Regulation of thyroid cell volumes and fluid transport: opposite effects of TSH and iodide on cultured cells

D. Cauvi1, C. Penel2, M. C. Nlend1, N. Venot1, C. Allasia3, and O. Chabaud1

1 Faculté de médecine, Institut National de la Santé et de la Recherche Médicale U38, 2 Faculté de pharmacie, Unité Propre de Recherche de l'Enseignement Supérieur Associée au Centre National de la Recherche Scientifique 6032, and 3 Centre de microscopie et d'analyse d'images, Faculté de médecine, Université de la Méditerranée, 13385 Marseille, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.

cell size; cAMP; thyrotropin control; iodide control


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

CELL VOLUME DEPENDS ON the equilibrium between the intracellular ion concentration and the transport of electrolytes, particularly inorganic ions (Na+, K+, and Cl-) and water. As a steady state of cell volume is essential to maintaining the metabolic functions of cells, any variations in ion and water transport that are liable to modify the cell volume will result in metabolic dysfunctions (18). Various regulatory mechanisms serve to prevent any excessively large changes in cell volume. The authors of several studies have described the effects of cell volume variations induced by changes in cell osmolarity triggered by anisotonic media and the resulting regulatory mechanisms (24). Furthermore, several effectors, including hormones and neurotransmitters, can also induce cell swelling or shrinkage via their effects on ion transport and the state of cellular hydration. Hormonal effects on cell volume have been described in detail in various tissues, especially in hepatocytes, where insulin leads to cell swelling whereas glucagon results in cell shrinkage (15). Moreover, in hyperthyroid states, thyroid hormones increase the size of cardiomyocytes (33) and induce renal hypertrophy (23). Some hormonal effects on cell volume are mediated by cAMP. However, depending on the target cells involved, an increase in the cAMP pool can lead to either a decrease in the cell volume, as observed in hepatocytes, MDCK cells, and pancreatic epithelial cells, or an increase, as is found to occur in sweat gland cells (24). Therefore, as suggested by Haussinger and Lang (17), the cell volume variations occurring in response to hormonal stimuli can be said to act like another second messenger in the hormonal processes.

In the thyroid gland, epithelial cell metabolism is modulated by thyrotropin (TSH) and iodide. Like other epithelial cells, thyroid cells transport Na+ in the apical-to-basal direction, as well as transporting K+ and Cl- in the opposite direction. This transport of electrolytes is regulated by TSH (28, 29, 30, 38) via the cAMP pathway (3), like most of the effects of TSH (12), especially the expression of the Na+/I- symporter (32). Variations in thyroid gland volume can be observed in some pathological states; the goiter resulting from hypertrophy and hyperplasia of the thyroid gland are often associated with a decrease in the serum iodide level (14) or overstimulation by excessive TSH effects (9). Experiments on rat gland have shown that a correlation exists between hypertrophy of the thyroid cells and the regulation of follicular volume by iodide (7, 31). The increase in thyroid cell volume observed in human pathologies and animal models is accompanied by a corresponding decrease in the colloidal space that is probably associated with fluid transport. On the other hand, atrophic thyroid glands are also known to occur when the effects of TSH on thyroid cells are either reduced or completely lacking. This pathological state is generally characterized by a decrease in thyroid cell volume.

