobulina/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/metabolismo , Tirotropina/farmacología , Tirosina/metabolismoRESUMEN
OBJECTIVE: To study the regulation of thyroglobulin sulfation by thyrotropin (TSH) and iodide. Sulfation, a widespread post-translational modification of proteins, is involved in various biological activities. Thyroglobulin has been reported to be sulfated but, to date, the role of sulfate residues in the metabolism and function of thyroglobulin is not known; moreover, the regulation of thyroglobulin sulfation has not been yet investigated. METHODS: The effect of TSH on thyroglobulin sulfation was studied in porcine thyroid cells cultured on porous collagen-coated filters. Cells cultured with or without TSH and with or without iodide (KI) were incubated for 4 days with radioactive sulfate. The specific radioactivity of thyroglobulin subunit (330kDa) was determined from apical media analyzed by electrophoresis. Enzymatic hydrolysates of the purified thyroglobulin were separated by oligosaccharide affinity chromatography and thin-layer chromatography; alkaline hydrolysates were analyzed only by thin-layer chromatography. RESULTS: Thyroglobulin secreted by TSH-stimulated cells incorporated about twofold less radioactive sulfate. Iodide slightly modified this incorporation. Enzymatic hydrolysates of purified thyroglobulin showed sulfate residues bound essentially to complex oligosaccharide units. Alkaline hydrolysis was necessary to release all sulfated amino acids (tyrosine and serine). In the absence of TSH the proportion of tyrosine sulfate was dramatically increased: 24% compared with 7% (+KI) or 5% (-KI). The ratio of specific radioactivity of thyroglobulin to the specific radioactivity of intracellular inorganic sulfate (determined in each culture condition) gave the number of sulfated residues incorporated: 46 (-TSH) and 31 (+TSH) per mol thyroglobulin. From this distribution, we deduced the number of residues bound to complex oligosaccharide units and to tyrosine. Thus TSH decreased the number of sulfate residues on tyrosine from 11 to 2 per mol thyroglobulin. CONCLUSIONS: TSH regulates the binding of sulfate groups to tyrosine residues. Iodide exerts a slight control over this process.
Asunto(s)
Sulfatos/metabolismo , Tiroglobulina/metabolismo , Tirotropina/farmacología , Tirosina/metabolismo , Animales , Células Cultivadas , Concanavalina A/metabolismo , Glicopéptidos/metabolismo , Yoduros/farmacología , Oligosacáridos/metabolismo , Sulfatos/análisis , Radioisótopos de Azufre , PorcinosRESUMEN
The regulation of thyroperoxidase (TPO) expression and of its intracellular distribution was studied in porcine thyroid cells cultured on porous bottom filters. Cells were cultured for 18 days in the absence or in the presence of thyrotropin (TSH) and with or without iodide. Microsomes were purified and analyzed by electrophoresis. TPO was detected by immunoblotting with polyclonal anti-porcine TPO antibodies and quantified by scanning the bands. The amount of TPO was increased 2-fold by TSH. High concentrations of iodide (1-50 microM, added daily) decreased the level of TPO. Confocal microscopy served to determine the intracellular localization of TPO and its quantitative distribution. Intracellular and surface-located TPO was detected by fluorescein-labeled antibodies on saponin-treated cells. Quantitative confocal microscopy showed that TSH increased the total amount of TPO 2-fold as for immunoblotting. The highest amount of TPO was found in the perinuclear area and between the nucleus and the Golgi apparatus. Only 4% of TPO was present on the apical surface and about 1% on the basolateral membrane; the remainder (about 95%) was inside the cells. TSH did not change these relative contents. TSH modified the intracellular distribution of the enzyme, increasing the TPO pool from the perinuclear area to apical membrane. This domain could be a site of storage of TPO. Adding a physiological concentration of iodide (0.5 microM, daily) did not influence the intracellular distribution of TPO. We concluded that chronic TSH stimulation 1) increased 2-fold the pool of TPO but did not change the relative proportion of TPO inside the cells and on the apical surface, and 2) modified the intracellular distribution of vesicular TPO, the major part of which was accumulated in the perinuclear and cytoplasmic area under the subapical domain of the polarized cells.
