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Endocrinology Vol. 143, No. 2 476-483
Copyright © 2002 by The Endocrine Society

TRH-TSH-THYROID

Thyroid Hormone Export Varies among Primary Cells and Appears to Differ from Hormone Uptake

Francisco A. R. Neves, Ralph R. Cavalieri1, Luiz A. Simeoni, David G. Gardner, John D. Baxter, Bruce F. Scharschmidt, Noureddine Lomri and Ralff C. J. Ribeiro2

Department of Pharmaceutical Sciences, University of Brasília (F.A.R.N., L.A.S., R.C.J.R.), Brasília, DF 70910-900, Brazil; Veterans Affairs Medical Center (R.R.C.) and Metabolic Research Unit (D.G.G., J.D.B.), University of California, San Francisco, California 94143; Chiron Corp. (B.F.S.), Emeryville, California 94608; and Department of Biology, University of Cergy-Pontoise (N.L.), Paris, France

Address all correspondence and requests for reprints to: Dr. Ralff C. J. Ribeiro, Campus Universitário Darcy Ribeiro, Asa Norte, Department of Pharmaceutical Sciences, University of Brasília, Brasília, DF 70910-900, Brazil. E-mail: ralff{at}unb.br


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We characterized T3 efflux in primary cultures of cells derived from human placenta, neonatal rat cardiac myocytes, and rat inner medullary collecting ducts (IMCD). The T3 efflux rate was highest in placenta cells, followed by ventriculocytes, atriocytes, and IMCD cells. Verapamil reversibly blocked [125I]T3 efflux in these cells in a manner that correlated with their T3 efflux rate. Thus, verapamil inhibition of [125I]T3 efflux in placenta cells led to a 432% increase in the [125I]T3 content compared with 33% increase in IMCD cells. Several unlabeled iodothyronines, but not TRIAC, differentially blocked [125I]T3 efflux such as (T4 > T3 > rT3 = D-T3 > D-T4) in placenta cells and (T4 > rT3 = D-T4 = T3 > D-T3) in ventriculocytes, suggesting tissue-specific differences in the carriers/transporters responsible for T3 efflux. This hypothesis draws further support from the fact that D-T3 inhibited [125I]T3 efflux in placenta cells, but not in ventriculocytes. TRIAC did not affect T3 efflux in ventriculocytes or placenta cells, but it greatly inhibited [125I]T3 uptake in these cells, suggesting that [125I]T3 uptake and efflux mechanisms are distinct and appear to be mediated by distinct carrier/transporter proteins. Collectively, these data suggest that differences in thyroid hormone transport in target cells may provide an important mechanism for regulating hormone action in a tissue-specific fashion.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONES (TH) have effects on nearly every cell and extensive influences on mammalian development, differentiation and metabolism (1, 2, 3, 4, 5). Most actions of these hormones are mediated by binding of T3 to TRs. The TRs are widely distributed in different tissues (6, 7, 8). The number of receptor molecules per nucleus is about 2,000–10,000, with the highest concentration found in TH-responsive tissues such as the pituitary, brown fat, liver, heart, placenta, and kidney (6, 8).

To bind to TRs, T3 must enter the target cells. Earlier studies indicated that steroid hormones could enter cells by passive diffusion, and this led to a prevalent idea that TH could also enter cells passively (9). However, a number of subsequent studies have demonstrated a saturable, stereo-specific, and energy-requiring uptake of T3 into target cells, supporting the idea that specific transport mechanisms mediate T3 entry (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26). Additional support for this hypothesis was recently provided by the demonstration that T3 uptake in Xenopus oocytes increases significantly after the injection of a specific fraction of rat liver mRNA, suggesting that synthesis of a membrane transport protein(s) from a RNA template included in this fraction can enhance T3 uptake (27). Moreover, recent evidence has been presented indicating that members of the organic anion transporter family (28, 29) or the Na+/taurocholate cotransporting polypeptide (29) are capable of mediating the cellular uptake of THs.

In contrast, few studies have investigated T3 efflux processes. The available data suggest that the T3 efflux is rapid, saturable, and stereospecific (30, 31, 32, 33). T3 efflux may be important for T3 action. We have shown that hepatoma cells acquire resistance to T3 through accelerated T3 export activity (34). Cellular resistance to the action of dexamethasone or steroid hormones (35, 36) may also be explained by an enhanced ability to export these ligands (37, 38, 39, 40). As the above studies were conducted in tumor-derived cell lines, it is possible that the higher efflux activity and hormone resistance in those cells are due to overexpression of transport proteins, such as the multidrug resistance P-glycoproteins (34, 36, 41). Thus, it is critical to examine T3 efflux in nontransformed cells to validate the physiological relevance of studies attempting to link T3 transport mechanisms to T3 action.