No data have been published so far, however, on the respective and/or correlated roles of TSH and iodide in the regulation of thyroid cell volume and the associated fluid transport nor are any quantitative data available to date on thyroid cell volume. The aims of the present study were, therefore, 1) to determine the volumes (total cell volume and cytosolic volume) of thyroid cells in the steady state and 2) to investigate the respective and combined effects of TSH and iodide on those volumes and on fluid transport. For this purpose, we used porcine thyroid cells cultured on collagen-coated filters in which all of the thyroid functions, even thyroid hormone synthesis (16), were kept. With this cell culture system, it was possible to study the effects of TSH and iodide separately after adding them on the basolateral side, as well as to measure the volumes of interest in living thyroid cells and in the colloid-like space. It was established here that TSH and iodide have opposite regulatory effects on thyroid cell volumes and on apical-to-basal net fluid transport. These antagonistic effects may be partly due to the fact that TSH and iodide affect the production of cAMP in opposite ways. These findings are discussed in relation to the data available on the regulation of ion exchanges and fluid transport in thyroid cells, as well as those on pathological states caused by TSH or iodide deficits.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Cell culture. Thyroid epithelial cells were isolated from porcine glands and cultured on porous filters coated with collagen, as previously described (5). The isolated cells, suspended (2.5 × 106/ml) in phenol red-free DMEM containing 1 g/l glucose (GIBCO BRL, Cergy Pontoise, France), were seeded into the apical compartment of chambers. The same medium was introduced into the basal compartment outside the insert. The culture was carried out at 37°C in a 7.5% CO2-92.5% air/water-saturated atmosphere. From day 4 onward, 10% newborn calf serum (Biomedia, Boussens, France) was added to the basal compartment whenever the media were changed. On day 6, the tightness of the monolayer to serum protein was checked in the apical media by performing a colorimetric protein assay, and only tight monolayers were kept (5). From day 6 onward, the basal media were supplemented, or not, with 100 µU/ml b-TSH (Calbiochem, San Diego, CA). From day 8 onward, potassium iodide was added daily, or not, to the basal media at 0.5 µM final concentration, and the apical media were replaced by B-DMEM (a saline medium DMEM devoid of amino acids and vitamins). Moreover, on the same day, an analog of cAMP, 8-chloro-cAMP (Roche Diagnostics, Meylan, France), was added at 0.5 mM final concentration in some experiments instead of TSH. Then, the basal media were subsequently changed every 3 days, maintaining the same TSH or cAMP concentration. From days 8 to 14, apical media were not changed.

Measurement of total cell volume by confocal laser-scanning microscopy. On day 14, apical media were removed, and cells were washed with B-DMEM and loaded with 20 µM octadecylrhodamine B (chloride form, Molecular Probe, Eugene, OR) in B-DMEM for 20 min at 37°C in a 7.5% CO2-92.5% air/water-saturated atmosphere. Then cells were washed three times with PBS. The filter was placed between lamina that were separated by spacers and mounted on the microscope stage.

A Leica system based on an inverted Leitz microscope (DMIRBE, Leica, Heidelberg, Germany) was used to perform confocal laser-scanning microscopy. Living cells labeled with octadecylrhodamine B were observed with a 40X apochromat lens (NA 1.0). Red fluorescence of octadecylrhodamine B, excited at 514 nm with an argon-krypton ion laser, was collected with a 590-nm long-pass filter. Serial optical slices through the cell thickness were obtained. Images (512 × 512 pixels) with a 0.49- × 0.49- × 0.9-µm voxel in size were collected and imported into a PC workstation equipped with MATROX image analysis cards (image series 640, Dozval, Canada). A homemade computer program was drawn up from 3-D reconstitution data (19) with the SAMBA software program (Unilog, Grenoble, France) and used to pile up n consecutive optical slices and then evaluate the total cell volume of each cell (in pl). The same computer program was also used to count the number of cells per culture filter.

Determination of the 3-O-methyl-D-glucose space. On day 14, cells were used to measure their cytosolic volume with the nonmetabolizable 3-O-methyl-D-glucose (3-OMDG). This was done when both the intracellular and the extracellular 3-OMDG (Sigma, St. Louis, MO) concentrations had reached equilibrium as previously described (21). Briefly, the apical and basal media were changed, the basal medium was adjusted with 1 mM 3-OMDG (final concentration), and the cells were incubated with 5 µCi/ml 3-O-methyl-D-[1-3H]glucose (5 Ci/mmol, Amersham France, Les Ulyss, France) for 30 min at 37°C in a 7.5% CO2-92.5% air/water-saturated atmosphere. By this stage, the equilibrium between the intracellular and extracellular concentration of 3-OMDG was reached for all of the culture conditions used. Cells were promptly washed three times with ice-cold PBS containing 0.6 mM phloretin (Sigma), a sugar transport inhibitor, to prevent the extrusion of 3-OMDG. Cells were then solubilized in a 1% SDS solution, and aliquots were counted in a liquid scintillation counter (Tricarb 2100TR, Packard Instruments, Meriden, CT). The 3-OMDG space was deduced from the radioactivity remaining in the cells (dpm/cell) and from the volumic radioactivity (dpm/µl) of the extracellular compartment (25).

Protein and DNA assays. Proteins were evaluated by micro-bicinchoninic acid protein assay (Pierce, Rockford, IL).

