Follicle-like structures formed by intestinal cell lines derived from the HT29-D4 adenocarcinoma cell line: morphological and functional characterization.

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, Ca2+).

Follicle-like structure and polarized monolayer: role of the extracellular matrix on thyroid cell organization in primary culture.

The thyroid follicle, the morphofunctional unit of thyroid gland, is a spheroidal structure formed by a monolayer of polarized cells surrounding a closed cavity in which thyroglobulin accumulates. Newly isolated porcine thyroid cells reorganize into two types of structures which differ by the orientation of cell polarity: in follicle-like structures, obtained in the presence of TSH, the apical pole delineates a closed cavity and cells express most parameters characteristic of thyroid function; in inside-out follicles the apical pole is oriented towards the culture medium and cells do not express properly the thyroid function. The organization of newly formed follicles can be modified by stimulation of cell migration or by interaction of their apical poles with a new cell environment. Seeded on a hard surface (glass, plastic), cells of follicle-like structures or inside-out follicles formed in suspension migrate giving a monolayer. On the contrary, cells organized into a monolayer treated with hexamethylene bisacetamide, reorganize into follicle-like structures. Inside-out structures reorganize upon interaction of their apical poles with collagen I gel, a coherent matrix, or with a reconstituted basement membrane (RBM), a soft matrix. Overlaid with collagen I, monolayers reorganize into follicles. Embedded in collagen I or in RBM, inside-out follicles reorient their polarity giving functional follicles. On the RBM surface, cells pull on the gel and embed themselves in the soft matrix gel, finally reorienting their polarity to inside-in polarity. When comparing thyroid cells with other epithelial cell types (mammary cells, Sertoli cells), it appears that the obtention in culture of follicle-like structures, i.e. closed inside-in polarized cell organization, is the best way to express in culture both morphology and function of any specific epithelial tissue, the polarized monolayer in porous bottom culture chamber coming just behind.

Characterization of an electrogenic sodium/glucose cotransporter in a human colon epithelial cell line.

In this study, we have characterized the Na/glucose transporter in polarized monolayers formed by the clonal human colon carcinoma cell line HT-29-D4. Isotopic tracer flux measurements show that differentiated HT-29-D4 cells possess a sodium-dependent alpha-methyl-D-glucopyranoside (AMG) uptake that is competed for by increasing concentrations of D-glucose, D-galactose, and phlorizin. This transport is exclusively localized on the apical side of the epithelium. Kinetic data demonstrate the existence of a single Michaelian sodium-dependent AMG transporter with a Km of 1.2 +/- 0.12 mM and a Vmax of 3.24 +/- 0.25 nmol/mg of protein per min. Hill analysis reveals a coefficient of 1.9 +/- 0.03, consistent with at least two sodium ions involved in AMG transport. Interestingly, the cotransporter function is not modulated by glucose in the culture medium. Transepithelial electrical parameter measurements show that the transepithelial potential difference (Vt) is glucose dependent and phlorizin sensitive. Antibodies directed against a peptide of the rabbit intestinal glucose cotransporter (Ser402-Lys420) recognize, in western blot experiments, the characteristic bands of the cotransporter on a crude membrane preparation of differentiated HT-29-D4 cells and react strongly with the apical domain of the monolayer in immunofluorescence experiments. We conclude that HT-29-D4 cells express the sodium/glucose cotransporter SGLT1 at their apical membrane and that this transporter generates the basal transepithelial potential difference.

Thyrotropin regulation of basolateral Cl- and I- effluxes in thyroid follicles in culture.

