The BeWo choriocarcinoma cell line as a model of iodide transport by placenta.

Cultured human choriocarcinoma cells of the BeWo line exhibited saturable accumulation of radioiodide. Inhibition by competing anions followed the affinity series perchlorate >> iodide > or = thiocyanate, consistent with uptake through the thyroid iodide transporter, NIS, whose messenger RNA was found in BeWo cells, and whose protein was distributed towards the apical pole of the cells. Efflux obeyed first order kinetics and was inhibited by DIDS, an antagonist of anion exchangers including pendrin, whose messenger RNA was also present. In cultures where iodide uptake through NIS was blocked with excess perchlorate, radioiodide accumulation was stimulated by exposure to medium in which physiological anions were replaced by 2-morpholinoethanesulfonic acid (MES), consistent with the operation of an anion exchange mechanism taking up iodide. Chloride in the medium was more effective than sulfate at inhibiting this uptake, matching the ionic specificity of pendrin. These studies provide evidence that the trophoblast accumulates iodide through NIS and releases it to the fetal compartment through pendrin.

Facilitation of renal autoregulation by angiotensin II is mediated through modulation of nitric oxide.

AIMS: This study was designed to investigate the influence of angiotensin II (Ang II) and nitric oxide (NO) on autoregulation of renal perfusion. METHODS: Autoregulation was investigated in isolated perfused kidneys (IPRK) from Sprague-Dawley rats during stepped increases in perfusion pressure. RESULTS: Ang II (75-200 pM) produced dose-dependent enhancement of autoregulation whereas phenylephrine produced no enhancement and impaired autoregulation of GFR. Enhancement by Ang II was inhibited by the AT1 antagonist, Losartan, and the superoxide scavenger, Tempol. Under control conditions nitric oxide synthase (NOS) inhibition by 10 microm N-omega-nitro-L-arginine methyl ester (L-NAME) facilitated autoregulation in the presence of non-specific cyclooxygenase (COX) inhibition by 10 microm indomethacin. Both COX and combined NOS/COX inhibition reduced the autoregulatory threshold concentration of Ang II. Facilitation by 100 pm Ang II was inhibited by 100 microm frusemide. Methacholine (50 nm) antagonised Ang II-facilitated autoregulation in the presence and absence of NOS/COX inhibition. Infusion of the NO donor, 1 microm sodium nitroprusside, inhibited L-NAME enhancement of autoregulation under control conditions and during Ang II infusion. CONCLUSIONS: The results suggest than an excess of NO impairs autoregulation under control conditions in the IPRK and that endogenous and exogenous NO, vasodilatory prostaglandins and endothelium-derived hyperpolarizing factor (EDHF) activity antagonise Ang II-facilitated autoregulation. Ang II also produced a counterregulatory vasodilatory response that included prostaglandin and NO release. We suggest that Ang II facilitates autoregulation by a tubuloglomerular feedback-dependent mechanism through AT1 receptor-mediated depletion of nitric oxide, probably by stimulating generation of superoxide.

Sodium iodide symporter (NIS) gene expression in human placenta.

The placenta must allow the passage of iodide from the maternal to the fetal circulation for synthesis of thyroxine by the fetal thyroid. The thyroid sodium iodide symporter (NIS) was cloned in 1996 and, although widely distributed among epithelial tissues, early studies failed to detect it in placenta. We demonstrated NIS mRNA in human placenta and in the human choriocarcinoma cell line, JAr. NIS protein was localized to trophoblasts, with a tendency to apical distribution, in sections of human placenta immunostained with a monoclonal antibody against hNIS. We conclude that NIS is expressed in placenta and may mediate placental iodide transport.

Microtubule integrity is essential for apical polarization and epithelial morphogenesis in the thyroid.