Asunto(s)
Células Epiteliales/enzimología , Yoduro Peroxidasa/metabolismo , Glándula Tiroides/enzimología , Tirotropina/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Células Cultivadas , Microscopía Confocal , PorcinosRESUMEN
We previously showed that thyroglobulin (Tg) glycosylation is enhanced 1.5-fold under thyrotropin (TSH) stimulation, corresponding to an increased number of oligosaccharide chains per molecule of Tg. Now the steps involving dolichol components and oligosaccharyltransferase activity have been studied. Porcine thyroid cells were cultured on porous bottom filters with or without TSH and incubated with [14C]mevalonate. Under TSH regulation, the level of the whole of dolichol components was increased 1.25-fold without modifying their distribution. Dolichol, and free and monosaccharide-linked dolichyl-phosphate, represented respectively 40% and 45% of total dolichol components while dolichyl-pyrophosphate-oligosaccharide represented 3% only. A marked enhancement (4.2-fold) of oligosaccharyltransferase activity occurred in stimulated cells, which could correspond to the addition of the two TSH effects: stimulation of Tg synthesis (3-fold) and of Tg glycosylation (1.5-fold). The amount of lipid carriers appeared to be insufficiently increased but no component is a limiting step, suggesting that the turnover of dolichol derivatives may be increased under TSH control through their use by higher amounts of Tg.
Asunto(s)
Dolicoles/metabolismo , Hexosiltransferasas , Proteínas de la Membrana , Glándula Tiroides/metabolismo , Tirotropina/farmacología , Transferasas/metabolismo , Animales , Células Cultivadas , Glicosilación , Ácido Mevalónico/metabolismo , Porcinos , Tiroglobulina/metabolismo , Glándula Tiroides/efectos de los fármacosRESUMEN
Porcine thyroid cells were cultured for 15 days on porous bottom chambers with or without different mixtures of hormones added to serum-free basal medium. Assays with 10% serum were also performed for comparison with previously published results. The effects of the hormones, particularly insulin, TSH and hydrocortisone, were studied on total RNA content, thyroglobulin mRNA level, the amount of thyroglobulin secreted into the apical medium and on glycosylation. Insulin and TSH similarly increased the total RNA content, and their effects were additive. Thyroglobulin mRNA content was increased twofold by insulin and threefold by TSH. When they were added simultaneously, the maximal level of thyroglobulin mRNA was reached, showing that TSH and insulin effects on thyroglobulin gene expression were additive. Hydrocortisone alone did not modify total RNA or thyroglobulin mRNA content but the hormone amplified total RNA when insulin and TSH were present together. The basal level of thyroglobulin secreted into the apical medium was increased threefold by insulin and fourfold by TSH. The effects of these two hormones added together appeared to be additive. Hydrocortisone had no effect alone or even when combined with insulin or TSH. However, when the three hormones were added together, the hormonal response was amplified. TSH effect and insulin effect on the incorporation of 3H-mannose into thyroglobulin as well as on the anionic residue content of the molecule were additive.
Asunto(s)
Hidrocortisona/fisiología , Insulina/fisiología , Tiroglobulina/biosíntesis , Glándula Tiroides/metabolismo , Tirotropina/fisiología , Animales , Células Cultivadas , Medio de Cultivo Libre de Suero , Glicosilación , Focalización Isoeléctrica , Manosa/metabolismo , ARN Mensajero/metabolismo , Porcinos , Tiroglobulina/genética , Tiroglobulina/metabolismo , Glándula Tiroides/ultraestructuraRESUMEN
Porcine thyroid cells were cultured in porous bottom chambers in the presence or in the absence of TSH added to the basal medium. Radiolabeled-sugar (3H-mannose) was added to the basal medium on day 11 for 4 days and the glycosylation of thyroglobulin (Tg), the major glycoprotein secreted into the apical medium, was studied. The incorporation of 3H-mannose per molecule of Tg was increased 1.5-fold by a 50 microU/ml minimal concentration of TSH. The distribution of glycopeptides (after pronase digestion) on concanavalin A sepharose column was not modified by the presence of TSH. However this distribution was different from that observed for Tg extracted from gland (more multiantennary units than biantennary units and polymannose units). After desialylation and desulfation, the sizes of the oligosaccharide chains analyzed on HPLC appeared similar when cells were cultured under stimulation or not. Thus TSH enhanced sugar incorporation without modifying either the distribution of the different oligosaccharide moieties or their sizes. Consequently the effect of TSH was a 1.5-fold increase in oligosaccharide chains linked to asparagine residues. 3H-Mannose-oligosaccharide chains were then analyzed on ion-exchange HPLC before and after desialylation and desulfation. The number of anionic residues per oligosaccharide unit (particularly sulfate residues) was higher in the absence of TSH than in the presence of TSH. Nevertheless, since TSH increased the number of carbohydrate units per molecule of Tg 1.5-fold, the total content of anionic residues bound to oligosaccharide units per molecule of Tg seems not to be modified by TSH.