This report describes chloride and iodide effluxes across the basolateral membrane of porcine thyroid follicles reconstituted in culture. Basolateral chloride efflux is activated by thyrotropin (TSH). TSH (10 mU/ml) induces a twofold increase in the initial rate of chloride efflux. Forskolin (FSK, 5 microM) which increases intracellular cAMP also stimulates the initial rate of chloride efflux 3.5-fold, whereas an increase in the free cytosolic Ca2+ with the ionophore A23187 or thapsigargin, fails to mimic the TSH effect. The chloride channel blocker 5-nitro-2(3-phenylpropylamino)benzoic acid (NPPB) dose dependently inhibits chloride efflux rates with the maximal and half maximal effects observed for 100 microM and 30 microM, respectively. Basolateral chloride efflux rates are also inhibited in the presence of the organic anion transporter blocker probenecid (5 mM) or the Cl-/HCO3- exchanger blocker 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS, 250 microM), respectively, by 60% and 40%, whereas it is not affected by ClO4 (100 microM). The initial rate of iodide efflux is weakly activated (1.4-fold) by TSH (10 mU/ml). TSH effect could be reproduced by agents known to activate Ca(2+)-dependent processes as A23187, ionomycin (1 microM), phorbol 12-myristate 13-acetate (TPA, 0.1 microM) and epidermal growth factor (EGF, 0.1 microM) which increase the initial rate of iodide efflux from 1.2- to 1.8-fold, whereas FSK is without effect. The chloride channel blocker NPPB (500 microM) is required to significantly inhibit the initial rate of iodide efflux by 30%. The initial rate of iodide efflux is also reduced by 30% in the presence of SITS (250 microM) or probenecid (5 mM) whereas it is activated by ClO4 (100 microM). We conclude that basolateral chloride and iodide effluxes are both regulated by TSH, using two different transduction pathways. Chloride efflux regulation may involve a cAMP transduction signal, whereas the regulation of iodide efflux may involve a Ca2+ signal. Furthermore, as the sensitivities of chloride and iodide effluxes for the anion transporter blockers (especially NPPB) are different, it seems likely that chloride and iodide use two different transport pathways.

Spatial and temporal thyrocyte response to TSH: a computer-assisted image analysis.

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.

Polarity reversal of inside-out thyroid follicles cultured on the surface of a reconstituted basement membrane matrix.

When cultured in suspension, epithelial thyroid cells organized into inside-out follicles. We studied the behavior of these structures after seeding on polystyrene, type I collagen, and reconstituted basement membrane (RBM) gel. When seeded on plastic, type I collagen or mixed type I collagen-RBM gel, inside-out follicles attached and spread, forming polarized cell monolayers. In contrast, on thick RBM gel, inside-out follicles attached penetrated into the gel, and reorganized into properly oriented follicular structures. Polarity of the cell layer was progressively inverted while, after adhesion, cells penetrated the soft RBM gel. In the process of reorientation, cells with hybrid polarity were observed. The fraction of the apical pole which was not yet in the gel showed an inside-out orientation, while a modified orientation was observed in contact with the RBM gel. Cells which had penetrated completely in the matrix formed a new apical pole and displayed an opposite orientation of their polarity. A continuous basement membrane was observed, lining the basal cell surface when native RBM gel was present in the substratum.

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.

Evidence for probenecid-sensitive organic anion transporters on polarized thyroid cells in culture.

Epithelial thyroid cells in primary cultures loaded with BCECF/AM rapidly released the impermeant fluorescent dye BCECF (bis(carboxyethyl)carboxyfluorescein) in the incubation medium. Cells organized into follicles rapidly cleared BCECF (80% within 10 min) whereas fluorescence microscopy did not show any fluorescence in the follicular cavity. Cells organized into monolayers on plastic exported BCECF into the medium (70% within 40 min) whereas fluorescence microscopy showed intense fluorescence under the domes. BCECF efflux was blocked by probenecid, one of the known inhibitors of organic anion transporters, with similar efficiency in both structures. Maximal and half-maximal effects were respectively observed for 5 mM and 0.4 mM probenecid. The polarity of BCECF efflux was studied by using monolayers on collagen-coated Nuclepore filters: 85% of BCECF released was found in the basal compartment and 15% in the apical compartment. These findings suggested that thyroid cells in culture expressed a transport mechanism for the anionic form of BCECF. Furthermore, the observed activation of the Na+/H+ exchanger by probenecid suggested that the presence of this blocker did not overcome problems arising in the use of BCECF as intracellular pH indicator for thyroid cells.

