Sulfate transport in porcine thyroid cells. Effects of thyrotropin and iodide

David Cauvi, Marie-Christine Nlend, Nicole Venot, and Odile Chabaud

Institut National de la Santé et de la Recherche Médicale U555, Faculté de Médecine, Université de la Méditerranée, 13385 Marseille, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In porcine thyroid cells, thyroglobulin sulfation is controlled by thyrotropin (TSH) and iodide, which contribute to regulating the intracellular sulfate concentration, as we previously established. Here, we studied the transport of sulfate and its regulation by these two effectors. Kinetic studies were performed after [35S]sulfate was added to either the basal or apical medium of cell monolayers cultured without any effectors, or with TSH with or without iodide. The basolateral uptake rates were about tenfold higher than the apical uptake rates. TSH increased the basolateral and apical uptake values (by 24 and 9%, respectively, compared with unstimulated cells), and iodide inhibited these effects of TSH. On the basis of results of the pulse-chase experiments, the basolateral and apical effluxes appeared to be well balanced in unstimulated cells and in cells stimulated by both TSH and iodide: ~40-50% of the intracellular radioactivity was released into each medium, whereas in the absence of iodide, 70% of the intracellular radioactivity was released on the basolateral side. The rates of transepithelial sulfate transport were increased by TSH compared with unstimulated cells, and these effects decreased in response to iodide. These results suggest that TSH and iodide may each control the sulfate transport process on two sides of the polarized cells, and that the absence of iodide in the TSH-stimulated cells probably results in an unbalanced state of sulfate transport.

apical and basolateral uptake; efflux; transepithelial transport; hormonal regulation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE TRANSPORT OF INORGANIC SULFATE has been studied in some epithelial tissues, particularly in the kidney, liver, small intestine, and endometrium, and it has been correlated with other ionic transport processes. The sulfate uptake and efflux processes occurring at the apical pole or on the basolateral side of epithelial cells serve to transport sulfate ions, either in the apical-to-basal direction or in the opposite direction.

Some studies have been carried out on the regulation of sulfate transport, but few data have been published so far showing the existence of a relationship between the regulation of sulfate transport and the regulation of macromolecule sulfation. In the normal cellular state, all of the anions and cations are balanced, and their transport, as well as their turnover, depends on various factors, such as variations in the cell volumes, the levels of circulating ions, and the hormonal stimulation exerted. The cotransporter Na/Si involved in renal reabsorption, which mainly contributes to maintaining the homeostasis of circulating sulfate, depends on some ionic serum levels: both dietary sulfate (24) and chronic K+ deficiency (25) are known to lead to a downregulation of the NaSi-1 expression. This transporter is subject to hormonal regulation (22, 27). It has been reported in some studies that insulin-like growth factor I (IGF-I) stimulates the sulfate uptake occurring in the branchial cartilage of the eel (15) and coho salmon (23), as well as both the uptake of sulfate and the sulfation of proteoglycans present in the cartilage of chicken embryos (20). Moreover, growth hormone intensifies the effects of IGF-I on the sulfate uptake without having any stimulating effects of its own. An increase in the sulfate uptake occurs in endometrial epithelial cells in response to progesterone in the presence of estradiol (3), and this response was found to be mediated by cAMP (5). This increase in the sulfate uptake was correlated with an increase in the protein sulfation levels (43). In a pathological state (type 1B achondrogenesis), an impaired sulfate transport process occurring in chondrocytes was correlated with an undersulfation of proteoglycans (37), but in cystic fibrosis airway epithelial cells, the hypersulfation of mucins was not due to a defective sulfate transport mechanism (26).

Only a few studies, therefore, have dealt so far with the hormonal regulation of both sulfate transport and macromolecule sulfation. However, we recently reported (31) that in porcine thyroid cells, the sulfation of thyroglobulin (Tg), the thyroid hormone precursor, and particularly that of tyrosine, are downcontrolled by thyrotropin (TSH) and that this effect was slightly regulated by iodide. The sulfated tyrosines have been found to be involved in thyroid hormone synthesis (30). TSH may therefore control thyroid hormone synthesis via the sulfation of hormonogenic tyrosines. Tg sulfation may in fact be regulated by TSH and iodide at each step in the sulfation process, and we observed that TSH and iodide control the intracellular sulfate concentration by regulating the cytosolic volumes (8). These results therefore suggest that the sulfate transport may also be regulated by TSH and iodide.