Quantitative determination of DNA was performed by measuring DNA fluorescence in the presence of Hoechtst 33258 (Roche Diagnostics, Meylan, France) by use of the procedure previously described by Teixeira et al. (37). Samples and standards were prepared in the same way, and the fluorescence was measured in 96-well plates with a PC-controlled fluorometer (Fluostar +, Salzburg, Austria) with the Biolyse 1.7 program.

Determination of net fluid transport. From the beginning of the culture, the volumes of apical (1 ml) and basal (2.3 ml) media were adjusted so as to preserve the hydrostatic equilibrium. On day 8, the apical (1 ml) and basal (2.3 ml) media were changed, and the apical volumes were measured carefully up to day 14. It was verified that the volume loss in the apical media was recovered in the basal medium; thus evaporation was found to be negligible. The net fluid transport was determined from the slopes of the linear regression curves and was expressed as microliters per hour per 106 cells.

Statistical analyses. The data presented here are expressed as means ± SE for n values. Variations between assays and control were shown to be significant using the Student's t-test with a 1% statistical significance limit. The data presented for the net fluid transport were calculated from the slopes of the linear regression curves ± SD determined with the ORIGIN Software program (Microcal Software, Northampton, MA).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In all experiments, porcine thyroid cells were cultured for 14 days as monolayers on permeable filters coated with collagen. This experimental culture system leaves the main features of thyroid cells intact, especially their functional bipolarity. In addition, it gives separate access to the apical and basal compartments and makes it possible to culture the thyroid cells without any hormones or other effectors. In contrast, functional, well oriented follicles can be obtained only in the presence of TSH or cAMP analog (27). Moreover, when cell monolayers are cultured on plastic supports, TSH and iodide are in contact only with the apical membranes, and this does not correspond to physiological conditions. Conversely, under the bicameral culture conditions used here, the TSH and iodide added to the basal compartment come into contact with the basolateral membranes on which the TSH receptor and the Na+/I- symporter are present. In this study, cells cultured with TSH and iodide were used as the control cells, because these conditions are similar to the in vivo ones. Three other conditions were then obtained by removing the iodide, the TSH, or both, which might mimic what occurs in some pathological states.

TSH and iodide regulate total volume of thyroid cells. To estimate the total cell volume, thyroid cells were loaded with octadecylrhodamine B, a fluorescent probe generally used to label cell membranes by intercalating it between the lipid bilayers. Optical slices were performed on the z axis with a confocal laser-scanning microscope. Median optical slices of control cells showed that each cell of the monolayer was labeled with octadecylrhodamine B (Fig. 1A). The absence of TSH and/or iodide did not give rise to any differences in the shape of the cells (Fig. 1, B, C, and D).


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Fig. 1.   Median optical slices of porcine thyroid cells cultured as monolayers on collagen-coated filters. Thyroid cells were cultured for 14 days. From day 6 onward, thyrotropin (TSH) (100 µU/ml) was added, or not, to the basal media, which were afterward changed every 3 days, maintaining the same TSH concentration. From day 8 onward, iodide (0.5 µM) was daily added, or not, to the basal media. On day 14, cells were labeled with 20 µM octadecylrhodamine B in B-DMEM (DMEM devoid of amino acids and vitamins), placed in the apical compartment for 20 min at 37°C, and washed three times with PBS. A filter was then placed between the lamina, which were separated by spacers and mounted on the microscope stage. Confocal laser-scanning microscopy was performed with a Leica system based on an inverted Leitz microscope. The fluorescence of octadecylrhodamine B, excited at 514 nm with an argon-krypton ion laser, was collected with a 590-nm long-pass filter. Serial optical cell sections were obtained. Cells were cultured with TSH plus iodide (control cells; A), with TSH and without iodide (B), without TSH and with iodide (C), and without TSH and without iodide (D). Bar = 19 µm.

Total cell volume was calculated by piling up n consecutive optical slices with a computer software program, and the volume amounted to 3.73 ± 0.06 pl (mean value ± SE) in the case of the control cells (Fig. 2A). When cells were cultured without iodide, the total cell volume increased by 36% compared with the control cells, whereas the total volume of cells cultured without TSH decreased by about 19% compared with the control cells. The total volume of cells cultured without TSH and without iodide decreased to a value very similar to that obtained on cells cultured only without TSH. These results show that TSH without iodide has a swelling effect and that iodide, but only in the presence of TSH, has a shrinking effect on the total volume of thyroid cells, which suggests that TSH and iodide have antagonistic effects on the total volume of thyroid cells.