For these experiments we examined primary cells from heart, placenta, and kidney tissues because the importance of T3 in the metabolism of these cells is well documented (42, 43, 44, 45, 46, 47, 48, 49). TH are critical for growth and development of the human fetus, and the placenta is an important link in the maternal-fetal communication network for THs, which is essential for the normal development and differentiation of the fetus. The placenta transports and metabolizes maternal TH and contributes to maintain low T3 serum concentrations during fetal life (44, 45, 46, 50). In the heart, TH regulate the production and activity of many enzymes, for example, myosin heavy chain (42, 43, 51), Na,K-adenosine triphosphatase (52, 53), and sarcoplasmic reticulum calcium-adenosine triphosphatase (47, 54). In the kidney, T3 also regulates Na,K-adenosine triphosphatase activity in the proximal and the collecting tubules (48, 49). Here, we report that T3 efflux is saturable, stereo-specific, and verapamil-inhibitable in a number of T3-responsive primary cells. T3 efflux appears to operate through mechanisms that are independent from those involved in T3 influx. Furthermore, the specificity of and capacity for T3 efflux vary significantly among different cell types.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents
[125I]T3 (2200 Ci/mmol) was obtained from NEN Life Science Products, L-T3, L-T4, rT3, D-T3, D-T4, TRIAC, and verapamil were obtained from Sigma (St. Louis, MO. DMEM (DME-H21), Ham’s F-12 medium, FCS, enriched calf serum (EC), glutamine, penicillin-streptomycin, fungizone, BSA, and collagenase were obtained from the Cell Culture Facility, University of California (San Francisco, CA).

Primary cell cultures
Ventricular and atrial myocytes were prepared from 1-d old Sprague Dawley rats as previously described (55). Briefly, cells were dispersed from neonatal rat hearts by alternate cycles of trypsin digestion and mechanical disruption. Ventriculocytes or atriocytes were separated from the mesenchymal cells (primarily fibroblasts) using a 30-min preplating step that fostered selective adherence of fibroblasts, but not myocytes, to the culture surface. Ventriculocytes and atriocytes were plated directly on plastic culture dishes in DME-H21 with 10% EC for 3 d before the experiments. Primary cultures of human chorionic laeve cells (chorion-decidual cells) were prepared as previously described (56), except that cells were grown in DME-H21 with 10% FCS. Briefly, placentas were obtained from normal pregnant women after delivery. The chorion was separated from other membrane layers and collected under sterile conditions in DME-H21 supplemented with antibiotics and glutamine. The chorion tissue was cut into small pieces with scissors at room temperature and digested with collagenase and trypsin. Cell suspensions were filtered through a 45-µm nylon mesh and centrifuged for 3 min at 1800 rpm. The supernatants were discarded, and cell pellets were resuspended in DME-H21 with 10% FCS supplemented with streptomycin, penicillin, fungizone, and glutamine and plated directly in culture dishes. Cells were confluent after 2–3 wk. Inner medullary collecting duct (IMCD) cells were collected from tissue between white inner core of papillary tissue and outer red medulla of adult rat kidney and cultured as previously described (57). Briefly, the tissue was minced into small pieces and digested with collagenase at 30 C. The cells were plated directly on the culture dishes (four to six kidneys for three six-well dishes). The cells were maintained in DME-H21 (50%) and Ham’s F-12 (50%) medium with 10% EC for 24 h. After 24 h the medium was changed to K-1 medium [DME-H21/Ham’s F-12 (1:1, vol/vol)] with 10% serum substitute (transferrin, T3, hydrocortisone, HEPES solution, and NaHCO3) and maintained for 7–10 d until the cells were 90% confluent.

Cell uptake
Measurements of T3 uptake were made in cells grown in monolayer on six-well plates to 80–100% confluence at 37 C. Thirty minutes before the start of the experiment, cells were preincubated with medium containing the test substance, TRIAC, or vehicle (90% ethanol/10% dimethylsulfoxide, control group). After the preincubation, cells were washed twice with PBS, prewarmed to 37 C, and incubated in DME-H21 medium containing 0.01% newborn bovine serum and 1 nM [125I]T3 (2200 Ci/mmol; NEN Life Science Products) with or without 10-4–10-6 M TRIAC. Uptake was stopped at 1 and 5 min by aspirating rapidly the medium and washing the cells six times with 1 ml each of ice-cold PBS (34). Previous experiments established that this washing procedure efficiently removes extracellular [125I]T3 while causing minimal loss of intracellular tracer. Cells were then harvested with 1 ml calcium/magnesium-free PBS/1 mM EDTA prewarmed to 37 C, and radioactivity and protein content were determined. Protein concentrations were measured by the method of Bradford, using BSA as the standard.

Efflux studies
[125I]T3 efflux from cells in monolayer culture was measured as previously described (34). Cells were preincubated for 3 h at 37 C in DME-H21 medium containing 0.01% FCS, [125I]T3 (1 nM) with or without verapamil (10-4 M), unlabeled T3, T4, and/or T3 analogs. After the 3-h preincubation, the cells were washed six times with ice-cold PBS, and efflux was begun by adding 1 ml/well prewarmed DME-H21 medium containing 10% FCS. The addition of 10% FCS to the medium effectively prevented reuptake of [125I]T3 during the efflux study (33). This medium was collected and replaced with fresh efflux medium at 5, 10, 30, and 60 min. After 60 min, cells were removed with 1 ml calcium- and magnesium-free PBS/1 mM EDTA, warmed to 37 C, and counted for radioactivity. Efflux curves were calculated using a curve-fitting algorithm (34), and rates of efflux and percentages remaining in cells are expressed as the mean ± SD.

Column chromatography
The 125I-labeled compounds in efflux medium were analyzed by column chromatography according to a previously described method (58). Briefly, Pasteur pipettes containing Sephadex LH-20 (0.5 x 4 cm) and plugged with cotton wool were equilibrated with 0.1 M HCl. One milliliter of efflux medium was acidified to pH 1.0 with HCl and applied to the column. Iodide was eluted with 3 x 1 ml 0.1 M HCl. Conjugates of T3 were eluted seven times with 1 ml H2O. Subsequently, T3 was eluted three times with 1 ml 0.1 M NaOH/ethanol (1:1, vol/vol).