Absence of fodrin (spectrin-like protein) under the pseudopod membrane in stimulated thyroid cells.

A fodrin-like protein purified from porcine thyroid cells and characterized by its properties identical to those of pig brain spectrin (F. Regnouf et al., Eur. J. Biochem. 153, 313-319 (1985)) has been localized by immunofluorescence and electron immunocytochemistry in porcine and rat thyroid. Fodrin-like polypeptides were detected in subplasmalemmal meshworks of microfilaments attached to isolated or in situ plasma membranes. In resting cells, fodrin was found under apical and basolateral membrane domains, whereas it was always absent under the pseudopod membrane domain induced by acute TSH stimulation in vitro, using monolayers of porcine cultured cells attached to collagen permeable substrates, as well as in vivo, using rats intravenously treated with TSH. Thyroid fodrin could be involved in exocytosis and membrane stabilization which occurs during the formation of pseudopods induced by TSH stimulation.

Suramin-induced differentiation of the human colic adenocarcinoma cell clone HT29-D4 in serum-free medium.

The clonal cell line HT29-D4 was able to grow in a completely defined medium containing EGF, selenous acid, and transferrin in the presence of the anti-helminthic drug suramin. In the absence of suramin, the kinetics of cell growth and the cell density obtained were dependent on the external EGF concentration. In the presence of suramin, cell density reached a plateau independent of EGF concentration above 50 ng/ml. At the morphological level, suramin allowed hemicyst formation in the cell monolayer. The cells were polarized with a well-ordered brush border facing the culture medium and mature junctional complexes that divided the cell membrane in two distinct domains. The carcinoembryonic antigen was found to be restricted to the apical membrane domain while the major histocompatibility molecules HLA-ABC were segregated within the basolateral domain. The electrical parameters of suramin-treated cells grown on permeable filters were measured and demonstrated that the cell monolayer was electrically active. These properties were never found in the absence of the drug. Moreover, the vasoactive intestinal polypeptide (VIP) was able to induce a dramatic increase in cAMP only when it was added, in agreement with the localization of the VIP receptor, in the lower compartment of the culture chamber. In conclusion we described for the first time conditions allowing the growth of functionally differentiated human colic cell monolayers in chemically defined medium. This model will contribute to a better understanding of suramin action and of the mechanisms involved in cell polarization.

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.

Suramin-treated HT29-D4 cells grown in the presence of glucose in permeable culture chambers form electrically active epithelial monolayers. A comparative study with HT29-D4 cells grown in the absence of glucose.

The clonal cell line HT29-D4 is able to differentiate by two different ways: i) by replacing glucose by galactose in the culture medium; ii) by addition of suramin (a drug known to interfere with the growth promoting activity of growth factors) in the medium. In both cases the transition in the organization of the cell monolayer occurred without cell loss. The two ways (i.e., glucose starvation or suramin addition) lead to polarized cells which generate electrically active cell monolayers (Fantini et al., Biol. Cell 65, 163-169 (1989) and this paper). Yet several important differences can be observed at the morphological or at the electrophysiological levels. 1) The suramin-treated cells (HT29-D4-S cells) organized into monolayers of high (40-50 microns) columnar cells while glucose-starved cells (HT29-D4-Gal cells) were rather cuboidal (20-25 microns). 2) HT29-D4-S cells were highly polarized; the nucleus was rejected at the basal side of the cell and lysosomes in the upper part of the cytoplasm. Numerous lipid-like droplets surrounded with glycogen were observed underneath the nucleus. HT29-D4-Gal cells never presented such a degree of organization. 3) The transepithelial resistance and the potential difference of HT29-D4-S monolayers reached values significantly higher than those for HT29-D4-Gal monolayers, reflecting a higher degree of organization. Specific proteins such as sucrase-isomaltase, alkaline phosphatase and carcinoembryonic antigen were localized exclusively on the apical membrane while human lymphocyte antigen (HLA) class I molecules were restricted to the basolateral membrane for both HT29-D4-S and HT29-D4-Gal cells. The present data demonstrate that the same cells can generate a different degree of cellular organization according to the experimental conditions of cell growth, the most elaborate state of differentiation being obtained in the presence of suramin.