In this study, we examined the contribution of microtubules to epithelial morphogenesis in primary thyroid cell cultures. Thyroid follicles consist of a single layer of polarized epithelial cells surrounding a closed compartment, the follicular lumen. Freshly isolated porcine thyroid cells aggregate and reorganize to form follicles when grown in primary cultures. Follicular reorganization is principally a morphogenetic process that entails the assembly of biochemically distinct apical and basolateral membrane domains, delimited by tight junctions. The establishment of cell surface polarity during folliculogenesis coincided with the polarized redistribution of microtubules, predominantly in the developing apical poles of cells. Disruption of microtubule integrity using either colchicine or nocodazole caused loss of defined apical membrane domains, tight junctions and follicular lumina. Apical membrane and tight junction markers became randomly distributed at the outer surfaces of aggregates. In contrast, the basolateral surface markers, E-cadherin and Na(+),K(+)-ATPase, remained correctly localized at sites of cell-cell contact and at the free surfaces of cell aggregates. These findings demonstrate that microtubules play a necessary role in thyroid epithelial morphogenesis. Specifically, microtubules are essential to preserve the correct localization of apical membrane components within enclosed cellular aggregates, a situation that is also likely to pertain where lumina must be formed from solid aggregates of epithelial precursors.

Different transporters for tri-iodothyronine (T(3)) and thyroxine (T(4)) in the human choriocarcinoma cell line, JAR.

We investigated transport systems for tri-iodothyronine (T(3)) and thyroxine (T(4)) in the human choriocarcinoma cell line, JAR, using a range of structurally similar compounds to determine whether these thyroid hormones are transported by common or different mechanisms. Saturable T(3) but not saturable T(4) uptake was inhibited by a wide range of aromatic compounds (nitrendipine, nifedipine, verapamil, meclofenamic acid, mefenamic acid, diazepam, phenytoin). Nitrendipine and diazepam were the most effective inhibitors of saturable thyroid hormone uptake. Nitrendipine decreased the K(m) for T(4) uptake from a control value of around 500 nM to around 300 nM (n=6). In contrast, the K(m) for T(3) uptake was increased from a control value of around 300 nM to around 750 nM (n=4). Diazepam had similar effects. This divergent shift in affinity for the uptake of T(3) and T(4) suggested that separate uptake systems exist for these two thyroid hormones. This provides evidence for at least two transporters mediating uptake of T(3) and T(4) in JAR cells: a specific T(4) transporter that does not interact with T(3) or structurally similar compounds; and a shared iodothyronine transporter that interacts with T(3), T(4), nitrendipine and diazepam.

Lack of membrane transport of l-thyroxine sulphate in the human choriocarcinoma cell line, JAr.

We examined uptake of l -thyroxine sulphate (T(4)S) and possible interactions between T(4)S and thyroxine (T(4)) uptake in the choriocarcinoma cell line JAr. Cells were incubated with 50 p m(125)I-T(4)S in the absence (total uptake) and in the presence (non-specific uptake) of 10 microm T(4)S. Cells were also incubated at 37 degrees C for 2 min with 50 p m(125)I-T(4)in the presence of an increasing amount of unlabelled T(4)(0-10 microm) or T(4)S (0-30 microm). There was negligible total uptake of(125)I-T(4)S (1.14+/-0. 05 fmol/mg cellular protein, mean+/-sem) and no specific uptake after 120 min incubation. Minor inhibition of(125)I-T(4)uptake by T(4)S could be explained entirely by a low level of residual T(4)(0. 2 per cent) in the T(4)S preparation. These findings indicate that T(4)S does not share the T(4)membrane transporter.

Comparison of mechanisms mediating uptake and efflux of thyroid hormones in the human choriocarcinoma cell line, JAR.

