In contact with a collagen gel, thyroid follicular cells form a basal pole. A monolayer, with the apical side facing the culture medium is formed when cells are seeded on a collagen layer. In contrast cells reorganize into follicle-like structures when they are embedded in the collagen gel. If vesicles which have their apical pole in contact with the culture medium are embedded in the gel, collagen-cell interaction induced the inversion of cell polarity giving follicular structures. In addition after being overlaid with a collagen layer, cells forming a monolayer reorgnaize into follicles. The apical pole is formed on the free surface of the monolayer or on the internal side of structures embedded in collagen which differentiate into follicles.
Confluent monolayers of cultured porcine thyroid cells transport fluid from the apical to the basal surface, forming circumscribed zones of detachment from the culture dish substrate (domes). The transepithelial potential (TEP), positive on the basal side, was 12.9 +/- 0.4 (S.E.M.) mV (n = 93) under control conditions, increasing to 38.9 +/- 0.3 mV (n = 281) when fluid transport was stimulated by prostaglandin E2 (PGE2; 1 mumol/l). Forskolin (1 mumol/l) and 8-(4-chlorophenylthio) adenosine 3',5'-cyclic monophosphate (0.5 mmol/l) were also effective in increasing TEP. Addition of amiloride in concentrations sufficient to block fluid transport (100 mumol/l) reduced the TEP to 5.8 +/- 0.3 mV (n = 76). Substitution of N-methyl-D-glucamine for sodium in the medium reduced the PGE2-stimulated TEP to 13.4 +/- 0.8 mV (n = 32). Substitution of gluconate for chloride increased the TEP to 40.3 +/- 0.4 mV (n = 160). Removal of bicarbonate or potassium from the medium, or addition of ouabain (200 mumol/l) were also effective in reducing the TEP. In media of low bicarbonate concentration (1 mmol NaHCO3/l), acetazolamide (1 mmol/l) reduced the TEP. Fluid transport by the monolayer as measured by the change in height of domes was increased by PGE2 (1 mumol/l). PGE2-stimulated fluid transport was inhibited by sodium or chloride ion substitution, bicarbonate removal or the addition of ouabain (200 mumol/l) or amiloride (100 mumol/l). It was concluded that fluid transport in thyroid monolayers is mediated by rheogenic sodium transport with chloride transport being passive, electrogenically coupled to sodium transport.(ABSTRACT TRUNCATED AT 250 WORDS)
Pseudopods at the apical pole of porcine thyroid monolayers in culture were considered as reflecting individual thyrocyte responses to thyrotropin (TSH) stimulation. Scanning electron microscopy and computer-assisted image analysis showed that whatever TSH stimulation was used, the pseudopods were characterized by two populations: P1 with small diameters (2 microns) and P2 with greater diameters (5 microns). The density of P1 rapidly increased to reach a plateau, while P2 continuously increased during stimulation. Two-dimensional pseudopod patterns were compared with random point distributions by means of two topographical parameters: the interpseudopod distances and angles. A factorial analysis of experimental distribution of pseudopods obtained after increasing stimulation times displayed a shift from a nonrandom (10-20 min) to a random (60-90 min) distribution. Clusters of three pseudopods characterized by short distances (6-9 microns) and equilateral organization (angles 40-60 degrees) were observed after a 10-min stimulation. These results suggested that early thyrocyte response to TSH stimulation is characterized by interrelations between three adjoining cells, with the thyrocyte response later appearing as random.
When cultured in high glucose containing medium, the human colon carcinoma cell line HT29‐D4 and a clone derived by transfection with the MDR1 cDNA (MDR31) form multilayers of unorganized cells which are not polarized and are linked by desmosomes. Within these multilayers appear spontaneously large multicellular follicle‐like‐structures (FLS) where polarized cells linked by tight junctional complexes surround a lumen. Electron microscopy showed that some FLS display well developed brush borders with densely packed microvilli. Others have irregularly oriented microvilli of various lengths or are even completely devoid of apical differentiation. The lumen contains a variable amount of amorphous osmiophilic material. The apical surface of FLS forming cells express dipeptidylpeptidase IV, carcinoembryonic antigen, the mucin MUC1 and for the transfected cells the gp‐170 protein. The organic anion fluorescein is transported from the cell to the lumen of FLS. Rhodamine 123, a substrate of the gp‐170 ABC transporter is also concentrated in the lumen formed by MDR31 cells. Verapamil and cyclosporine A inhibited this last transport. Cyclic AMP stimulates the formation of these structures since treatment of post‐confluent multilayers dramatically increased the number of FLS in HT29‐D4 and MDR31 cell cultures within 24 h. The spontaneous formation of these morphologically and functionally polarized structures appeared at random and might respond to the coincidence of fluctuating parameters of the regulatory pathways (cAMP, Ca 2+ ).
Thyroid cell cyclic AMP synthesis is stimulated by beta-adrenergic agonists. We have characterized this sensitivity on cultured porcine thyroid cells and have studied its modulation by chronic treatment with thyrotrophin. The synthesis of cyclic AMP in intact porcine thyroid cells in primary culture was stimulated by the beta-adrenergic agonist, isoproterenol. This stimulation was dose-dependent and was inhibited by the beta-adrenergic antagonists propranolol and alprenolol. The cell responsiveness (i.e. the response elicited by 5 microM-isoproterenol after 5-min stimulation) was increased when the cells were cultured in the absence of thyrotrophin. Thyrotrophin, when present in the culture medium at the onset of culturing, inhibited this increase. A concentration of 100 microunits. thyrotrophin/ml was sufficient to reduce the cyclic AMP response to 15% of its control value. Prostaglandin E2 or dibutyryl cyclic AMP did not mimic the effect of thyrotrophin. The low sensitivity of thyrotrophin-treated cells to beta-adrenergic agonists could be explained by a decreased number of beta-adrenergic receptors. [125I]Iodohydroxybenzyl pindolol specific binding was ten times greater in membrane preparations of control cells than in membranes derived from thyrotrophin-treated cells. The beta-adrenergic sensitivity of cultured thyroid cells was also decreased after long-term treatment by terbutaline. A time- and dose-dependent desensitization was observed.
The influence of collagen gels on the orientation of the polarity of epithelial thyroid cells in culture was studied under four different conditions. (a) Isolated cells cultured on the surface of a collagen gel formed a monolayer. The apical pole was in contact with the culture medium and the basal membrane was attached to the substratum. (b) Isolated cells embedded inside the gel organized within 8 into follicles. The basal pole was in contact with collagen and the apical pole was oriented towards the interior of the follicular lumen. (c) Cells were first organized into floating vesicles, structures in which the apical surface is in contact with the culture medium, and the vesicles were embedded inside the collagen gel. After 3 d, cell polarity was inverted, the apical pole being oriented towards the cavity encompassed by cells. Vesicles had been transformed into follicles. (d) Monolayers formed on collagen gels as in a were overlaid with a second layer of collagen, which was polymerized in contact with the apical cell surface. A disorganization of the continuous pavement occurred within 24 h; cells attached to the upper layer of collagen and reorganized into follicles in the collagen sandwich within 4-8 d. A similar process occurred when the monolayer was grown on plastic and overlaid with collagen, or grown on collagen and covered with small pieces of glass cover slips. No reorganization was observed between two glass surfaces. In conclusion, first, a basal pole was always formed in the area of contact between the cell membrane and an adhesive surface and, second, the interaction of a preformed apical pole with an adhesive surface was not compatible with the stability of this domain of the plasma membrane. The interaction of the cell membrane with extracellular components having adhesive properties appears to be a determinant factor in the orientation and stabilization of epithelial cell polarity.