Statistics
Data were analyzed by one-way ANOVA complemented with the Newman-Keuls multiple comparison test for all comparisons except unpaired t test, which was used to compare the net accumulation of [125I]T3 by ventriculocytes during the 3-h preincubation period. P < 0.05 was considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
To study the T3 efflux in primary cells, we preloaded the cells with [125I]T3 and measured their [125I]T3 content after a 60-min efflux period. Figure 1Go shows that the capacity to export [125I]T3 varies significantly among different types of TH-responsive cells (chorion-decidual > ventriculocyte > atriocyte > IMCD cells; P < 0.001). Thus, after 60 min of efflux, human chorion-decidual cells were the most active in exporting [125I]T3, retaining only 8.3 ± 0.7% [125I]T3 (n = 9), followed by ventriculocytes (14.4 ± 2.0%; n = 6), atriocytes (18.8 ± 1.6%; n = 6), and kidney IMCD cells (47.6 ± 6.1%; n = 9).



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  		Figure 1. Efflux of [125I]T3 from primary cultures of different cell types. The [125I]T3 efflux of human chorion-decidual cells, neonatal rat atrial or ventricular myocytes, and rat renal IMCD cells was studied as described in Materials and Methods. {square}, [125I]T3 content retained within each of these cells after the 60-min efflux period. {blacksquare}, Effect of verapamil (10-4 M) present in the cultures solely during the efflux period. The results are representative of at least six independent experiments and show the mean ± SD. *, P < 0.01, verapamil vs. vehicle.

 
We previously showed that verapamil, a drug that has been used as a probe for monitoring the activity of cell membrane export systems (59), blocked T3 efflux in rat hepatocarcinoma cells (HTC-R) that displayed both resistance to T3 and accelerated T3 export, but did not block T3 export in rat hepatocarcinoma parental cells (HTC) that exhibit lower capacity for T3 export (34). As shown in Fig. 1Go, verapamil ({blacksquare}) added during the efflux period significantly inhibited [125I]T3 efflux in all cell types in a manner that correlated with the cellular capacity to export T3 (P < 0.001, verapamil vs. vehicle in all cell types). Indeed, verapamil greatly inhibited [125I]T3 export in human chorion-decidual cells (n = 9) and ventriculocytes (n = 6), but had only a minor, although significant, effect on blocking [125I]T3 efflux in IMCD cells (n = 9). Because the degree of verapamil inhibition of T3 efflux varied according to the cell type, the [125I]T3 content after verapamil (44.2 ± 1.8%) increased 432% in chorion-decidual cells, 193% (42.1 ± 3.7%) in ventriculocytes, 168% (50.3 ± 4.2%) in atriocytes, and 33% (63.2 ± 10.9%) in IMCD cells. Thus, after verapamil, the final [125I]T3 content became equivalent in chorion-decidual cells and ventriculocytes, which were still lower than those seen in atriocytes and IMCD cells (chorion-decidual = ventriculocyte < atriocyte < IMCD cells; P < 0.001).

In addition, verapamil inhibited significantly [125I]T3 efflux (P < 0.001, verapamil vs. vehicle) whether it was added during preincubation and efflux periods (+/+) or only during the efflux period (-/+) in chorion-decidual cells (Fig. 2AGo; n = 3 and n = 9 for +/+ and -/+ periods, respectively) and ventriculocytes (Fig. 2BGo; n = 6 for +/+ and -/+ periods). Because such inhibition was effective immediately after verapamil addition (-/+), we suggest that verapamil may be directly inhibiting the T3 transport system. Removal of verapamil after the preincubation period (+/-) completely reversed the inhibition by this drug of [125I]T3 efflux in chorion-decidual cells (n = 3) and ventriculocytes (n = 6). Thus, at the end of 60-min efflux period, there was no difference between verapamil and vehicle (Fig. 2Go, A and 2BGo, respectively; P > 0.05). This reversibility argues against verapamil-induced cellular toxicity as an explanation for reduced efflux. The verapamil effect was dose dependent. In atrial myocytes, addition of 10-4 M (n = 6) or 10-5 M (n = 3) verapamil significantly increased the [125I]T3 content to 50.3% and 29%, respectively (P < 0.001), compared with both verapamil (10-6 M; n = 3) and vehicle, which were similar to each other (P > 0.05, 10-6 M verapamil vs. vehicle). As shown previously for a variety of cells (e.g. HTC, HTC-R, FRTL-5, and hepatocytes) (33, 34), chromatographic analysis of the 125I-labeled compounds in the efflux medium from ventriculocytes at 180 min showed that less than 2.0% of the total 125I was in the form of iodide, and more than 94% was in the form of T3. These results indicate that [125I]T3 in the cells or medium did not undergo 3'-deiodination to a significant extent during the period of efflux.



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  		Figure 2. Effects of verapamil on efflux of [125I]T3 in human chorion-decidual cells (A) and rat ventriculocytes (B). Human chorion-decidual cells and rat ventriculocytes were isolated as described in Materials and Methods. The efflux of [125I]T3 was measured in the presence of vehicle ({blacksquare}) or 10-4 M verapamil, which was added either during the 3-h preincubation and efflux periods (+/+, {blacktriangleup}), during the preincubation period only (+/-, {bullet}), or during the efflux period only (-/+, {circ}). These results are representative of at least three independent experiments.