We compared the specificities of transport mechanisms for uptake and efflux of thyroid hormones in cells of the human choriocarcinoma cell line, JAR, to determine whether triiodothyronine (T3), thyroxine (T4) and reverse T3 (rT3) are carried by the same transport mechanism. Uptake of 125I-T3, 125I-T4 and 125I-rT3 was saturable and stereospecific, but not specific for T3, T4 and rT3, as unlabelled L-stereoisomers of the thyroid hormones inhibited uptake of each of the radiolabelled hormones. Efflux of 125I-T3 was also saturable and stereospecific and was inhibited by T4 and rT3. Efflux of 125I-T4 or 125I-rT3 was, in contrast, not significantly inhibited by any of the unlabelled thyroid hormones tested. A range of compounds known to interfere with receptor-mediated thyroid hormone uptake in cells inhibited uptake of 125I-T3 and 125I-rT3, but not 125I-T4. We conclude that in JAR cells uptake and efflux of 125I-T3 are mediated by saturable and stereospecific membrane transport processes. In contrast, the uptake, but not the efflux, of 125I-T4 and 125I-rT3 is saturable and stereospecific, indicating that uptake and efflux of T4 and rT3 in JAR cells occur by different mechanisms. These results suggest that in JAR cells thyroid hormones may be transported by at least two types of transporters: a low affinity iodothyronine transporter (Michaelis constant, Km, around 1 microM) which interacts with T3, T4 and rT3, but not amino acids, and an amino acid transporter which takes up T3, but not T4 or rT3. Efflux of T4 and rT3 appears to occur by passive diffusion in these cells.

Uptake of L-triiodothyronine sulphate by human choriocarcinoma cell line, JAr.

This study investigated uptake of triiodothyronine sulphate (T3S) and interactions between uptake of T3S and triiodothyronine (T3) using the human choriocarcinoma cell line (JAr) as a model of placental transport. Cells were incubated at 37 degrees C with 30 pM 125I-T3 for 2 min with unlabelled T3 (0-30 microM) or T3S (0-1 mM). Addition of an excess unlabelled T3 (30 microM) or T3S (1 mM) reduced the initial rate of 125I-T3 uptake by 69.3+/-3.6 per cent (P<0.0001) and 52.9+/-7.8 per cent (P<0.0001), respectively. The calculated Michaelis constant (Km) for T3 uptake was 0.378+/-0.133 microM (n = 3) with a corresponding maximum velocity (Vmax) of 15.4+/-6.9 pmol/min/mg protein. Uptake of 125I-T3 was inhibited in a dose-dependent way by the addition of unlabelled T3S (0-1 mM). The calculated inhibition constant (Ki) for the inhibition of 125I-T3 uptake by T3S was 121.8+/-35.2 microM (n = 6). Saturable uptake of 125I-T3S by JAr cells was negligible. The T3S preparation incubated with the cells contained about 0.1 per cent T3, sufficient to explain the apparent inhibition of 125I-T3 uptake by unlabelled T3S. These results suggest that, in contrast to T3 uptake in these cells, JAr cells do not have a saturable uptake mechanism for T3S, and that T3S does not interact with the T3 transporter in these cells.

Uptake of reverse T3 in the human choriocarcinoma cell line, JAr.

The uptake and efflux of reverse triiodothyronine (rT3) in JAr cells were investigated. Uptake of 125I-rT3 was time dependent and reversible with a saturable component of around 70 per cent of total uptake after 30 min of incubation. Efflux was not saturable. Kinetic analysis of the initial specific uptake rates revealed an uptake process with a Michaelis constant of 3.04+/-0.53 microM (mean+/-SEM, n=15) and a corresponding maximum velocity of 9.65+/-2.49 pmol/min/mg protein (n=15). Uptake of rT3 was stereospecific, but not specific for rT3, as unlabelled L stereoisomers of thyroid hormone analogues were more effective as inhibitors of 125I-rT3 uptake than rT3. Unlabelled T3 and thyroxine (T4) (10 microM) reduced cellular uptake of 125I-rT3 by around 82 and 74 per cent, respectively. The calculated inhibition constants Ki were 1.23+/-0.29 microM (n=4) and 0.66+/-0.19 microM (n=4) for T3 and T4, respectively. Similarly, rT3 reduced cellular uptake of 125I-T3 and 125I-T4 by 34 and 23 per cent, respectively. The calculated inhibition constants Ki were 1.75+/-0.55 microM (n=8) and 1.08+/-0.36 microM (n=8) for the inhibition of 125I-T3 and 125I-T4 uptake, respectively. Reverse T3 inhibited efflux of 125I-T3 from the cells by around 20 per cent, but did not inhibit efflux of 125I-T4. These results suggest that uptake of rT3 in JAr cells may occur via a single, saturable membrane carrier, which also interacts with T3 and T4, while efflux of rT3 may occur by passive diffusion.