Small conductance chloride channels in the apical membrane of thyroid cells.

A small conductance chloride channel has been identified on the apical membrane of porcine thyroid cells using the patch-clamp technique. In cell attached membrane patches with NaCl in the pipette, the single channel conductance is 5.5 pS. The channel is highly selective for chloride over gluconate and iodide, and is impermeable to Na+, K+ and tetraethylammonium ions. The open state probability of the channel is not affected by voltage. The channel activity disappears after excision of the patch. The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) did not affect the activity of the thyroid Cl- channels. Treatment of thyroid cells with 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (8-chloro-cAMP) (0.5 mM) prior to giga-seal formation increased Cl- channel activity in the apical membrane of thyroid cells.

The thyroid cell monolayer in culture. A tight sodium absorbing epithelium.

When cultured on collagen coated nitrocellulose filters, thyroid epithelial cells form morphologically and functionally polarized monolayers. The bioelectric parameters of these monolayers were measured after mounting in Ussing chambers; transepithelial potential (Vab), short circuit current (Isc) and transepithelial resistance were respectively 12 +/- 1 mV (apical side negative), 3.8 +/- 0.2 microA cm-2 and 3250 +/- 214 omega cm2 (mean +/- SEM, n = 75). Eighty two percent of the short circuit current was related to sodium absorption as shown by inhibition by apical amiloride (Km = 0.2 microM) and by basal ouabain (K1/2 = 0.3 microM). Amphotericin B (5-25 micrograms/ml) added to the apical bath increased Isc suggesting an apical rate-limiting step. Step by step replacement of choline by Na+ in a Na+-free medium resulted in a progressive increase in Vab and Isc with half maximal effect at 20 +/- 1 mM Na+. Thyrotropin (TSH) increased Isc and Vab in a biphasic way with a transient maximum after 5 min and a plateau after 20 min (about four times the basal level at 100 microU/ml TSH). This increase in sodium transport was also inhibited by apical amiloride. Thus, in culture, the thyroid cell monolayer behaves as a tight sodium absorbing epithelium controlled by TSH, with a rate limiting apical sodium channel as the entry mechanism and a basolateral Na+, K+-ATPase as the electromotive force.

Identification and properties of a novel type of Na+-permeable amiloride-sensitive channel in thyroid cells.

Amiloride-sensitive cationic channels are present in the apical membrane of porcine thyroid cells in primary culture. An amiloride-sensitive (K0.5 = 150 +/- 28 nM where K0.5 is the concentration of unlabelled ligand which reduces the specific binding of the same labelled ligand by 50%) 22Na+-flux component (Km for Na+ at 18 mM) has been identified which was also blocked by the potent amiloride derivative phenamil (K0.5 = 47 +/- 21 nM). The most potent inhibitor of Na+/H+ exchange, ethylisopropyl-amiloride, hardly inhibited this 22Na+-influx component at a concentration of 21 microM. Amiloride binding sites were characterized using [3H]phenamil. The tritiated ligand binds to a single family of binding sites in thyroid membranes with a Kd value of 50 +/- 10 nM and a maximal binding capacity of 5 +/- 1 pmol/mg protein. Patch-clamp experiments have directly demonstrated the existence of a phenamil- and amiloride-sensitive cationic channel, with a conductance of 2.6 pS, which is permeable to sodium, but not very selective (PNa+/PK+ = 1.2). This channel is an important element in the regulation of the resting membrane potential of thyroid cells.