Sulfate transport has not been studied so far to our knowledge in the thyroid gland, although pendrin, which has been identified as a sulfate transporter by its sequence signature, has been described in thyroid cells (17). However, the pendrin located on the apical membrane of cells (6, 38) has been found to take up iodide and chloride but not sulfate (40).

In the present study, we investigated the sulfate transport process in thyroid cells and its regulation by TSH and iodide, given that the absence of either iodide, or both TSH and iodide, corresponds to pathological thyroid states involving variations in the cell volumes and fluid transport (9). These studies were carried out on porcine thyroid cells cultured on collagen-coated filters, a culture system in which all of the specific thyroid functions are present: we previously established that TSH regulates the Tg mRNA levels and the rate of protein synthesis (12) via cAMP control (10) as well as Tg exocytosis into the apical medium (11), Tg iodination (18), and hormonosynthesis (19). The rate of protein synthesis decreased in response to supraphysiological concentrations of iodide (18, 19).

The results of this study show that TSH and iodide control the sulfate uptake and efflux processes at the apical pole and on the basolateral side, as well as its transepithelial transport, via their opposite effects on these processes. Only the absence of iodide gave rise to an unbalanced efflux state: the apical efflux decreased, while the basolateral efflux increased, as did the apical-to-basolateral transport, which was correlated with the net fluid transport occurring in the apical-to-basal direction under the same culture conditions.


    MATERIALS AND METHODS
TOP
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 (12). The isolated cells were suspended (2.5 × 106/ml) in phenol red-free DMEM containing 1 g/l glucose (GIBCO-BRL, Cergy Pontoise, France), and the cellular suspension (1 ml) was put inside the insert, corresponding to the apical compartment when the epithelial cell monolayer was established. The same medium was introduced outside the insert into a well of a 6-well plate corresponding to the basal compartment (see this culture system illustrated in Fig. 7). The cell cultures were 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 monolayers to serum protein was checked in the apical media by performing a colorimetric protein assay, and only tight monolayers were kept (12). From day 6 onward, the basal media were supplemented or not with 100 µU/ml thyrotropin (b-TSH, Calbiochem, San Diego, CA). From day 8 onward, potassium iodide (KI) 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) (19). Up to day 14, only the basal media were changed every 2 or 3 days; the sulfate concentration was ~0.8 mM in the apical medium (the usual culture medium concentration) and 1.2 mM in the basal medium (additional sulfate was provided by serum, antibiotics, and vitamins). On day 14, the apical and basal media were changed: the sulfate concentration was decreased to 0.4 mM in each medium (and checked as indicated in Ref. 8) to keep a physiological concentration of external sulfate and prevent any excessive isotopic dilution. MgSO4 was therefore partly replaced by MgCl2 so as to maintain the same Mg2+ concentration. At this stage, the cells were used to study the sulfate uptake and efflux as well as the transepithelial transport.

Basolateral and apical uptake of [35S]sulfate. Cells were incubated with inorganic [35S]sulfate in aqueous solution (1,000 Ci/mmol Sulphur-35, Amersham, Little Chalfont, UK) at a concentration of 20 µCi/ml added to the basal or apical medium for various times up to 60 min. At each uptake time, the apical and basal media were removed, and cells were washed promptly three times with ice-cold PBS (containing 1 mM sodium sulfate) and stored at -20°C. Cells were then solubilized in a 1% SDS solution, and the radioactivity of aliquots was measured in a liquid scintillation counter (Tricarb 2100TR, Packard Instrument, Meriden, CT). Results were expressed as dpm/106 cells or as dpm/mg of protein.

Uptake of [35S]sulfate in the presence of inhibitors. Experiments were performed on TSH-stimulated cells cultured either with or without iodide, as indicated above, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) was added to either the basal or the apical medium at a final concentration of 1 mM just before the [35S]sulfate was added to the same compartment. Sodium thiosulfate (1, 3, and 5 mM, final concentrations) was also used to inhibit the sulfate uptake under the same conditions as with DIDS.