Thyroid hormone efflux from placental tissue is not stimulated during cell volume regulation.

The effects of cell swelling induced by hyposmotic shock on efflux of hybrid hormones and selected amino acids from human placental tissue were examined. Decreasing the osmolarity of external medium from 290 to 140 mOsm/kg stimulated release of taurine, tryptophan and glutamine from placental tissue fragments. The efflux rate constant for taurine increased from 0.0069 +/- 0.0012/min to 0.0646 +/- 0.0217/min (n = 6) (P < 0.001), for tryptophan from 0.016 +/- 0.0010/min to 0.0295 +/- 0.0016/min (n = 6) (P < 0.001), and for glutamine from 0.0267 +/- 0.0027/min to 0.0659 +/- 0.0043/min (n = 4) (P < 0.001). In contrast, hyposmotic challenge did not affect release of triiodothyronine, thyroxine and leucine. These results indicate that transport processes involved in the regulation of cellular volume are unlikely to facilitate efflux of thyroid hormones from placental tissue, and therefore are unlikely to mediate transfer of thyroid hormones across the placenta. In addition, it is unlikely that the transport system facilitating the release of amino acids from placental tissue during regulatory volume decrease is one of the known amino acid carriers.

Protein tyrosine phosphorylation influences adhesive junction assembly and follicular organization of cultured thyroid epithelial cells.

The follicular histoarchitecture of the thyroid forms the anatomical basis for thyroid physiology and is commonly disturbed in diseases of the thyroid. We have used cultured porcine thyroid cells to study thyroid epithelial morphogenesis and its regulation. When cultured in the presence of TSH, freshly isolated thyroid cells reorganize to form follicles within three-dimensional cell aggregates. However, when established follicles are washed into TSH-free medium, thyroid cells spread and migrate to convert follicles into confluent epithelioid monolayers, activating morphogenetic mechanisms, such as cell locomotility, that may be relevant to thyroid inflammation and tumor invasiveness. The phenomenon of follicle to monolayer conversion, therefore, provides an opportunity to identify morphogenetic mechanisms that 1) must be tonically inhibited to maintain follicular organization and 2) may contribute to pathogenetic disturbances of follicular architecture when functioning aberrantly. In this study we found that follicle to monolayer conversion is associated with an increase in cellular phosphotyrosine. This was particularly evident at nascent focal adhesions (cell-substrate adhesive junctions) and later at cell-cell junctions. Focal adhesion assembly was accompanied by reorganization of the actin cytoskeleton, with the appearance of prominent stress fibers. Genistein, a potent inhibitor of protein tyrosine kinases, inhibited the accumulation of phosphotyrosine, focal adhesion assembly, and follicle to monolayer conversion. We conclude that tyrosine phosphorylation exerts an important influence on thyroid epithelial organization in culture, at least partly mediated through regulation of focal adhesion assembly.

Sodium channel heterogeneity in the apical membrane of porcine thyroid epithelial cells.

Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and secrete Cl-. Two types of Na+ channels were found in cell-attached patches of apical membrane. A low conductance Na+ channel (conductance g = 4 picosiemens (pS)) remained open for seconds and showed a high selectivity for Na+ compared with K+. In contrast, a high conductance Na+ channel (g = 10 pS) flickered rapidly and had reduced selectivity. Both types of Na+ channel became more prevalent when the cells were exposed to Na(+)-free medium, though only the high conductance channel increased in prevalence on addition of prostaglandin E2, a stimulator of adenylate cyclase which increases Na+ absorption in this cultured epithelium. Two minority types of channel were also found: a non-selective small conductance cation channel which had been reported previously, and an intermediate conductance channel found only in Na(+)-free medium. It was concluded that passage of Na+ across the apical membrane of thyroid cells is mediated by typical epithelial Na+ channels, but that the two types of channel are differentially regulated.

Chloride channels in the apical membrane of thyroid epithelial cells are regulated by cyclic AMP.