 
To further characterize the mechanisms of T3 efflux in these primary cultures, we studied the effects of excess unlabeled T3, T4, rT3, and TRIAC on [125I]T3 efflux in rat ventriculocytes and human chorion-decidual cells. Because the addition of unlabeled iodothyronines solely during the efflux period (-/+) produced a minor inhibition of [125I]T3 efflux (data not shown), we decided to perform the efflux studies with the inclusion of unlabeled T3, T4, rT3, and TRIAC during both preincubation and efflux periods. Here, the addition of excess unlabeled T4, T3, or rT3 induced a significant, but differential, inhibition of [125I]T3 efflux in rat ventriculocytes and human chorion-decidual cells. T4 was the most efficient inhibitor in chorion-decidual cells (n = 3) and ventriculocytes (n = 8), but the ranking order of inhibition varied such that T3 (n = 3) > rT3 (n = 3) > vehicle (n = 3) in chorion-decidual cells (P < 0.001), whereas in ventriculocytes rT3 (n = 9) and T3 (n = 8) were equally efficient but more effective (P < 0.001) than vehicle (n = 9) in inhibiting [125I]T3 efflux. In contrast, TRIAC had no effect on [125I]T3 efflux in either cell type (P > 0.05; Fig. 3Go, A and B). Thus, in the presence of excess T4, the percentage of [125I]T3 remaining in chorion-decidual cells was 64.4 ± 1.0% compared with 11.4 ± 1.4% in the controls (P < 0.001). Likewise, excess T4 increased the level of [125I]T3 remaining in ventriculocytes to 46.6 ± 6.7% compared with 21.4 ± 3.3% in the control group (P < 0.001). T3 was significantly more effective than rT3 in blocking [125I]T3 efflux from chorion decidual cells (43.1 ± 3.7% vs. 37.5 ± 3.6%; P < 0.05), whereas rT3 (41.9 ± 4.5%) was as effective as T3 (37.0 ± 5.3%) in blocking [125I]T3 efflux on ventriculocytes (P > 0.05). These increments in cellular content of [125I]T3 are not simply due to reuptake of [125I]T3 displaced from TH-binding sites in serum proteins by unlabeled iodothyronines, because cellular [125I]T3 reuptake is negligible in the presence of 10% FCS (33). Interestingly, in contrast to other unlabeled iodothyronines, TRIAC (n = 8) did not modify [125I]T3 export in rat ventriculocytes (21.9 ± 3.0% vs. 21.4 ± 3.3% in the control; n = 9; P > 0.05) or in chorion-decidual cells [9.0 ± 0.2% (n = 3) vs. 11.4 ± 1.4% in the control (n = 3); P > 0.05].



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  		Figure 3. Effect of excess unlabeled iodothyronines (T3, T4, rT3, and TRIAC) on [125I]T3 efflux from human chorion-decidual cells (A) and rat ventriculocytes (B). The efflux of [125I]T3 was measured in the presence of vehicle ({blacksquare}) or iodothyronines (10-4 M) added during the 3-h preincubation and efflux periods. T3 ({square}), T4 ({diamond}), rT3 ({oplus}) and TRIAC (X). These results are representative of at least three independent experiments.

 
Next, we investigated the stereospecificity of [125I]T3 efflux by incubating the cells with unlabeled D-T3 or D-T4 at 10-4 M (Fig. 4Go, A and B). In the absence of D-T3 or D-T4, ventriculocytes retained 21.4 ± 3.3% (n = 9) at the end of the 60-min efflux period. The addition of D-T4 to ventriculocytes (n = 8) provoked a very effective inhibition of [125I]T3 efflux (40.0 ± 3.6%), which was significantly greater than that seen with D-T3 (29.6 ± 3.3%; n = 8), such as D-T4 > D-T3 > vehicle (P < 0.001). In contrast, with chorion-decidual cells, which retained 11.4 ± 1.4% at the end of the 60-min efflux period (n = 3), D-T3 (n = 3) was very effective in blocking [125I]T3 efflux (36.1 ± 1.2%) whereas D-T4 (n = 3) had a significantly smaller inhibitory effect (28.2 ± 2.4%), such as D-T3 > D-T4 > vehicle (P < 0.001). These results suggest that the mechanisms governing [125I]T3 efflux have stereospecificity, as L-T3 was more effective than D-T3 (43.1 ± 3.7% vs. 36.1 ± 1.2% in chorion-decidual cells, P < 0.01; 37.0 ± 5.3% vs. 29.6 ± 3.3% in ventriculocytes, P < 0.01), and LT4 was more effective than D-T4 (64.4 ± 0.9% vs. 28.2 ± 2.4% in chorion-decidual cells, P < 0.001; 46.6 ± 6.7% vs. 40.0 ± 3.6% in ventriculocytes, P < 0.05) in reducing [125I]T3 efflux. Furthermore, the collective results using iodothyronines to block [125I]T3 efflux provide a distinct ranking order for inhibition in placenta cells (T4 > T3 > rT3 = D-T3 > D-T4) compared with ventriculocytes (T4 > rT3 = D-T4 = T3 > D-T3), indicating that T3 efflux mechanisms may differ among tissues.