Basolateral and apical efflux of [35S]sulfate. Cells were incubated with [35S]sulfate, which was added at a concentration of 70 µCi/ml to the basal medium for 60 min (pulse). The apical and basal media were then removed, and cells were washed promptly three times with ice-cold PBS. Some inserts were extracted (as described in the uptake experiments) to determine the intracellular radioactivity at zero efflux time. The other cells were incubated with the usual apical (B-DMEM) and basal media (DMEM with 10% calf serum), each containing 0.4 mM sulfate. Aliquots were taken from the two media at various chase times, and the radioactivity was counted. The effluxes were then expressed as the percentage of radioactivity released into either the apical or basal medium at time t vs. the cellular radioactivity present at time 0.

Transepithelial [35S]sulfate transport. Cells were incubated with [35S]sulfate (20 µCi/ml), added either on the basolateral side or to the apical pole. At various incubation times up to 60 min, aliquots were taken from the opposite medium (the apical one when radioactive material was added to the basal medium and vice versa), and the radioactivity was counted. Results were expressed as picomoles of sulfate/106 cells released into the media.

DNA and protein assays. DNA content was evaluated (42) by measuring the DNA fluorescence (Hoechst 33258, Roche Diagnostics, Meylan, France). Samples and standards were both prepared in the same way, and the fluorescence was measured in 96-well plates with a PC-controlled fluorometer (Fluostar+, Salzburg, Austria) by the Biolyse 1.7 program. The number of cells per filter was deduced from these data on the basis of the fact that 7.8 µg of DNA corresponded to 106 cells (9).

Protein was quantified by performing the micro-bicinchoninic acid protein assay (Pierce, Rockford, IL).

Statistical analysis. The number of picomoles of sulfate was calculated from the specific radioactivity (dpm/pmol) determined in each medium to which the radioactivity had been added. The data, expressed as picomoles of sulfate per 106 cells or as picomoles per milligram of protein, are means ± SE for n values. The initial values of the uptake and efflux rates were determined using nonlinear regression analysis (Graph Pad Prism, version 2). Student's t-test was carried out using a 5% statistical significance limit.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Sulfate transport was studied in porcine thyroid cells cultured on collagen-coated filters. This culture chamber system made it possible to have access independently to the apical pole and to the basolateral side of polarized epithelial cells. The basolateral and apical transport of sulfate was investigated under three different conditions of stimulation by TSH and iodide, corresponding to three culture conditions: unstimulated cells (-TSH -KI), TSH-stimulated cells (+TSH -KI), and TSH/KI-stimulated cells (+TSH +KI). [35S]sulfate was added to either the basal or apical medium in the culture chambers, and the sulfate uptake and efflux processes were studied, as well as the transport processes from the basal to apical medium and vice versa.

Basal and apical uptakes. When [35S]sulfate was in contact with the basolateral membranes, the sulfate uptake values measured in the cell monolayers plateaued from an incubation time of 10 min onward, but 80-90% of this quantity was already present in the cell monolayers after 2 min (Fig. 1A), whereas when [35S]sulfate had access to the apical membranes, a linear pattern of uptake occurred from incubation times of 2-60 min (Fig. 1B) and probably extended beyond this time. On both sides of the cells, the uptakes were faster between 0- and 2-min incubation time than subsequently. However, lower sulfate uptakes were recorded on the side of the apical membrane than on that of the basolateral membrane.


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Fig. 1.   Basolateral and apical sulfate uptake by porcine thyroid cells. Thyroid cell cultures forming monolayers on collagen-coated filters were prepared. From day 6 onward, thyrotropin (TSH) was added (100 µU/ml), or not, to the basal media, which were subsequently changed every 3 days with maintenance of the same TSH concentration. From day 8 onward, potassium iodide (KI, 0.5 µM) was added daily, or not, to the basal media. On day 14, the media were changed, and cells were incubated with [35S]sulfate (20 µCi/ml) added either to the basal media (A) or to the apical media (B). Kinetic experiments were performed up to an incubation time of 60 min. At each time, cells were rapidly washed 3 times with ice-cold PBS. They were then solubilized in a 1% SDS solution, and the radioactivity of aliquots was counted. Results were expressed as pmol sulfate/106 cells. , Unstimulated cells; , TSH-stimulated cells; open circle , TSH/KI-stimulated cells. A: basolateral uptake; values are means ± SE (n = 15, 5 experiments); B: apical uptake; values are means ± SE (n = 9, 3 experiments). A:  and open circle  did not differ significantly, 0.05 < P < 0.2. B:  and  differed significantly, P < 0.01.