Porcine thyroid epithelial cells cultured as a monolayer with their apical membranes facing the medium are known to absorb Na+ and to secrete the anions Cl- and HCO3-. Chloride channels were found in the apical membrane, and displayed a reversal potential close to the resting membrane potential, linear current-voltage relationships, a conductance at physiological temperature of 6.5 pS, and a small but significant permeability to HCO3-. Stimulation of ion transport with prostaglandin E2 or 8-(4-chlorophenylthio) adenosine 3':5'-cyclic monophosphate promoted activation of Cl- channels in cell-attached patches, and excised patches were reactivated by exposure of their cytoplasmic surface to protein kinase A and ATP. Physiological temperatures were necessary for activation of Cl- channels in cell-attached patches. The channels exhibited sub-states with a conductance exactly half that of the full unit conductance, suggesting a dual-barrelled channel structure. It is concluded that the apical membrane of thyroid epithelial cells contains cyclic AMP-activated Cl- channels controlling anion transport.

Cadherin-mediated adhesion and apical membrane assembly define distinct steps during thyroid epithelial polarization and lumen formation.

The biogenesis of follicles from aggregates of precursor cells is an important morphogenetic process in thyroid embryology. It necessitates the creation of a polarized cell phenotype, assembly of specialized cell-cell junctions, and generation of follicular lumena. In this study we sought to investigate the relationship between cell polarization and lumen formation by studying the cell surface events that occurred when freshly isolated adult porcine thyroid cells reorganized to form follicles in primary culture. Follicular reorganization entailed the initial formation of solid three-dimensional cell aggregates and the subsequent appearance of lumena within aggregates. During the initial stage of cell aggregation, the adhesion molecule, E-cadherin, became expressed at all surfaces involved in cell-cell contact. Aggregation was inhibited by monoclonal antibodies that block cadherin function, indicating directly that E-cadherin is a dominant initial cell-cell adhesion molecule. Cell aggregation was also associated with the recruitment to the cell surface of ZO-1, a tight junction-associated protein, and Na+/K(+)-adenosine triphosphatase. These proteins were initially found throughout regions of cell-cell contact and only subsequently redistributed to their mature locations in tight junctions and the basolateral cell surface, respectively. In contrast, components associated with the apical membrane were first detected within large intracellular vacuoles, which subsequently fused with the cell surface between maturing tight junctions to yield the apical membrane domain and nascent follicular lumena. Follicle formation occurred independently of basal lamina assembly and TSH, although maintenance of follicular architecture required the presence of this hormone. These findings indicate that cultured follicles form in two distinct stages: 1) initial aggregation mediated by E-cadherin and associated with recruitment of components of both tight junctions and the basolateral membrane domain, and 2) subsequent formation of a specialized apical membrane domain by coordinated fusion of intracellular vacuoles at sites of the cell surface where tight junctions are maturing. We propose that follicular morphogenesis may arise as a consequence of epithelial cell polarization within coherent three-dimensional cell aggregates.

Uptake of thyroxine in the human choriocarcinoma cell line JAR.

We have studied the uptake of 125I-thyroxine (125I-T4) in the human choriocarcinoma cell line JAR. Uptake of 125I-T4 was time-dependent, stereospecific and reversible, with a saturable component of 33% after 120 min of incubation. Kinetic analysis of the initial specific uptake rates indicated the presence of a single uptake process with a Michaelis constant of 59.4 +/- 13.9 nM (n = 12) and maximum velocity of 0.29 +/- 0.06 pmol/min per mg protein. Uptake was dependent on intracellular energy as, in the presence of 2 nM potassium cyanide, saturable uptake was reduced to 60.6 +/- 8.5% (n = 4) of control uptake. Uptake was also temperature-dependent. Saturable 125I-T4 uptake after 60 min of incubation was 26.1 +/- 3.0% at 25 degrees C (n = 6) and 27.3 +/- 5.7% at 4 degrees C of control uptake at 37 degrees C. Ouabain did not inhibit 125I-T4 uptake indicating that the uptake was independent of the Na+ gradient across the cell membrane. Although T4 uptake was stereospecific, as D-T4 failed to inhibit 125I-L-T4 uptake, it was not specific for T4, as tri-iodothyronine (T3) and reverse T3 also inhibited 125I-T4 uptake. We conclude that JAR cells have a saturable, stereospecific and reversible membrane transport mechanism for T4 which is dependent on intracellular energy, but independent of the Na+ gradient across the cell membrane.