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  		Figure 4. Effect of excess unlabeled D-T3 or D-T4 (10-4 M) on [125I]T3 efflux from human chorion-decidual cells (A) and rat ventriculocytes (B). The efflux of [125I]T3 was measured in the presence of vehicle ({blacksquare}) or with D-T3 ({diamond}) or D-T4 ({square}) added during the 3-h preincubation and efflux periods. These results are representative of at least three independent experiments.

 
The relative rates of influx and efflux, the rate of intracellular production of T3 and T4, and the rate of metabolic disposal of T3 were used to determine the steady state concentration of T3 within cells. To test whether the mechanisms of [125I]T3 influx differ from those that mediate efflux, we tested whether TRIAC, which does not inhibit [125I]T3 efflux in these cells, has an effect on T3 influx. First, we calculated net accumulation of [125I]T3 by ventriculocytes during the 3-h preincubation period by adding the amount of [125I]T3 exported during the efflux period to the [125I]T3 retained by the cells at the end of efflux. Total [125I]T3 accumulation in ventriculocytes was reduced approximately 2-fold in the presence of TRIAC compared with vehicle [131 ± 6.9 (n = 9) vs. 62.8 ± 4.5 (n = 9) fmol/million cells; P < 0.03, by unpaired t test). Because TRIAC does not inhibit [125I]T3 efflux, these results suggest that the reduced accumulation of [125I]T3 is due to inhibition of [125I]T3 uptake. To address this issue more directly, we measured [125I]T3 uptake in human chorion-decidual cells and rat ventriculocytes at 1 and 5 min after incubation with [125I]T3 and different concentrations of TRIAC. As shown in Fig. 5Go, A and B, TRIAC produced a concentration-dependent reduction of T3 uptake in human chorion-decidual cells, such that 10-4 M TRIAC (n = 5) was similar to 10-5 M TRIAC (n = 5), which were significantly greater than 10-6 M TRIAC (n = 5) and vehicle (n = 5; P < 0.05). In rat ventriculocytes 10-4 M TRIAC (n = 5) was similar to 10-5 M (n = 4) and 10-6 M (n = 4) TRIAC, but was greater than that in vehicle (n = 4; P < 0.05). To exclude the possibility that TRIAC did not inhibit efflux because it was rapidly metabolized during prolonged incubation, we took the medium in which 10-4 M TRIAC was incubated with for 3 h with ventriculocytes and showed that it inhibited the cellular influx of [125I]T3 in a manner similar to that shown in Fig. 5BGo (data not shown). This result indicates that TRIAC inhibition of influx, but not [125I]T3 efflux, is not due to metabolism of this compound during the time intervals used in this study. Collectively, these findings suggest that T3 entry occurs through a TRIAC-sensitive mechanism, whereas T3 efflux is TRIAC insensitive.



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  		Figure 5. Uptake of [125I]T3 by human chorion-decidual cells (A) and rat ventriculocytes (B) in the presence of unlabeled TRIAC. Cellular uptake was measured (counts per min/mg cellular protein) at 37 C in DME-H21 medium containing 0.01% newborn calf serum and [125I]T3 (1 nM) in the absence or presence of increasing concentrations (10-6–10-4 M) of TRIAC. Uptake at 5 min for the various conditions is expressed as the percentage of [125I]T3 uptake relative to the control group (vehicle treated). *, P < 0.05 for differences between TRIAC and control group (vehicle treated).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
There are extensive studies of TH transport across cell membranes, but only a few of them have investigated the mechanisms of T3 export (30, 31, 32, 33, 34). The previous studies suggest that T3 influx is regulated and plays an important role in maintaining the overall T3 content in cells. There are other factors, such as T3 production through T4 deiodination and cellular T3 metabolism, that determine steady state T3 levels in the cell. In the present study we present evidence that cellular T3 efflux is also an important determinant of T3 content in primary cultures of T3-sensitive cells such as cardiac, placenta, and kidney cells.

We show that T3 efflux rates vary considerably among primary cells, such as rat atrial and ventricular myocytes, kidney IMCD cells, and human placenta cells. Whereas human placenta cells export more than 90% of the cellular [125I]T3 after a 60-min period of efflux, rat IMCD cells export only 50%. Therefore, we argue that although T3 uptake is an important factor controlling the T3 content in cells, the tissue-to-tissue variability in cellular T3 efflux rates may also be important in determining differences in intracellular T3 content that have been noted in different tissues (60, 61). Although our present studies involved T3 taken up by cells from the medium, it is likely that export of T3 produced by 5'-deiodination of T4 within the cell is subject to the same regulatory control. In normal rats, the concentration of T3 in kidney cells is 5 and 3 times higher than that in plasma or heart, respectively (61). The cell to plasma concentration ratio for TH is dependent on multiple rates, including the rate of cellular uptake, the rate of efflux, cellular hormone binding capacity, and metabolism of the hormone. Thus, differences in efflux capability in different tissues may offer an additional explanation for the coexistence of low, normal, or high TH levels in tissues of hypothyroid animals supplemented with T4, because these results cannot be explained by differences in deiodinase activities (61). Likewise, an enhanced T3 efflux rate may also offer an explanation for TH resistance syndromes that occur in the absence of TR mutations (62, 63). Because a number of individuals in these kindreds had normal gene sequences of TR{alpha}1 TRß1, TRß2, coactivators, corepressors, and RXR{gamma}, it has been suggested that other mechanisms, such as defects in TH transport, may be involved in the genesis of TH resistance in these families (64). Disturbances in T3 transport may also contribute to the heterogeneity of tissue responsiveness to TH in patients with subclinical hypothyroidism or thyroid hormone resistance (65, 66, 67).