Both the basal sulfate uptake (Fig. 1A), expressed as picomoles of sulfate per 106 cells, and the apical uptake (Fig. 1B) increased more in the cells exposed to TSH than in the unstimulated cells. When iodide was added to TSH-stimulated cells, the sulfate uptake returned on the basolateral side to the unstimulated cell level (80% of the TSH-stimulated cell level, Fig. 1A) and also decreased on the apical side (75% of the TSH-stimulated cell level, Fig. 1B), reaching a level below that of unstimulated cells.

The amount of cellular protein present in thyroid cells depends on the stimulation conditions: TSH increases protein synthesis, whereas iodide decreases this effect (9). Consequently, expressing the uptakes as picomoles of sulfate per milligram of protein takes the effects of TSH and iodide on protein synthesis into account. The basal sulfate uptake, for example, was found to decrease in the presence of TSH to ~80% of the unstimulated cell value and was not affected by iodide (Fig. 2A). The apical uptake expressed per milligram of protein also decreased in response to TSH and decreased slightly more in response to iodide (Fig. 2B). This method of expressing the data showed that the basal and apical uptakes were greater in unstimulated cells than in TSH-stimulated or TSH/KI-stimulated cells. The control exerted by TSH and iodide on both apical and basal sulfate uptake may therefore be partly correlated with the regulation of protein synthesis.


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Fig. 2.   Basolateral and apical sulfate uptake by porcine thyroid cells. Procedures and data are as in Fig. 1, but results are expressed as pmol sulfate/mg cellular protein. , Unstimulated cells; , TSH-stimulated cells; open circle , TSH/KI-stimulated cells. A: basolateral uptake; values are means ± SE (n = 15, 5 experiments); B: apical uptake; values are means ± SE (n = 9, 3 experiments). A:  and open circle  did not differ significantly, 0.05 < P < 0.2.

Basal and apical effluxes. The effluxes were studied in pulse-chase experiments. After a 60-min pulse in the presence of [35S]sulfate added to the basal medium, cells were washed rapidly and incubated without radioactive sulfate. The radioactive sulfate released into the basal and apical media was measured after several chase times. The radioactivity released was expressed as a percentage of the intracellular radioactivity present after a 60-min pulse.

After a 10-min chase time, the basal efflux plateaued at 45 and 40% of the intracellular radioactivity recorded with unstimulated cells and TSH/KI-stimulated cells, respectively (Fig. 3A). In the case of TSH-stimulated cells, this equilibrium was reached after a 20-min chase time, when 70% of the [35S]sulfate had been released (Fig. 3A).


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Fig. 3.   Basolateral and apical sulfate efflux. Cell cultures were prepared as in Fig. 1. On day 14, media were changed, and cells were incubated with [35S]sulfate (70 µCi/ml) added to the basal media. After 60 min of pulse labeling, cells were washed with ice-cold PBS. Cells were then incubated with the usual apical and basal media, each containing 0.4 mM sulfate. Aliquots were taken from the two media at various chase times, and radioactivity was counted. Effluxes were expressed as percentage of radioactivity released into either the apical or basal medium at time t vs. cellular radioactivity present at time 0. , Unstimulated cells; , TSH-stimulated cells; open circle , TSH/KI-stimulated cells. A: basal efflux; values are means ± SE (n = 15, 5 experiments). B: apical efflux; values are means ± SE (n = 15, 5 experiments). A:  and open circle  differed significantly, P < 0.05. B:  and open circle  differed significantly, P < 0.05, except for the 20-min release time (P > 0.05).

In the case of the apical efflux, the equilibrium states were reached later (Fig. 3B): 45% of the [35S]sulfate present in unstimulated cells and 50% of the [35S]sulfate present in TSH/KI-stimulated cells were released after 20 and 30 min of chase, respectively. In TSH-stimulated cells, only 25% of the intracellular [35S]sulfate was released after a 60-min chase (Fig. 3B).

Similar proportions of sulfate were therefore released in the same way by both unstimulated cells and TSH/KI-stimulated cells through the basolateral and apical membranes. Only TSH-stimulated cells released different amounts of intracellular sulfate on the apical and basolateral sides, showing a higher rate of sulfate efflux in the basal direction. The absence of iodide, therefore, seems to have disturbed the balance between the two sulfate efflux processes.