Asunto(s)
Oligosacáridos/metabolismo , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Animales , Aniones/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Cromatografía Líquida de Alta Presión , Cámaras de Difusión de Cultivos , Glicosilación , Manosa/metabolismo , Porcinos , Tiroglobulina/química , Glándula Tiroides/citología , Tirotropina/farmacologíaRESUMEN
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).
Asunto(s)
Glándula Tiroides/enzimología , Activador de Tejido Plasminógeno/metabolismo , Animales , Células Cultivadas , Medios de Cultivo , Ensayo de Inmunoadsorción Enzimática , Células Epiteliales , Epitelio/efectos de los fármacos , Epitelio/enzimología , Cinética , Porcinos , Glándula Tiroides/citología , Glándula Tiroides/efectos de los fármacos , Tirotropina/farmacología , Activador de Tejido Plasminógeno/análisisRESUMEN
The long-term iodination of thyroglobulin secreted into the apical medium of thyroid cells cultured as monolayers on porous bottom chambers reached 5.87 +/- 1.66 atoms of iodine/mol thyroglobulin after 11 days incubation in the presence of TSH (0.1 mU/ml) and iodide (0.5 microM) in the basal medium. This iodinated thyroglobulin contained thyroid hormones (T3 + T4) which involved 22.7% of the thyroglobulin iodine content. The iodoamino acid content was, in residues per mole, 2.2 +/- 0.35 for monoiodotyrosine, 0.74 +/- 0.04 for diiodotyrosine, 0.23 +/- 0.04 for T4, and 0.098 +/- 0.02 for T3. Kinetic studies showed that a minimal level of iodination (2.05 +/- 0.26 atoms iodine/mol thyroglobulin) was necessary for hormonogenesis. A maximal level of iodination and hormonogenesis was obtained with 0.5 microM iodide added daily to the basal medium. In these conditions, hormonogenesis efficiency reached about 40% (a value close to this one observed in vivo). Above 0.5 microM iodide, both iodination and T4 synthesis were inhibited (28.3% and 73.9%, respectively, for 1 microM iodide). Our culture system makes it possible to demonstrate that this high iodide concentration in the basal medium did not increase apical iodide concentration above 10 microM but decreased apical thyroglobulin concentration. The inhibitory effect of iodide on hormonogenesis cannot be due to a competition with tyrosine residues of thyroglobulin for their binding to thyroperoxidase although it could be related, at least in part, to a decrease in protein synthesis.
Asunto(s)
Yoduros/farmacología , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Tiroxina/biosíntesis , Triyodotironina/biosíntesis , Animales , Células Cultivadas , Yodo/metabolismo , Cinética , PorcinosRESUMEN
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.
Asunto(s)
Glándula Tiroides/metabolismo , Hormonas Tiroideas/biosíntesis , Tirotropina/farmacología , Animales , Células Cultivadas , Medios de Cultivo , Cámaras de Difusión de Cultivos , Regulación de la Expresión Génica/fisiología , Yoduros/farmacología , Radioisótopos de Yodo , Porosidad , Propiedades de Superficie , Porcinos , Tiroglobulina/genética , Tiroglobulina/metabolismo , Glándula Tiroides/citologíaRESUMEN
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.
Asunto(s)
Polaridad Celular/fisiología , Yodo/metabolismo , Fenolsulfonftaleína/farmacología , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Animales , Células Cultivadas , Células Epiteliales , Epitelio/metabolismo , Yoduros/análisis , Peroxidasa/metabolismo , Porcinos , Glándula Tiroides/citologíaRESUMEN
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.
Asunto(s)
Radioisótopos de Yodo/metabolismo , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Tirotropina/fisiología , Animales , Autorradiografía , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Radioisótopos de Yodo/farmacología , Cinética , Porcinos , Glándula Tiroides/citologíaRESUMEN
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.