We showed previously that HTC-R cells display an enhanced rate of T3 export compared with HTC parental cells, which increases their resistance to T3 action through a mechanism that is saturable and verapamil inhibitable (34). In fact, verapamil treatment increased significantly the T3 sensitivity of HTC-R cells that partially reversed their state of T3 resistance (34). Our present findings indicate that T3 efflux in primary cells is also saturable, verapamil-inhibitable, and stereospecific, but its capacity differs according to cell type. Verapamil-dependent inhibition of T3 efflux in primary cells does not appear to be due to cellular toxicity, because it is effective immediately after addition of the drug, does not require preincubation, and is completely reversible after withdrawal. On the other hand, we showed that unlabeled T3 and analogs including T4, rT3, D-T4, and D-T3 require preincubation with cells to inhibit T3 efflux. This suggests that T3 analogs need to reach a critical intracellular concentration to show inhibition of T3 efflux. The rank order of potency of unlabeled T3 analogs was T4 > T3 > rT3 > D-T3 > D-T4 in chorion-decidual cells and T4 > rT3 > D-T4 > T3 > D-T3 in ventriculocytes, suggesting tissue-specific differences in the efficacy of the individual analogs. In fact, the addition of excess D-T3 inhibited T3 efflux in a cell type-specific manner, as it blocked [125I]T3 efflux in human chorion-decidual cells but not in rat ventriculocytes. Because unlabeled T4 was more effective in inhibiting [125I]T3 than unlabeled T3, one may argue that this is indeed a T4 efflux system. However, our competition data indicate that the efflux transport system we have studied serves to carry both T3 and T4 out of the cell. Further studies are needed to distinguish whether there are also specific carrier/transport proteins for each hormone. It is also worth mentioning that because inhibition of [125I]T3 efflux required high concentrations of TH analogs, one cannot, at this time, ascertain the physiological role of such a mechanism in regulating TH cellular content and function. Collectively, these data suggest that there may be more than one carrier/transporter regulating T3 efflux, as analogs blocked the [125I]T3 efflux rate differentially in different cell types. These results are also consistent with the presence of a single carrier/transporter with different affinities for the individual analogs that are differentially expressed in target tissues. Of greatest importance, the fact that [125I]T3 efflux may be regulated differentially in diverse tissues raises the possibility of finding or designing tissue-specific inhibitors (themselves devoid of TH activity) of T3 efflux that may increase T3 content selectively in target tissues.

Another intriguing inference of our results is that the T3 transporter(s)/carrier(s) responsible for T3 influx differs from that which mediates T3 efflux. This interpretation is based on TRIAC’s ability to inhibit T3 influx, but not T3 efflux, in human chorion-decidual cells and rat ventriculocytes. In both cell types TRIAC-impaired T3 uptake in a dose-dependent manner, indicating that T3 entry occurs through a TRIAC-sensitive mechanism. These results are supported by the other studies showing that TRIAC reduced T3 uptake in cultured anterior pituitary cells (68). Because the influx studies were shorter, the lack of a TRIAC effect on T3 efflux could be explained if it were degraded during the 3-h incubation. However, incubation of TRIAC with ventriculocytes for 3 h did not affect its capacity to inhibit [125I]T3 uptake in ventriculocytes. Moreover, the net accumulation of [125I]T3 after the 3-h incubation was greatly inhibited by TRIAC, which demonstrates the effectiveness of TRIAC during the time interval covered by the efflux studies. In addition, we found that T3 efflux is most effectively inhibited by T4 in both placenta cells and neonatal rat ventriculocytes, whereas T4 did not inhibit T3 uptake in neonatal rat cardiac myocytes (69). These findings support the idea that T3 efflux occurs by a mechanism distinct from that which mediates T3 uptake. The same phenomenon is observed with T4 transport. In human choriocarcinoma cells, unlabeled L-stereoisomers of TH inhibited uptake, but not efflux, of T4, suggesting that uptake and efflux of T4 occur by different mechanisms (32).

Taken together, these findings represent the first demonstration that T3 efflux mechanisms vary both quantitatively and qualitatively among different TH-responsive cells in primary culture. They also suggest that the mechanism(s) responsible for T3 entry differ from that governing T3 export and indicate that the regulation of these phenomena may provide novel ways to control intracellular hormone concentration and function in a tissue-specific fashion.


   Footnotes
 
This work was supported by fellowships from the Brazilian Research Council, CNPq (to F.A.R.N., L.A.S. and R.C.J.R.), University of California STAR Biotechnology Program Award S96-14 (to R.C.J.R. and N.L), and a V.A. Medical Research Grant (to R.R.C). J.D.B. has proprietary interests in, and serves as a consultant and Deputy Director to Karo Bio AB, which has commercial interests in this area of research.

1 R.R.C. is deceased. Back

2 N.L. and R.C.J.R. are senior authors. Back

Abbreviations: EC, Enriched calf serum; IMCD, inner medullary collecting ducts; TH, thyroid hormones.

Received June 11, 2001.