Sulfate uptake in the presence of anion inhibitors. Anion inhibitors and radioactive sulfate were added to the same compartment at the same time. The effects of DIDS on the uptake of [35S]sulfate were studied in TSH-stimulated and TSH/KI-stimulated cells. The basal uptakes were not affected by 1 mM DIDS under these two culture conditions (Fig. 4, A and B). However, 1 mM DIDS increased (about twofold) the apical uptakes in TSH-stimulated cells cultured in either the absence (Fig. 4C) or the presence (Fig. 4D) of iodide. In Fig. 4C, the effects of DIDS can be seen to have appeared after an incubation period of 5 min, which suggests that the absence of iodide might affect the action of DIDS. DIDS therefore did not inhibit the basal and apical sulfate uptake, which suggests that, in the thyroid gland, the basal and apical sulfate transporters might be a Na-dependent sulfate transporter, such as NaSi-1 in kidney, which is not inhibited by DIDS but by sodium thiosulfate, as observed by Perego et al. (35).


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Fig. 4.   Effect of DIDS on basolateral and apical sulfate uptake. Cell cultures were prepared as in Fig. 1. On day 14, media were changed and cells were incubated with DIDS (1 mM) and [35S]sulfate (20 µCi/ml) added to either the basal medium (A and B) or the apical medium (C and D). Kinetic experiments were performed for 30 min. At each incubation time, cells were rapidly washed 3 times with ice-cold PBS. They were then solubilized in a 1% SDS solution, and the radioactivity of aliquots was counted. Results were expressed as pmol sulfate/106 cells. , TSH-stimulated cells; triangle , TSH-stimulated cells + DIDS; open circle , TSH/KI-stimulated cells; black-triangle, TSH/KI-stimulated cells + DIDS. A and B: basolateral uptakes; values are means ± SE (n = 10, 3 experiments). C and D: apical uptakes; values are means ± SE (n = 6, 2 experiments). A:  and triangle  did not differ significantly, P > 0.9. B: open circle  and black-triangle did not differ significantly, 0.05 < P < 0.1.

To check this possibility, several concentrations of sodium thiosulfate were used to inhibit the sulfate uptake. The basal uptakes by unstimulated cells were not inhibited, even by 5 mM thiosulfate (Fig. 5A). A slight enhancement, which was not significant, was observed with 1 mM thiosulfate in TSH-stimulated cells and with 3 mM in TSH/KI-stimulated cells (Fig. 5A). The apical uptakes by unstimulated cells were not affected by 1 mM thiosulfate (Fig. 5B). However, with this concentration, a slight increase in the apical uptakes was observed in TSH-stimulated as well as in TSH/KI-stimulated cells (Fig. 5B).


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Fig. 5.   Effect of sodium thiosulfate on basolateral and apical sulfate uptake. Cell cultures were prepared as in Fig. 1. On day 14, media were changed, and cells were incubated with sodium thiosulfate and [35S]sulfate (20 µCi/ml) added to either the basal media (top) or the apical media (bottom). Cells were rapidly washed 3 times with ice-cold PBS. They were then solubilized in a 1% SDS solution, and radioactivity of aliquots was counted. Results were expressed as pmol sulfate/106 cells. Top: values are means ± SE (n = 10, 3 experiments); they did not differ significantly. Bottom: values are means ± SE (n = 8, 2 experiments); they differ significantly, $P < 0.01; §P < 0.1.

No inhibition of the sulfate uptake was therefore observed with either DIDS or thiosulfate. The increase observed in the apical uptake may have been due either to the inhibition of the apical sulfate efflux or to the blocking of the transport of other anions possibly involved in the sulfate uptake process.