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Fig. 2.   A: determination of total volumes of porcine thyroid cells cultured as monolayers on collagen-coated filters. KI, potassium iodide. Cell culture and confocal laser-scanning microscopy were performed as in Fig. 1, and serial optical slices were collected and imported into a PC workstation equipped with image analysis cards. A homemade computer program was used to pile up n consecutive optical slices and evaluate the total volume of each cell in pl. Data are means ± SE (n = 105). B: determination of heights of porcine thyroid cells cultured as monolayers on collagen-coated filters. Cell heights were determined by adding together all the serial optical slices obtained by confocal laser-scanning microscopy from the same experiments as in Fig. 2A. Data are means ± SE (n = 28). * Values differ significantly vs. control, P < 0.01.

As is shown in Fig. 1, the cross-sectional areas of the cells were similar under all of the culture conditions studied. The variations in total cell volume observed were therefore caused mainly by an increase (without iodide) or a decrease (without TSH) in the cell heights (Fig. 2B). Moreover, the number of cells counted was the same on each filter, and the amount of DNA evaluated was similar (Table 1) under all of the culture conditions studied. Our results on the DNA content per cell are extremely similar to previous data on porcine thyroid cells cultured as follicles (6).

                              
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Table 1.   Amount of DNA per porcine thyroid cell cultured as monolayers on collagen-coated filters

To investigate whether the control of the total cell volume by TSH was mediated by cAMP, basal medium prepared without TSH was supplemented with the cAMP analog, 8-chloro-cAMP, for 5 days. When cells were cultured with both 8-chloro-cAMP and iodide, the total cell volume was 3.88 ± 0.06 pl, compared with 4.73 ± 0.09 pl when cells were cultured with 8-chloro-cAMP without iodide (Fig. 3). These values are similar to those obtained from the cells cultured with TSH with or without iodide. We concluded that the effects of TSH on total cell volume are mediated by cAMP.


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Fig. 3.   Effect of cAMP on total volume of porcine thyroid cells cultured as monolayers on collagen-coated filters. Cells were cultured as indicated in Fig. 1 legend. Furthermore, on day 8, basal media with or without iodide but without TSH were supplemented with cAMP analog (8-chloro-cAMP, 0.5 mM at final concentration) for 6 days. Total volume of cells was then calculated in pl, as described in Fig. 2A legend. Data are means ± SE (n = 120); ns, differences between mean values not significant.

Thus we established that TSH (via cAMP) and iodide (but only in the presence of TSH) had antagonistic effects on the total volume of porcine thyroid cells cultured as monolayers on collagen-coated filters and that these effects resulted mainly in changes in the cell heights.

TSH and iodide control the cytosolic volume of thyroid cells. We measured the cytosolic portion of thyroid cells, which is the soluble part of the cytoplasm and represents 50-55% of cell volume in most cell types. The remaining volume includes the nucleus and the intracellular organelles, and their variations under each culture conditions may be evaluated in part by the determination of protein content. The cytosolic portion has been shown to be the space occupied by the nonmetabolizable sugar, 3-OMDG, in the cases of leucocytes (13) and glioma cells (20). The 3-OMDG probably did not enter into the nucleus and cytoplasmic organelles (13). Thus, to evaluate the cytosolic volume, experiments were first performed either with the usual media (5.6 mM glucose) or with glucose-depleted media. There was no difference between these two conditions as far as the 3-OMDG equilibrium time and the intracellular space occupied by the 3-OMDG were concerned (data not shown), as was previously observed in glial cells (25). Media containing 5.6 mM glucose were chosen because they correspond to the physiological concentrations used in our culture media. The effects of TSH and iodide on the 3-OMDG space of thyroid cells were studied under each of the culture conditions (Table 2). In the absence of iodide or TSH, the 3-OMDG space increased, whereas it decreased clearly when the two effectors were absent. The proportion of the space occupied by 3-OMDG in relation to the total cell volume was calculated (Table 2). In the control cells, this proportion was ~54%, which shows that, in our cells, the 3-OMDG probably accounted for the cytosolic volume of the cell. This proportion decreased in the absence of iodide but increased in the absence of TSH (Table 2). TSH and iodide, therefore, had antagonistic effects on the proportional size of the cytosolic fraction in thyroid cells.