Microtubule integrity is necessary for the epithelial barrier function of cultured thyroid cell monolayers.

Vectorial transport in the thyroid epithelium requires an efficient barrier against passive paracellular flux, a role which is principally performed by the tight junction (zonula occludens). There is increasing evidence that tight junction integrity is determined by integral and peripheral membrane proteins which interact with the cell cytoskeleton. Although the contribution of the actin cytoskeleton to tight junction physiology has been intensively studied, less is known about possible interactions with microtubules. In the present study we used electrophysiological and immunohistochemical approaches to investigate the contribution of microtubules to the paracellular barrier in cultured thyroid cell monolayers which displayed a high transepithelial electrical resistance (6000-9000 ohm.cm2). Colchicine (1 microM) caused a progressive fall in electrical resistance to < 10% of baseline after 6 h and depolarization of the transepithelial electrical potential difference consistent with a significant increase in paracellular permeability. The effect of colchicine on TER was not affected by agents which inhibit the major apical conductances of thyroid cells but was reversed upon removal of the drug. Immunofluorescent staining for tubulin combined with confocal laser scanning microscopy demonstrated that thyroid cells possessed a dense microtubule network extending throughout the cytoplasm which was destroyed by colchicine. Colchicine also produced changes in the localization of the tight junction-associated protein, ZO-1: its normally continuous junctional distribution was disrupted by striking discontinuities and the appearance of many fine strands which extended into the cytoplasm. A similar disruption in E-cadherin staining was also observed, but colchicine did not affect the distribution of vinculin associated with adherens junctions nor the integrity of the perijunctional actin ring. We conclude that microtubules are necessary for the functional and structural integrity of tight junctions in this electrically tight, transporting epithelium.

Characterization of cell polarity and epithelial junctions in the choriocarcinoma cell line, JAR.

In the placenta the trophoblast cell layer separates maternal and fetal circulations and is involved in the active transport of selected substances across this barrier. We have used the JAR choriocarcinoma cell line to study aspects of trophoblast membrane transport. To determine whether JAR cells could be used in studies of vectorial transepithelial transport it was necessary to determine whether these cells were polarized and assembled tight junctions. In the present study we investigated JAR cells using a range of markers for specific cell surface domains combined with confocal laser scanning microscopy. Freshly isolated cells initially formed a confluent epithelial monolayer with recruitment of a tight junction-associated protein, ZO-1, and a cell adhesion molecule, E-cadherin, to the surface at sites of cell-cell contact. They did not, however, display cell surface polarization, as NaK-ATPase was not segregated in the basolateral domain, and a differentiated apical cell surface was not assembled. The monolayer stage was also unstable, as continued proliferation resulted in the formation of multilayered aggregates where ZO-1 and E-cadherin were lost from the cell surface. These results suggest that the JAR cell line is unlikely to be a suitable model for studies of transepithelial transport in the placenta.

Vinculin localization and actin stress fibers differ in thyroid cells organized as monolayers or follicles.