Accepted for publication October 15, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Oppenheimer JH, Schwartz HL, Mariash CN, Kinlaw WB, Wong NC, Freake HC 1987 Advances in our understanding of thyroid hormone action at the cellular level. Endocr Rev 8:288–308[Medline]
  2. Weiss RE, Refetoff S 1996 Effect of thyroid hormone on growth. Lessons from the syndrome of resistance to thyroid hormone. Endocrinol Metab Clin North Am 25:719–730[CrossRef][Medline]
  3. Kohrle J 2000 Thyroid hormone metabolism and action in the brain and pituitary. Acta Med Austr 27:1–7[CrossRef]
  4. Hulbert AJ 2000 Thyroid hormones and their effects: a new perspective. Biol Rev Camb Philos Soc 75:519–631[Medline]
  5. Thompson CC, Potter GB 2000 Thyroid hormone action in neural development. Cereb Cortex 10:939–945[Abstract/Free Full Text]
  6. Oppenheimer JH, Schwartz HL, Surks MI 1974 Tissue differences in the concentration of triiodothyronine nuclear binding sites in the rat: liver, kidney, pituitary, heart, brain, spleen, and testis. Endocrinology 95:897–903[Medline]
  7. Ribeiro RCJ, Apriletti JW, Wagner RL, West BL, Feng W, Huber R, Kushner PJ, Nilsson S, Scanlan TS, Fletterick RJ, Schaufele F, Baxter JD 1998 Mechanisms of thyroid hormone action: insights from x-ray crystallographic and functional studies. Recent Prog Horm Res 53:351–394
  8. Shahrara S, Drvota V, Sylven C 1999 Organ specific expression of thyroid hormone receptor mRNA and protein in different human tissues. Biol Pharm Bull 22:1027–1033[Medline]
  9. Utiger RD 1995 The thyroid: physiology, thyrotoxicosis, hypothyroidism, and the painful thyroid. In: Felig PF, Baxter JD, Frohman CA, eds. Endocrinology and metabolism. New York: McGraw-Hill; 435–519
  10. Rao GS, Eckel J, Rao ML, Breuer H 1976 Uptake of thyroid hormone by isolated rat liver cells. Biochem Biophys Res Commun 73:98–104[Medline]
  11. Krenning EP, Docter R, Bernard HF, Visser TJ, Hennemann G 1978 Active transport of triiodothyronine (T3) into isolated rat liver cells. FEBS Lett 91:113–116[CrossRef][Medline]
  12. Eckel J, Rao GS, Rao ML, Breuer H 1979 Uptake of L-tri-iodothyronine by isolated rat liver cells. A process partially inhibited by metabolic inhibitors; attempts to distinguish between uptake and binding to intracellular proteins. Biochem J 182:473–491[Medline]
  13. Holm AC, Wong KY, Pliam NB, Jorgensen EC, Goldfine ID 1980 Uptake of L-triiodothyronine into human cultured lymphocytes. Acta Endocrinol (Copenh) 95:350–358[Medline]
  14. Krenning EP, Docter R, Bernard B, Visser T, Hennemann G 1982 Decreased transport of thyroxine (T4), 3,3',5-triiodothyronine (T3) and 3,3',5'-triiodothyronine (rT3) into rat hepatocytes in primary culture due to a decrease of cellular ATP content and various drugs. FEBS Lett 140:229–233[CrossRef][Medline]
  15. Oppenheimer JH, Schwartz HL 1985 Stereospecific transport of triiodothyronine from plasma to cytosol and from cytosol to nucleus in rat liver, kidney, brain, and heart. J Clin Invest 75:147–154
  16. Blondeau JP, Osty J, Francon J 1988 Characterization of the thyroid hormone transport system of isolated hepatocytes. J Biol Chem 263:2685–2692[Abstract/Free Full Text]
  17. Movius EG, Phyillaier MM, Robbins J 1989 Phloretin inhibits cellular uptake and nuclear receptor binding of triiodothyronine in human hep G2 hepatocarcinoma cells. Endocrinology 124:1988–1997[Abstract]
  18. Topliss DJ, Kolliniatis E, Barlow JW, Lim CF, Stockigt JR 1989 Uptake of 3,5,3'-triiodothyronine by cultured rat hepatoma cells is inhibitable by nonbile acid cholephils, diphenylhydantoin, and nonsteroidal antiinflammatory drugs. Endocrinology 124:980–986[Abstract]
  19. Goncalves E, Lakshmanan M, Pontecorvi A, Robbins J 1990 Thyroid hormone transport in a human glioma cell line. Mol Cell Endocrinol 69:157–165[CrossRef][Medline]
  20. Mitchell AM, Manley SW, Mortimer RH 1992 Uptake of L-tri-iodothyronine by human cultured trophoblast cells. J Endocrinol 133:483–486[Abstract]
  21. Lakshmanan M, Goncalves E, Pontecorvi A, Robbins J 1992 Differential effect of a new thyromimetic on triiodothyronine transport into myoblasts and hepatoma and neuroblastoma cells. Biochim Biophys Acta 1133:213–217[Medline]
  22. de Jong M, Visser TJ, Bernard BF, Docter R, Vos RA, Hennemann G, Krenning EP 1993 Transport and metabolism of iodothyronines in cultured human hepatocytes. J Clin Endocrinol Metab 77:139–143[Abstract]
  23. Riley Jr WW, Eales JG 1994 Characterization of 3,5,3'-triiodo-L-thyronine transport into hepatocytes isolated from juvenile rainbow trout (Oncorhynchus mykiss), and comparison with L-thyroxine transport. Gen Comp Endocrinol 95:301–309[CrossRef][Medline]
