PubMed Health⌕ Search

Biomedical subjects

J Mauchamp

Publications and source records attributed to J Mauchamp.

At least 37 records · Page 2Linked to original sources
PubMed1988

Thyrotrophin and cyclic AMP regulation of thyroglobulin gene expression in cultured porcine thyroid cells.

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.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1988

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)

Action Potentials↗
PubMed1988

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.

Animals↗
PubMed1987

Synthesis and apical and basolateral secretion of thyroglobulin by thyroid cell monolayers on permeable substrate: modulation by thyrotropin.

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.

Animals↗
PubMed1987

Epithelial cell polarization in culture: orientation of cell polarity and expression of specific functions, studied with cultured thyroid cells.

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.

Animals↗
PubMed1987

Polarized properties of thyroid cells: a study with cultured porcine cells.

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.

Adenylyl Cyclases↗
PubMed1986

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: an ultrastructural study.

Inside-out porcine thyroid follicles in culture undergo polarity reversal after being embedded in collagen gel. The newly-formed follicles reexpress some specific thyroid functions lost in inside-out follicles (Chambard et al., 1984. We present here an ultrastructural study of the inversion of polarity in this model system. This process takes place within 24 to 48 hr, without any opening of the original tight junctions, as shown by fixation in the presence of ruthenium red. A general shrinkage of cellular aggregates was noted soon after embedding. At the apical pole, three different modifications were observed: structural changes appeared in the kinocilium, microvilli and underlying cytoskeleton as early as 10 min after embedding, mainly when the apical pole of the cells was in close contact with the collagen fibers; large cytoplasmic lamellipod- or pseudopod-like extensions, covering the adjacent apical domain, protruded from outer apical regions; some other apical areas invaginated and formed channels inside the aggregates. The last two processes prevented close contact between apical cell surfaces and collagen fibers and allowed a persistence of the initial polarity in some of the cells. Newly-formed lumens were closed 24 hr after embedding in gel and the outer surface of the cellular aggregates in close contact with collagen fibers looked like a basal membrane. These mechanisms proceeded at different rates and involved different numbers of cells, but they all appeared to be related to the transformation of inside-out follicles into follicular structures.

Animals↗
PubMed1985

Regulation by thyrotropin of acyl-CoA: cholesterol acyltransferase in cultured thyroid cells.

ACAT activity measured in microsomes of thyroid cells cultured for 4 days in the presence of TSH (1 mU/ml) was two or three times lower than that of the control cells cultured for the same period in the absence of TSH. The pool of cellular cholesterol which served as the ACAT substrate, was not exchangeable with exogenous cholesterol provided in the form of liposomes. However, the incubation of microsomes with liposomes made it possible to increase the cholesterol content in the microsomes by 35%, and this resulted in the activation of ACAT activity. Nevertheless, maximum activity measured after activation in the microsomes of the control cells remained higher than that of the microsomes of cells cultured in the presence of TSH. These findings would suggest that TSH acted by diminishing the cellular content in ACAT enzyme, as well as modifying the distribution of cholesterol in the intracellular membranes.

Animals↗
PubMed1985

Effects of prostaglandin E2 and cholera toxin on apical sodium uptake in thyroid epithelial cells: role of cAMP.

When cultured in tissue culture polystyrene dishes, porcine thyroid cells formed polarized monolayers. Their apical pole was oriented towards the culture medium. Influx of sodium 22 (5 min) through the apical surface was partially inhibited by amiloride. Amiloride-sensitive Na uptake was increased about 3-fold by prostaglandin E2 (1 microM, 30 min) and by cholera toxin (0.01 micrograms/ml, 1 h). This increase, which was also obtained with forskolin and 8-(4-chlorophenylthio)adenosine-3':5'-monophosphate, cyclic (8-chloro-cAMP), is probably a consequence of the increase of the intracellular cAMP level by prostaglandin and cholera toxin.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1985

Localization of the Na+/K+-ATPase and of an amiloride sensitive Na+ uptake on thyroid epithelial cells.

The Na+/K+-ATPase was localized using purified specific antibodies, on the basolateral membranes of rat thyroid epithelial cells and of cultured porcine thyroid cells, by immunofluorescence and immunoelectron microscopy. No staining was observed on the apical membranes. When cultured cells formed monolayers, with their apical pole in contact with the culture medium, 22Na+ uptake was inhibited by amiloride. Inhibition was dependent upon extracellular Na+ concentration, half maximal inhibition was obtained with 0.7 microM amiloride in the presence of 5 mM Na+. Ouabain was ineffective on Na+ uptake into intact monolayers. A brief treatment of the monolayers with ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) opened the tight junctions and allowed the access of ouabain to the basal pole of the cells. In this condition ouabain increased Na+ uptake. When cells were reorganized into follicle-like structures, with their basal pole in contact with the culture medium, Na+ uptake was not modified by amiloride but was increased by ouabain. We conclude that in thyroid cells, the Na+/K+-ATPase is present on the basolateral domain of the plasma membrane whereas an amiloride sensitive sodium uptake occurs at the apical surface.

Amiloride↗
PubMed1984

Chronic regulation by thyrotropin of arachidonic acid incorporation in cholesteryl esters of cultured thyroid cells.