Asunto(s)
Exocitosis/efectos de los fármacos , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Tirotropina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Células Cultivadas , Toxina del Cólera/farmacología , Medios de Cultivo , Relación Dosis-Respuesta a Droga , Cinética , Porcinos , Tiroglobulina/genética , Glándula Tiroides/citologíaRESUMEN
The effects of thyrotrophin, cholera toxin and 8-chloro-cyclic AMP on thyroglobulin gene expression in cultured porcine thyroid cells were compared. Cells organized either into monolayers in culture chambers with porous bases or into inside-out follicles in suspension were used. Thyroglobulin mRNA content was measured by dot-blot hybridization, and thyroglobulin synthesis rate was determined by immunoprecipitation of [35S]thyroglobulin after 2 h labelling with [35S]methionine. Cholera toxin and 8-chloro-cyclic AMP increased the thyroglobulin mRNA content and thyroglobulin synthesis rate in the same ratio (approximately 3) as did thyrotrophin, showing that cyclic AMP mediates the effect of thyrotrophin on thyroglobulin gene expression. The culture chamber system provides for contact of the effectors with either the apical or the basolateral membrane. The addition of 0.02-0.1 mU thyrotrophin/ml on the basolateral side of the cell layer gave a maximal response whereas this response was not reached on the apical side even with the addition of 5 mU/ml. In contrast, cholera toxin and 8-chloro-cyclic AMP stimulated thyroid cells equally on both sides.
Asunto(s)
AMP Cíclico/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Tiroglobulina/genética , Glándula Tiroides/citología , Tirotropina/farmacología , 8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Células Cultivadas , Toxina del Cólera/farmacología , ARN Mensajero/análisis , Porcinos , Tiroglobulina/biosíntesis , Glándula Tiroides/efectos de los fármacos , Glándula Tiroides/ultraestructuraRESUMEN
In the thyroid glands, thyroglobulin (Tg) is specifically synthesized by follicular cells and then secreted into the apical lumen where it is concentrated and used as a substrate for thyroid hormone synthesis. The presence of Tg in the circulation has been reported in normal and pathological situations. To determine the domains of the plasma membrane, apical and/or basolateral, involved in Tg secretion, porcine thyroid epithelial cells were cultured as a monolayer on the porous bottom of a culture chamber in which both apical and basal media are independently accessible. Control experiments using labeled Tg ascertained the tightness of the monolayer and showed that within 48 h only 0.2-0.5% of the Tg introduced in the apical medium was transferred through the cell layer into the basal compartment. For kinetic studies of Tg synthesis and secretion, monolayers were cultured for up to 72 h in the presence of 35S-methionine and with or without 100 microU/ml thyrotropin (TSH) in the basal medium. Labeled Tg was measured by double immunoprecipitation and by fluorography of polyacrylamide gel electrophoresis. We showed that 80-95% of total secreted Tg was recovered in the apical medium. The remainder was secreted through the basolateral membranes in the basal medium. The amount of tg secreted into the apical compartment was stimulated two- to threefold by TSH whereas no TSH effect was observed on secretion in the basal compartment. Moreover, measuring apical and basal volumes, we observed a net water flow from the apical to the basal side. It was stimulated threefold by TSH, increasing the Tg concentration in the apical compartment of the stimulated cell layer. During the culture time, the amount of Tg synthesized and secreted was increased by TSH, as was the Tg mRNA content, as determined by the dot-blot hybridization method.
Asunto(s)
Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Tirotropina/farmacología , Animales , Membrana Basal/metabolismo , Proteínas Sanguíneas/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Metionina/metabolismo , Microscopía de Contraste de Fase , Procesamiento Proteico-Postraduccional , ARN Mensajero/análisis , Porcinos , Tiroglobulina/genética , Glándula Tiroides/efectos de los fármacosRESUMEN
In primary culture porcine cells form polarized cell layers. We have designed culture conditions in which we can have access to only one side of the cell layer, either the apical or the basal surface. In addition, using culture chambers with permeable bottom we can have access to either side of the cell layer which separates two compartments. Using these organized systems we have shown that the iodide concentrating mechanism and the TSH-receptor adenyl cyclase complex are localized on the basolateral domain of the thyroid cell plasma membrane. We also demonstrated the existence on the apical surface of an amiloride sensitive sodium uptake. Finally we observed that about 10% of newly synthesized thyroglobulin appears to be secreted directly into the basal compartment, 90% being secreted in the apical compartment.