                              
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Table 2.   Determination of cytosolic volume of porcine thyroid cells cultured as monolayers on collagen-coated filters

Moreover, the changes in the proportion of the cytosolic volume induced by TSH and iodide were correlated with the variations of the amount of total protein under each of the culture conditions. In the control cells, the total proteins amounted to 120.9 ± 2.8 pg (Fig. 4). The amount of total proteins increased in the absence of iodide and decreased in the absence of TSH, particularly when iodide was present. Thus TSH and iodide had antagonistic effects also on the protein content, which varied inversely with the proportion of cytosolic volume. However, when cytosolic volume was expressed as microliters per milligram of proteins, similar values were found for control cells and cells cultured in the absence of iodide or both TSH and iodide (~16 µl/mg), whereas they were increased twofold in the absence of TSH alone (32 µl/mg), corresponding to a 75% cytosolic proportion. Thus the absence of TSH in the presence of iodide modified the equilibrium between cytosol and protein content.


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Fig. 4.   Determination of protein content per porcine thyroid cell cultured as monolayers on collagen-coated filters. Cells were cultured as indicated in Fig. 1 legend. On day 14, the monolayers were scraped off and solubilized with 1% SDS, and the proteins were evaluated by performing a micro-bicinchoninic acid protein assay. Results are expressed in pg/cell. Data are means ± SE (n = 26). * Values differ significantly vs. control, P < 0.01.

TSH and iodide regulate apical-to-basal fluid transport in thyroid cells. The apical volumes decreased differently depending on the culture conditions; these decreases in the apical volumes corresponded to a net fluid transport in the apical-to-basal direction. We carefully measured the apical media in the culture chambers under each of the culture conditions for 6 days. In the control cells, the volume of apical medium decreased from 1 ml to 356 ± 19 µl. In the case of cells cultured in the absence of iodide, the volume of apical medium was lower (-37%) than that with the control cells, whereas in the absence of TSH, the volume of the apical medium was greater either with (37%) or without (46%) iodide than that in the control cells. Such changes in the volume of the apical medium were also observed when 8-chloro-cAMP was added to the basal medium instead of TSH (results not shown), as was previously reported (5). After 5 days, the volumes of apical media became stabilized, which shows that the effects of TSH and iodide on the net fluid transport reached equilibrium. We determined the fluid flux rate (µl · h-1 · 106 cells-1) corresponding to the slopes obtained by performing linear regression analysis on the time-dependent apical volumes (Fig. 5). In the absence of iodide, this rate increased by 27%, whereas in the absence of TSH, it decreased by 27% with iodide and by 37% without iodide. These results show that the absence of iodide increases net fluid transport, whereas the absence of TSH decreases it. These findings demonstrate that TSH and iodide have antagonistic effects on apical-to-basal net fluid transport.


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Fig. 5.   Determination of net fluid transport in the apical-to-basal direction in porcine thyroid cells cultured as monolayers on collagen-coated filters. Cells were cultured as indicated in Fig. 1 legend. On day 8, apical media were changed: 1 ml of B-DMEM was introduced into each apical compartment while 2.3 ml of DMEM was placed in each basal compartment. From day 8 onward, apical media were carefully measured after 24, 48, 72, and 120 h in cells cultured with TSH and iodide (), with TSH and without iodide (black-triangle), without TSH and with iodide (), without TSH and without iodide (triangle ). Linear regression analysis of time-dependent apical volumes, 5 days. Rates of fluid transport (f, in µl · h-1 · 106 cells-1) are expressed as slopes ± SD near each curve (n = 16 for each time).

As a further investigation, the correlations between the results were summed up and expressed as percentages of the control values (Table 3). Table 3 clearly shows that, in the absence of iodide but with TSH, the total cell volume, cytosolic volume, and protein level increased, as did the net fluid transport. In the absence of TSH but with iodide, only the cytosolic volume increased, whereas the other parameters decreased, particularly the protein content. Interestingly, in the absence of both TSH and iodide, the cell parameters amounted to ~80% of the control values, probably reflecting the existence of a "quiescent state"; in this state, the concentration of protein per picoliter of total cell volume did not differ from that recorded in the control cells (~32 pg/pl).