During short term incubations, radioactive arachidonic acid and palmitic acid were incorporated in the cholesteryl ester fraction of the lipids of cultured thyroid cells. Three times more arachidonic than palmitic acid was incorporated and the incorporation of both was dependent upon the culture conditions: the presence of 1 mU/ml thyrotropin in the culture medium during four days almost completely inhibited the subsequent incorporation of the two fatty acids in the cholesteryl ester fraction whereas the total cholesterol and cholesteryl ester content of the cells was not affected.

Animals↗
PubMed1984

Polarity reversal of inside-out thyroid follicles cultured within collagen gel: reexpression of specific functions.

Isolated porcine thyroid cells cultured in suspension in Eagle Minimum Essential Medium supplemented with calf serum (5-20%) reorganize to form vesicles, i.e. closed structures in which all cells have an inverted polarity as compared to that found in follicles: the apical membranes are bathed by the culture medium. Under these conditions, cells neither concentrate iodide nor respond to acute thyrotropin (TSH) stimulation. When embedded in collagen gel, these vesicles undergo polarity reversal to form follicles. We describe here the change in the orientation of cell polarity and the subsequent reappearance of specific thyroid functions. Six hr after embedding, membrane areas in contact with collagen fibers show basal characteristics. At this time, cells begin to concentrate iodide and to respond to acute TSH stimulation (iodide efflux and increased cAMP levels). Most cells form follicles 24 hr after embedding, but 48 hr are required for the transformation of all vesicles into follicles. This occurs without opening of the tight junctions. Iodide organification is detected 24 hr after embedding, when periodic acid-Schiff positive material, identified as thyroglobulin by immunofluorescence, accumulates in the lumen. Iodide concentration and organification, as well as response to TSH stimulation reach maximal levels after 3 days in the collagen matrix. After a 5-day culture in the collagen matrix in the absence of TSH, cell activity can be stimulated by chronic treatment with low hormone concentrations (10-100 microU/ml). As shown with thyroid cells grown in monolayer on permeable substrates (Chambard M., et al., 1983, J. Cell Biol. 96, 1172-1177), iodide uptake and cAMP-mediated TSH responses are expressed when the halogen and the hormone have direct access to the basal membrane. Organification, on the contrary, requires a closed apical compartment.

Animals↗
PubMed1983

Polarization of thyroid cells in culture: evidence for the basolateral localization of the iodide "pump" and of the thyroid-stimulating hormone receptor-adenyl cyclase complex.

When cultured in collagen gel-coated dishes, thyroid cells organized into polarized monolayers. The basal poles of the cells were in contact with the collagen gel, whereas the apical surfaces were facing the culture medium. Under these culture conditions, thyroid cells do not concentrate iodide nor respond to acute stimulation by thyroid-stimulating hormone (TSH). To allow the free access of medium components to the basal poles, the gel was detached from the plastic dish and allowed to float in the culture medium. After release of the gel, the iodide concentration and acute response to TSH stimulation were restored. Increased cAMP levels, iodide efflux, and formation of apical pseudopods were observed. When the thyroid cells are cultured on collagen-coated Millipore filters glued to glass rings, the cell layer separates the medium in contact with the apical domain of the plasma membrane (inside the ring) from that bathing the basolateral domain (outside the ring). Iodide present in the basal medium was concentrated in the cells, whereas no transport was observed when iodide was added to the luminal side. Similarly, an acute effect of TSH was observed only when the hormone was added to the basal medium. These results show that the iodide concentration mechanism and the TSH receptor-adenylate cyclase complex are present only on the basolateral domain of thyroid cell plasma membranes.

Adenylyl Cyclases↗
PubMed1982

Chronic and acute effects of thyrotropin on phosphatidylinositol turnover in cultured porcine thyroid cells.

The 32P incorporation into phospholipids of isolated porcine thyroid cells, cultured for 1-4 days, has been studied in subsequent 2-h incubations. Along with culture ageing, decreased 32P incorporation into total phospholipid of control cells was observed. The presence of 40 munits/ml TSH during the 2 h incubation yielded a relative increase in labelling of phosphatidylinositol, named 'acute phospholipid effect'. A chronic treatment of the cells with TSH concentration ranging from 0.1 to 10 munits/ml ensured the maintenance of a high turnover rate of total phospholipids. The analysis of individual phospholipids revealed that 1-day culture cells in the presence of 0.1 munits/ml TSH presented a strong increase of phosphatidylinositol labelling. This 'chronic phospholipid effect' of TSH can be reproduced by a chronic treatment with dibutyryl cyclic AMP (10(-3)M) or prostaglandin E2 (10(-6)M), which did not evoke a classical phospholipid effect in a 2 h incubation. If TSH (40 munits/ml) is added to the cells in a 2 h incubation, control cells show the classical phospholipid effect whereas cells chronically treated with TSH, dibutyryl cyclic AMP or prostaglandin E2 presented a 'reverse phospholipid effect' i.e. a relative decrease in phosphatidylinositol labelling. 10(-4)M cycloheximide presence during the last 12 h of culture prevented the establishment of the 'chronic phospholipid effect' and of its consequence, 'the reverse phospholipid effect'. On the basis of these results a scheme is proposed in keeping with current hypotheses concerning phosphatidylinositol metabolism.

Animals↗