Transepithelial sulfate transport. Transepithelial transport involves both the paracellular transport through the tight junctions and the transcellular transport, which includes the apical and basolateral uptake and efflux. To study the transepithelial transport of sulfate from the basal to apical medium and vice versa, kinetic studies were performed in which [35S]sulfate was added to either the basal or apical medium, and the radioactivity was measured in the opposite medium. The release of sulfate into the apical medium (Fig. 6A) as well as into the basal medium (Fig. 6B) gave a linear curve as a function of the incubation time. A continuous flow therefore occurred, but the rates of release differed, depending on the culture conditions (Fig. 6, A and B). The lowest rate of sulfate transport was observed in unstimulated cells. The release into the apical medium increased by 120% in TSH-stimulated cells compared with unstimulated cells, and this effect of TSH decreased by 28% in the presence of iodide; the difference was more marked in the case of the release into the basal medium, which increased by 440% in the presence of TSH, and this effect decreased by 68% in response to iodide. These results suggest that the sulfate turnover was enhanced by TSH in the absence of iodide. In addition, whereas both unstimulated cells and TSH/KI-stimulated cells released very similar amounts of sulfate into the opposite media, TSH-stimulated cells released more sulfate (2.3 times more) into the basal medium than into the apical medium.


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Fig. 6.   Transepithelial transport of sulfate. Cell cultures were prepared as in Fig. 1. On day 14, media were changed, and cells were incubated with [35S]sulfate (20 µCi/ml) added to either the basal medium (A) or the apical medium (B). Kinetic experiments were performed, and aliquots were taken from the medium into which the radioactive material had been released and were counted. , Unstimulated cells; , TSH-stimulated cells; open circle , TSH/KI-stimulated cells. A: apical efflux; values are means ± SE (n = 15, 5 experiments). B: basal efflux; values are means ± SE (n = 9, 3 experiments).

Estimated rate of sulfate transport. Initial rate values were calculated in all of the kinetic experiments performed (Table 1). The basal uptake was found to be the faster process under all of the culture conditions. The apical uptake was slower (~15-20 times less) than the basal uptake. In the pulse-chase experiments, the initial rates of the basal efflux were faster than those of the apical efflux: about three times faster in the case of both unstimulated and TSH/KI-stimulated cells, and ~15 times faster in that of TSH-stimulated cells. Only in TSH/KI-stimulated cells did apical uptake and apical efflux show similar initial rate values, suggesting that the apical exchanges might be controlled by iodide. Moreover, the initial rate values of the effluxes were higher in unstimulated cells than in TSH-stimulated cells cultured with or without iodide. Because the flow rates varied according to the stimulation conditions and the membranes of the polarized cells involved, the steady states of sulfate exchanges were therefore reached at various times (Fig. 1 and 3), except for the apical uptakes in which equilibrium was never reached within 60 min.

                              
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Table 1.   Initial rates and steady-state values of basolateral and apical uptake, efflux, and transepithelial transport of sulfate

At steady states, the basal uptake was still higher than the basal efflux, and apical efflux became greater than the apical uptake (Table 1). The effluxes on the basolateral side and at the apical pole became well balanced in unstimulated cells and scarcely greater at the apical pole in TSH/KI-stimulated cells, whereas in the case of the TSH-stimulated cells, the effluxes remained imbalanced after plateauing (Table 1, Fig. 3).

As far as the transepithelial sulfate transport between the two media was concerned, the initial efflux rates were slower than in the medium-to-cell and cell-to-medium exchanges (Table 1), except in the case of TSH-stimulated cells. However, because the flows were continuous and no equilibrium states had been reached within 60 min, much greater amounts of radioactive sulfate accumulated in the media in these experiments than in the pulse-chase experiments (Table 1).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In porcine thyroid cells cultured on collagen-coated filters, we studied the regulation of the sulfate uptake and efflux processes exerted by TSH and iodide on the basolateral side and at the apical pole, as well as their effects on the transepithelial transport process. Depending on the process and on the membrane involved, the sulfate transport was controlled differently by TSH and iodide (Fig. 7). These processes were found to be well balanced in both cells costimulated by TSH and iodide and unstimulated cells. When the cell cultures were exposed only to TSH, an imbalance occurred in the sulfate transport process characterized by a decrease in the apical efflux and an increase in the basolateral efflux, as well as the transepithelial transport in the apical-to-basal direction (Fig. 7).


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Fig. 7.   Sulfate transports in porcine thyroid cells cultured on porous filters. The 3 culture conditions were shown with variations in height of cells, as well as in apical medium volume, previously observed in Ref. 9. Various movements of inorganic sulfate were summarized. : Apical uptake; : basolateral uptake; : apical efflux; : basolateral efflux; : transepithelial transport in basal-to-apical direction; : transepithelial transport in apical-to-basal direction. Width of arrows mimicked the relative amounts of sulfate transported.