In epithelial cells interactions between the actin cytoskeleton and cell-cell junctions regulate paracellular permeability and participate in morphogenesis. We have studied the relationship between supracellular morphology and actin-junction interactions using primary cultures of porcine thyroid cells grown either as three-dimensional follicles or as open monolayers. Regardless of morphology, thyroid cells assembled occluding and adhesive junctions containing ZO-1 and E-cadherin, respectively, and showed F-actin staining in apical microvilli and a perijunctional ring. In monolayers, actin stress fibers were also observed in the apical and basal poles of cells, where they terminated in the vinculin-rich zonula adherens and in cell-substrate focal adhesions, respectively. Surprisingly, we were unable to detect vinculin localization in follicular cells, which also did not form stress fibers. Immunoblotting confirmed significantly greater vinculin in triton-insoluble fractions from monolayer cells compared with follicular cells. Incubation of monolayers with 8 chloro(phenylthio)-cyclic AMP decreased the level of immunodetectable vinculin in the zonula adherens, indicating that junctional incorporation of vinculin was regulated by cyclic AMP. In monolayer cultures, cytochalasin D (1 microM) cause actin filaments to aggregate associated with retraction of cells from one another and the disruption of cell junctions. Despite morphologically similar perturbations of actin organization in follicular cultures treated with cytochalasin D, junctional staining of ZO-1 and E-cadherin was preserved and cells remained adherent to one another. We conclude that in cultured thyroid cells structural and functional associations between actin filaments and cellular junctions differ depending upon the supracellular morphology in which cells are grown. One important underlying mechanism appears to be regulation of vinculin incorporation into adhesive junctions by cyclic AMP.

Thyrotropin inhibits the intrinsic locomotility of thyroid cells organized as follicles in primary culture.

The regulation of cell locomotion is a fundamental determinant of tissue architecture. Even in solid tissues of adult organisms cells often retain an intrinsic locomotor capacity which is activated during wound healing or tumor metastasis. In this study we have examined the role of cell locomotion in an in vitro model of thyroid epithelial pattern generation. Primary cultures of adult porcine thyroid cells reorganize to form follicles within three-dimensional cell aggregates when stimulated by thyrotropin (thyroid-stimulating hormone, TSH). Removal of TSH from the culture medium caused established follicles to reorganize into a confluent, two-dimensional epithelioid monolayer. The earliest observed change was the appearance of spreading cells at the peripheries of aggregates. These cells displayed broad lamellipodia whose formation was associated with the redistribution of microfilaments and microtubules and the accumulation of myosin. Spreading cells could migrate into, and fill, artificial wounds several millimeters wide without evidence of cell proliferation, indicating that cells became locomotile as they spread from follicles to form monolayer. Both spreading and migration were inhibited by cytochalasin B. In contrast, cells spread in the presence of colchine, but failed to migrate subsequently. Thyroid cell locomotility from follicles was inhibited by TSH, a cAMP analog, and a cell-free membrane fraction. However, migration from established monolayer cultures was not affected by these regulatory agents. This indicated that cell spreading was an important regulatory locus in thyroid cell patterning. We conclude that the tonic inhibition of thyroid cell locomotility contributes to the maintenance of follicular architecture in vitro. TSH and cell-cell contact may inhibit locomotion by preventing follicular cells from spreading, the earliest step in the morphogenetic conversion of follicles to monolayer.

Thyroid epithelial morphogenesis in vitro: a role for bumetanide-sensitive Cl- secretion during follicular lumen development.

The structural and functional unit of the thyroid gland is the follicle, consisting of a closed lumen surrounded by a single layer of polarized epithelial cells. In this paper we have attempted to characterize the process of lumenal development when primary cultures of porcine thyroid cells reorganized to form follicles. Cells incubated with the loop diuretic, bumetanide, an inhibitor of NaK2Cl cotransport, aggregated but failed to form normal follicles. Laser scanning confocal microscopy combined with immunohistochemical markers of thyroid cell-surface proteins demonstrated that in the presence of bumetanide cells polarized and assembled ZO-1-containing tight junctions separating their apical and basolateral membrane domains. Cultures formed small lumena but their subsequent growth was inhibited by bumetanide. Electrophysiological studies confirmed that bumetanide-sensitive Cl- transport was the major contributor to the transepithelial electrical potential difference across the follicular wall after 48 h incubation. Other potential mechanisms did not contribute significantly to follicular lumenal growth. In particular, bumetanide did not affect cell proliferation and, in contrast to tissue follicles, thyroglobulin could not be detected within the lumena of cultured follicles. We conclude that thyroid follicular reorganization involves two distinct and separate phases of lumenal development: initial lumen formation which probably reflects the assembly of a specialized apical membrane domain; and subsequent lumenal growth which is mediated by the inward transport of Cl- by polarized epithelial cells.