                              
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Table 3.   Summary of results


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Porcine thyroid cells cultured as monolayers on permeable collagen-coated filters were used to study cell volume regulation by TSH and iodide, the two main effectors of thyroid cells. In cells obtained in this way, both the epithelial phenotypes and the metabolic functions are preserved compared with in vivo thyroid cells. This culture system was suitable for this study, because it makes it possible to observe the effects of TSH deprivation on the physiology of thyroid cells and gives access to the apical medium (the colloid-like space). It was used here to measure the volume of living cells by performing confocal laser-scanning microscopy and computer-assisted 3-D reconstitution. We had first used calcein as a marker to label the total intracellular space. Unfortunately, calcein was very rapidly extruded from the TSH-stimulated cells. It has been reported that calcein was also actively extruded from other cell types by transporters that carry organic anions such as multidrug resistance-associated protein (MRP), and this ABC transporter was expressed in thyroid follicular cells (35). Thus calcein was not suitable for measuring the total volume of thyroid cells. We therefore used octadecylrhodamine B, which labels plasma membranes, to determine the total volume of living cells.

In this study, we quantified TSH and iodide regulation of thyroid cell volumes. This is the first time that the values of the total volume of living thyroid cells have been determined under a variety of stimulation conditions. The results clearly show that, under our culture conditions, only the cell height varied. The values of the cell heights obtained on cells cultured with TSH and without iodide were similar to those recorded in vivo on rat thyroid follicular cells under iodide deficiency (31): 16.5 vs. 20 µm, respectively. Those authors observed a decrease in cell height when rats were refed with iodide and therefore were in a state corresponding to our culture conditions with TSH and iodide. The absence of TSH decreased the cell volume in the same way whether or not iodide was present. When TSH was replaced by a cAMP analog, similar variations in cell volume were observed. TSH and iodide were also found to regulate the cytosolic volumes and the total amount of proteins without affecting the number of cells. In the absence of iodide, the absolute cytosolic volume increased, but its proportion vs. the total cell volume decreased, showing an increase in the protein content when TSH stimulation was applied alone, partly by increasing the endoplasmic reticulum and the intracellular organelles (10, 31). In the absence of TSH but with iodide, the decrease in protein content was associated with an increase in the absolute cytosolic volume and its proportion vs. the total cell volume. Now it is well known that, in the presence of TSH, iodide decreases TSH-stimulated protein synthesis via the effects of 2-iodoaldehydes, which decrease adenylylcyclase activity (2). However, we also observed a decrease in the protein level in the presence of iodide and the absence of TSH, which was found to be necessary for the Na+/I- symporter to be activated and, consequently, for the formation of the 2-iodoaldehydes to occur. It is possible that, under our culture conditions, iodide influx may have occurred only when traces of TSH were present in the serum of the basal medium, and this hypothesis still remains to be investigated. Taking our data as a whole, two states were found to be relatively "well-balanced", that occurring when the cells were regulated either by both TSH and iodide (control cells) or without TSH and without iodide; this last state was characterized by values of the parameters amounting to ~80% of those recorded in the control cells. The other two states, namely that without iodide or that without TSH, which may correspond to pathological conditions, can be said to be "unbalanced" states, because the proportions of the cytosolic volumes and protein content were inverted, particularly in the absence of TSH. All these findings suggest that iodide and TSH control the total cell and cytosolic volumes, as well as the protein content, by exerting antagonistic effects.

In comparison with the control cells, the apical medium decreased in the absence of iodide and increased in the absence of TSH, which shows that the net fluid transport was also regulated in opposite ways by iodide and TSH. The changes in apical volume occurred during the first few days of TSH and iodide action, probably before reaching equilibrium. Our results on the effects of TSH are in good agreement with previous data showing that both TSH and the cAMP analog increased the height of the domes that developed when cell monolayers were cultured on a plastic support (3). This dome formation process was also dependent on fluid transport in the apical-to-basal direction. Although the cultured thyroid epithelium is tight (29), a slight fluid transport at the paracellular level is not excluded. Moreover, transcytosis, which was previously observed to be weak (1% during 48 h) in TSH-stimulated cells (5), could represent only a minor part of the apical-to-basal fluid transport.