In TSH/KI-stimulated cells (the normal state in vivo), the basal uptake was much higher than the apical uptake (Fig. 7). Most of the intracellular [35S]sulfate was therefore provided by the circulating sulfate, which entered the cells via the basolateral membranes. The intracellular sulfate subsequently left the cells via both the apical and basolateral membranes at different initial rates, but close amounts were released in the steady state. It was observed that the transepithelial sulfate transport from the basal-to-apical medium and from the apical-to-basal medium gave a linear curve, which increased without reaching a plateau within 1 h. The intracellular sulfate was therefore rapidly balanced, and the equilibrium between the two media was reached at a much later stage (~40 h; unpublished results). Equivalent amounts of sulfate were released into the basal and apical media in the pulse-chase experiments, as well as in the studies on transepithelial sulfate transport. These findings suggest that, in vivo, the sulfate present in colloid might be balanced with the circulating sulfate, possibly via both the transcellular and the paracellular pathways.

When the steady states were reached, the sulfate uptakes at the basolateral side, as well as the sulfate effluxes into the apical and basal media observed in unstimulated cells, were similar to those observed in the case of TSH/KI-stimulated cells. Therefore, when added together, TSH and iodide did not appear to really modify these processes.

The most striking features observed here focused on the cells stimulated only by TSH in the absence of iodide. The basal and apical uptakes (expressed as pmoles/106 cells) increased slightly in response to TSH compared with unstimulated cells, and iodide decreased these TSH-induced effects by ~30%. The effects of TSH on the basolateral uptake in the absence of iodide may be due either to short-term effects of the increase in the cAMP levels or to its effects mediated by the rate of protein synthesis. When the basolateral uptake was expressed as picomoles of sulfate per milligram of protein, a downregulation by TSH was observed. The constitutive proteins acting as sulfate transporters on the basolateral side may therefore be expressed relatively more in unstimulated cells, because TSH enhances the protein synthesis, especially that of the proteins specifically involved in thyroid function, such as Tg (12) via the cAMP production (10). It is well known that iodide decreases the TSH-stimulated cAMP level via the effects of 2-iodoaldehydes, which decrease adenylyl cyclase activity (33), and this emerged clearly from the pattern of protein synthesis observed under our culture conditions (9). When the basolateral uptakes were expressed in picomoles of sulfate per milligram of protein, no significant differences were observed between the two conditions of stimulation by TSH (with and without iodide). These data suggest that in the absence of iodide, TSH, by enhancing the protein synthesis, may also increase the number of sulfate transporters and appears to upregulate the sulfate uptake expressed per 106 cells. Iodide was found to decrease the apical uptake in the steady state (with both methods of data expression) as well as the initial rate values, and its effect on protein synthesis does not suffice to explain the differences observed. This suggests that iodide may either compete with sulfate for access to the cells at the apical pole or regulate this access. However, the possibility that a short-term effect of cAMP on sulfate transport may have been involved cannot be ruled out: a short-term cAMP-mediated regulation of sulfate uptake was previously observed in glandular epithelial cells of guinea pig endometrium stimulated by progesterone in the presence of estradiol (5).

In addition, the equilibrium observed between the basal and apical efflux in TSH/KI-stimulated cells was disturbed in TSH-stimulated cells: in the pulse-chase experiments, when steady states were reached, approximately three times more sulfate was released into the basal medium than into the apical medium. The apical and basal transepithelial transport increased conspicuously in TSH-stimulated cells compared with unstimulated and TSH/KI-stimulated cells. Therefore, both the intracellular and paracellular transport processes may have been enhanced, probably due to an enhancement of the sulfate turnover. Hence, in the TSH-stimulated cells, the absence of iodide tended to disturb the balance in each of the two sulfate transport systems. TSH decreases the transepithelial resistance (28, 34) via the action of cAMP (29), resulting in an increase in the permeability of the tight junctions. Because the paracellular transport might increase in the same way in both directions, the imbalanced state of the sulfate transport might be due to variations in the transcellular process. These data are in line with previous findings on the same cell culture system (9, 12). In the absence of iodide, the net waterflux was increased (by ~30%) in the apical-to-basal direction (9), as was the basal efflux and the transepithelial sulfate transport in this direction. The net fluid transport in the apical-to-basal direction was regulated in opposite ways by TSH and iodide (9), likely via their opposite effects on the cAMP level (33). Moreover, in thyroid gland, TSH via cAMP increases the bidirectional transport of ions, i.e., the Na+ transport occurring via both the apical Na+ channel conductance (7, 34) and the basolateral Na+-K+-ATPase activity (32, 36), resulting in the apical uptake of Na+ ions and their extrusion from the cells on the basolateral side and the Cl- transport in the opposite direction (2). When sulfate was transferred from the apical to the basal medium, it may thus have become part of the net fluid transport process and have been cotransported with Na+ ions.