Asunto(s)
Glándula Tiroides/citología , Adenilil Ciclasas/metabolismo , Animales , Células Cultivadas , Receptores de Tirotropina/metabolismo , Porcinos , Tiroglobulina/metabolismo , Glándula Tiroides/metabolismo , Glándula Tiroides/fisiología , Hormonas Tiroideas/metabolismoRESUMEN
Isolated porcine thyroid cells reorganize in culture into various types of multicellular structure, which differ in the orientation of cell polarity and in the surface of the cell layer accessible to molecules present in the culture medium. The types of structure are: (1) follicles: the basal pole is oriented toward the medium; (2) inside-out follicles or monolayers: the apical pole is facing the culture medium; (3) monolayers on a permeable substratum: both sides of the cell layer are accessible to the medium. Follicles can be transformed into inside-out follicles or monolayers and vice versa by manipulation of the external cell environment and without dissociating the cells. Cells concentrate iodide and respond to acute stimulation by thyroid-stimulating hormone (TSH) when the basal pole is accessible, and organification occurs only when cells form a closed follicular lumen. In porous-bottomed culture chambers monolayers are formed with the basal surface accessible to the medium and the apical compartment separated from the medium. Under these conditions 85-95% of the thyroglobulin produced is secreted apically and 5-15% basally. Thyrotropin stimulates (X3) apical accumulation without modifying secretion in the basal compartment. Sodium transport across the cell layer has been characterized. An amiloride-sensitive influx occurs at the apical pole whereas the Na+/K+-ATPase, localized in the basolateral membrane, mediates ouabain-sensitive efflux at the basal pole. The thyroid epithelium in culture appears therefore as a Na+-absorbing epithelium. The role of this transport in the stabilization of cell polarity is discussed.
Asunto(s)
Glándula Tiroides/citología , Animales , Células Cultivadas , Células Epiteliales , Epitelio/fisiología , Glándula Tiroides/fisiologíaRESUMEN
We report on the 466 nucleotides long, extreme 3' end of the ovine thyroglobulin (Tg) mRNA. The nucleotide sequence and the deduced carboxyl terminal region of the protein have been compared with those reported in other species. Comparison with hog peptides known to contain all the triiodothyronine (T3) of the mature Tg suggests that the antepenultimate amino acid, a tyrosine residue could be involved in hormone formation. This also agree with the data reported for bovine and murine Tg.
Asunto(s)
ARN Mensajero/ultraestructura , Tiroglobulina/análisis , Animales , Secuencia de Bases , Bovinos , ADN/ultraestructura , Datos de Secuencia Molecular , Terminación de la Cadena Péptídica Traduccional , Ratas , Ovinos , Especificidad de la Especie , Porcinos , Tiroglobulina/biosíntesis , Triyodotironina/biosíntesisRESUMEN
Triiodothyronine (T3) effects on the activity, rate of synthesis and mRNA content of the key lipogenic enzyme, fatty acid synthetase, were studied in differentiating ob17 preadipocytes cloned from ob/ob mouse epididymal adipose tissue. During differentiation in the presence of insulin, a 6-10-fold increase in both fatty acid synthetase specific activity and synthesis rate were reproducibly observed and occurred concomitantly. The relative synthesis rate exhibited a progressive elevation from 0.5% at confluence to a maximum level of 2% in the presence of insulin. The rate of the enzyme degradation determined by pulse-chase experiments was similar in differentiating cells and insulin-untreated cells of the same age (t 1/2, 40-42 h). Furthermore, the increase in the enzyme synthesis rate was preceded by a progressively elevating amount of mRNA encoding for this protein as detected by translation in a reticulocyte lysate cell-free system. It is thus suggested that the increment in total and neosynthesized fatty acid synthetase in essentially due to an increased enzyme synthesis, reflecting an increased relative content of its specific mRNA. T3 included at a physiological concentration (1.5 nM) in the culture medium enhanced significantly both enzyme synthesis and its specific mRNA. The most important T3 effect was an acceleration of both processes, a stimulation of the mRNA level being detected as early as day 3 post-confluence and maximum at day 5 when the effect on the synthetase synthesis rate and activity began to be enhanced. This suggests that T3 would mainly affect fatty acid synthetase as a pretranslational level.