The changes in cell volume occurring in response to hormones and/or other effectors are correlated with the regulation of water transport and closely associated ion exchanges through both the apical and the basolateral membranes of epithelial cells (15, 17). More specifically, Na+, K+, and Cl- transport are involved in the changes in cell volume (24). In the thyroid gland, the regulation of cell volume as well as colloidal space effected by TSH and iodide may also be mediated via Na+, K+, and Cl- exchanges; TSH via cAMP increases basolateral Na+-K+-ATPase activity (28, 30) and apical Na+ channel conductance (3, 29), resulting in the extrusion of Na+ ions from the cells and the uptake of K+ ions on the basolateral side. TSH also increases the secretion of Cl- ions, mainly by controlling the basolateral Na+-K+-2Cl- symporter (4, 38) and apical Cl- channels (4). At the apical membrane, Cl- and Na+ exchanges may also be regulated by the cystic fibrosis transmembrane conductance regulator (CFTR) via cAMP, as previously described in airway and colonic epithelial cells (26, 34), because it has been established that CFTR is present in thyroid cells (11). The movement of Na+ in the apical-to-basal direction was probably associated with the basal-to-apical secretion of Cl- as well as with the transepithelial apical-to-basolateral fluid transport (4, 38). Moreover, the fluid transport may also depend on the expression of water channels called aquaporin-2, as observed in kidney epithelial cells, where this protein is regulated by vasopressin via cAMP (22). Hormonal control of these ionic and water transport processes has been found to be cAMP dependent in several types of epithelial cells. In our thyroid cells, any changes in the intracellular cAMP pool triggered by TSH or iodide may therefore affect the total cell volume, the proportion of cytosol, and the protein level, as well as the size of the colloid-like space. Consequently, the opposite effects of TSH and iodide on the parameters studied here may be mediated via control of adenylylcyclase activity.

Antagonistic effects of TSH and iodide on thyroid cell and colloid volumes have also been observed in in vivo studies on both induced animal pathologies and human pathologies. Eliminating iodide from the diet of rats resulted in the development of goiter caused not only by the absence of iodide but also by an increase in the serum TSH level. This goiter was accompanied by a decrease in the colloidal space, which may have reflected an increase in the net fluid transport. Restoring iodide reduced the hypertrophy of the cells and increased the colloidal volume, probably by decreasing the net fluid transport (7, 31). In human pathologies, numerous authors have reported that goiter size can be reduced by application of an iodide treatment, which results in changes in the size of cells and follicles (14).

When our cells were cultured without TSH, the total cell volume decreased, showing that the absence of TSH had an atrophic effect on the thyroid cells, whereas the volume of the apical media increased. In human pathological states, this atrophic effect may also be due to the TSH effects being either decreased or abolished; these can occur for two major reasons, namely a dysfunction of the hypothalamic-pituitary axis (8) or a dysfunction of the TSH receptor induced either by gene mutations (1) or by the presence of autoantibodies blocking this receptor (36). The absence of either iodide or TSH in our cultured cells can therefore be said to have mimicked the state of imbalance present in pathological thyroid conditions.

In conclusion, the present results show that TSH and iodide regulate thyroid cell volumes and fluid transport in opposite ways, probably via up- and downregulation of adenylylcyclase activity, respectively. However, interrelations exist between these antagonistic effects; by upregulating the iodide uptake and transport, TSH controls its own antagonist, which in turn slows down the effect of TSH. TSH therefore exerts self-control via the iodide regulation process. In vivo, these regulatory mechanisms might maintain a steady state of the intracellular concentration of ions and other molecules through the control of the cell volumes as well as of the fluid transport.


    ACKNOWLEDGEMENTS

The authors thank Dr. P. Carayon for interest in this study and the Institut Federatif de Recherche. Jean Roche is thanked for the use of the confocal microscope. D. Cauvi is the recipient of a grant from the Ministère de l'Education Nationale de la Recherche et de la Technologie.


    FOOTNOTES

This study was supported by INSERM (U38), CNRS (SDI 401038, UPRESA 6032) and the Université de la Méditerranée.

Address for reprint requests and other correspondence: O. Chabaud, INSERM U38, Faculté de médecine, 27 Bd Jean Moulin, 13385 Marseille, cedex 05, France (E-mail: Odile.Chabaud{at}medecine.univ-mrs.fr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 2 December 1999; accepted in final form 6 April 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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