The noninhibitory effects of DIDS on the sulfate uptake suggest that the sulfate uptake may depend on Na+ ions, as was found to occur in the case of the NaSi-1 sulfate cotransporter (35). However, this cotransporter was not found to be present in the thyroid gland (4), and in addition, the sulfate uptake was not inhibited by sodium thiosulfate, contrary to what occurred in the case of NaSi (14). The Na+/iodide symporter (NIS) present on the basolateral membranes of thyroid cells (13) was not inhibited by DIDS (1), but the hypothesis that iodide and sulfate might both be transported by NIS can be ruled out, because NIS was found to carry only traces of sulfate when it was expressed in Xenopus laevis oocytes (16). Recent works on lactating rat mammary explants or acini showed that iodide increased in a dose-dependent manner the sulfate efflux, which was sensitive to DIDS, suggesting the presence of a sulfate-iodide exchange system likely located on the basolateral side of mammary epithelium (41). In thyroid cells, the basolateral sulfate uptake, as well as probably the efflux, was not sensitive to DIDS, suggesting that such an anion exchanger might not be detected on the basolateral membrane in our culture conditions, either with or without iodide.

The apical uptake was increased approximately twofold by DIDS in TSH-stimulated cell cultures with and without iodide, possibly due to the inhibition exerted by DIDS on the apical sulfate efflux. In a similar porcine thyroid cell culture system, DIDS has been found to increase the apical uptake of iodide approximately twofold and to inhibit its apical efflux (1). These findings suggest that iodide and sulfate might have a common apical transporter. However, DIDS may have increased the apical sulfate uptake by affecting other anion transport processes involved in the transport of sulfate.

The sulfate uptake and efflux processes occurring on the basolateral side therefore depend on basolateral transporters, which are probably different from the apical transporters. Pendrin, a thyroid-specific protein located on the apical membrane (6, 38), shows a sequence that is specific to sulfate transporters (17). In Xenopus laevis oocytes, the pendrin cDNA expressed a protein that allowed the entry of iodide and chloride but not that of sulfate (40), and this protein is also able to mediate chloride/formate exchanges (39). In addition, the apical sulfate uptake is not impaired in Pendred syndrome thyrocytes (21).

All of these results suggest that TSH and iodide, which regulate the sulfation of Tg (29), the thyroid hormone precursor, may control the influx and efflux of sulfate, probably so as to maintain the intracellular sulfate concentration (8) required for efficient tyrosine sulfation, because sulfated Tg tyrosines are involved in hormone synthesis (30). Moreover, the absence of iodide in TSH-stimulated cells, which occurs in a well known thyroid pathology, leads to a state of imbalance in the thyroid volumes, the sulfate transport processes, and the intracellular sulfate concentration, resulting in a final change in the level of Tg sulfation.


    ACKNOWLEDGEMENTS

We thank Prof. P. Carayon for interest shown in this study and Dr. C. Penel for helpful discussions and critical advice on this manuscript.


    FOOTNOTES

D. Cauvi is the recipient of a grant from the Ministère de l'Education Nationale de la Recherche et de la Technologie.

Present address of M.-C. Nlend: University of Miami, Pulmonary Division (R-47), 1600 NW 10th Avenue (Rosenstiel Medical and Science Bldg. R-7063A), Miami, FL 33136.

This study was supported by Institut National de la Santé et de la Recherche Médicale (U555), Centre National de la Recherche Scientifique (401038), and the Université de la Méditerranée.

Address for reprint requests and other correspondence: O. Chabaud, INSERM U555, Faculté de Médecine, Université de la Méditerranée, 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. Section 1734 solely to indicate this fact.

Received 7 March 2001; accepted in final form 20 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 281(3):E440-E448
0193-1849/01 $5.00 Copyright © 2001 the American Physiological Society




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