In vitro differentiated HT 29-D4 clonal cell line generates leakproof and electrically active monolayers when cultured in porous-bottom culture dishes.

HT 29-D4 is a clonal cell line, derived from the human colon adenocarcinoma cell line HT 29, which can be induced to differentiate into enterocyte-like cells by replacing glucose with galactose in the culture medium (Fantini et al. [1986], J. Cell Sci. 83, 235-249). Both undifferentiated and differentiated HT 29-D4 cells have been successfully grown to confluency in Costar Transwell permeable chambers. Only HT 29-D4 cells grown in glucose-free, galactose-containing medium were able to form leakproof monolayers, as demonstrated by their ability to prevent diffusion of serum proteins. These monolayers consist of highly polarized epithelial-like cells with a well organized apical brush border. Transepithelial electrical parameters have been measured under sterile conditions for both types of monolayer. Only HT 29-D4 monolayers cultured in glucose-free, galactose-containing medium were electrically active, with a transepithelial resistance R = 172 +/- 46 omega.cm2, a potential difference PD = 0.35 +/- 0.05 mV, apical negative and a short-circuit current Isc = 2.0 +/- 0.4 microA.cm-2. Apical addition of amphotericin B induced a rapid and considerable increase in Isc and PD, which was abolished by basal ouabain. In contrast, HT 29-D4 cells grown in glucose-containing medium did not generate any potential difference (PD = 0 mV) and their resistance was very low (R = 34.1 +/- 0.9 omega.cm2). It is concluded from these studies that HT 29-D4 cells grown in glucose-free, galactose-containing medium acquire functional characteristics of epithelia, compared to HT 29-D4 cells grown in glucose-containing medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophysiological correlates of fluid transport in cultured porcine thyroid cells.

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)

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: structure of the junctions assessed by freeze-fracture and lanthanum permeability.

The organization of tight junctional complexes (TJs) was studied in cultured porcine thyroid cells during the inversion of polarity induced by collagen-embedding of inside-out follicles, using freeze-fracture replicas and lanthanum penetration. During the early steps of polarity reversal, freeze-fractures showed that TJs generally persisted. They increased in width and progressively branched out into the basolateral surfaces, towards the basal pole. Later, the number of TJ strands decreased and gap junctions inserted within TJ networks were found between cells in reversed follicles, in the same manner as in typically polarized follicles, embedded in collagen or in suspension. The de novo formation of TJ complexes was rarely found in the reversing structures. Despite the heterogeneity of TJs assessed by freeze-fracture, impermeability to lanthanum tracer was noted in inside-out structures. During the reversal process, some TJs remained unstained, whereas others displayed permeability to lanthanum. This heterogeneity might be due to the "opening" of a small number of junctions (perhaps only one by aggregate). When the process was achieved after 48 hr in collagen, the tightness of the junctions was complete, confirmed by the absence of lanthanum in luminal cavities of newly formed follicles.

Cholesterol-rich microdomains in rat and porcine thyroid membranes involved in TSH-induced endocytotic processes.

Filipin, a polyene antibiotic, was used to detect cholesterol in thyroid membranes in vivo and in culture during TSH stimulation. We found that apical and basolateral plasma membranes were heterogeneously modified by filipin which induced abundant lesions in apical membranes, whereas Golgi apparatus, endoplasmic reticulum, nuclear membranes were unmodified. Small apical vesicles and colloid droplets were generally highly enriched in these complexes, suggesting a high cholesterol concentration in their membranes. Pseudopod membranes, known to be highly specialized domains in the apical plasma membrane, appeared enriched in cholesterol. Consequently, we suggest that an increased cholesterol content may be involved in the stabilization of thyroid membranes during endocytotic processes.