  24. Chantoux F, Blondeau JP, Francon J 1995 Characterization of the thyroid hormone transport system of cerebrocortical rat neurons in primary culture. J Neurochem 65:2549–2554[Medline]
  25. McLeese JM, Eales JG 1996 3,5,3'-Triiodo-L-thyronine and L-thyroxine uptake into red blood cells of rainbow trout, Oncorhynchus mykiss. Gen Comp Endocrinol 102:47–55[CrossRef][Medline]
  26. Powell KA, Mitchell AM, Manley SW, Mortimer RH 2000 Different transporters for tri-iodothyronine (T3) and thyroxine (T4) in the human choriocarcinoma cell line, JAR. J Endocrinol 167:487–492[Abstract]
  27. Docter R, Friesema EC, van Stralen PG, Krenning EP, Everts ME, Visser TJ, Hennemann G 1997 Expression of rat liver cell membrane transporters for thyroid hormone in Xenopus laevis oocytes. Endocrinology 138:1841–1846[Abstract/Free Full Text]
  28. Abe T, Kakyo M, Sakagami H, Tokui T, Nishio T, Tanemoto M, Nomura H, Hebert SC, Matsuno S, Kondo H, Yawo H 1998 Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transports thyroid hormones and taurocholate and comparison with oatp2. J Biol Chem 273:22395–22401[Abstract/Free Full Text]
  29. Friesema EC, Docter R, Moerings EP, Stieger B, Hagenbuch B, Meier PJ, Krenning EP, Hennemann G, Visser TJ 1999 Identification of thyroid hormone transporters. Biochem Biophys Res Commun 254:497–501[CrossRef][Medline]
  30. Osty J, Zhou Y, Chantoux F, Francon J, Blondeau JP 1990 The triiodothyronine carrier of rat erythrocytes: asymmetry and mechanisms of trans-inhibition. Biochim Biophys Acta 1051:46–51[Medline]
  31. Benvenga S, Robbins J 1998 Thyroid hormone efflux from monolayer cultures of human fibroblasts and hepatocytes. Effect of lipoproteins and other thyroxine transport proteins. Endocrinology 139:4311–4318[Abstract/Free Full Text]
  32. Mitchell AM, Rowan KA, Manley SW, Mortimer RH 1999 Comparison of mechanisms mediating uptake and efflux of thyroid hormones in the human choriocarcinoma cell line, JAR. J Endocrinol 161:107–113[Abstract]
  33. Cavalieri RR, Simeoni LA, Park SW, Baxter JD, Scharschmidt BF, Ribeiro RC, Lomri N 1999 Thyroid hormone export in rat FRTL-5 thyroid cells and mouse NIH-3T3 cells is carrier-mediated, verapamil-sensitive, and stereospecific. Endocrinology 140:4948–4954[Abstract/Free Full Text]
  34. Ribeiro RCJ, Cavalieri RR, Lomri N, Rahmaoui CM, Baxter JD, Scharschmidt BF 1996 Thyroid hormone export regulates cellular hormone content and response. J Biol Chem 271:17147–17151[Abstract/Free Full Text]
  35. Kralli A, Bohen SP, Yamamoto KR 1995 LEM1, an ATP-binding-cassette transporter, selectively modulates the biological potency of steroid hormones. Proc Natl Acad Sci USA 92:4701–4705[Abstract/Free Full Text]
  36. Kralli A, Yamamoto KR 1996 An FK506-sensitive transporter selectively decreases intracellular levels and potency of steroid hormones. J Biol Chem 271:17152–17156[Abstract/Free Full Text]
  37. Ueda K, Okamura N, Hirai M, Tanigawara Y, Saeki T, Kioka N, Komano T, Hori R 1992 Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J Biol Chem 267:24248–24252[Abstract/Free Full Text]
  38. Wolf DC, Horwitz SB 1992 P-glycoprotein transports corticosterone and is photoaffinity-labeled by the steroid. Int J Cancer 52:141–146[Medline]
  39. Nelson EJ, Hinkle PM 1992 Characterization of multidrug-resistant pituitary tumor cells. Endocrinology 130:3246–3256[Abstract]
  40. van Kalken CK, Broxterman HJ, Pinedo HM, Feller N, Dekker H, Lankelma J, Giaccone G 1993 Cortisol is transported by the multidrug resistance gene product P-glycoprotein. Br J Cancer 67:284–289[Medline]
  41. Bourgeois S, Gruol DJ, Newby RF, Rajah FM 1993 Expression of an mdr gene is associated with a new form of resistance to dexamethasone-induced apoptosis. Mol Endocrinol 7:840–851[Abstract]
  42. Green NK, Franklyn JA, Ahlquist JA, Gammage MD, Sheppard MC 1989 Differential regulation by thyroid hormones of myosin heavy chain {alpha} and beta mRNAs in the rat ventricular myocardium. J Endocrinol 122:193–200[Abstract]
  43. Ojamaa K, Klemperer JD, MacGilvray SS, Klein I, Samarel A 1996 Thyroid hormone and hemodynamic regulation of ß-myosin heavy chain promoter in the heart. Endocrinology 137:802–808[Abstract]
  44. Kilby MD, Verhaeg J, Gittoes N, Somerset DA, Clark PM, Franklyn JA 1998 Circulating thyroid hormone concentrations and placental thyroid hormone receptor expression in normal human pregnancy and pregnancy complicated by intrauterine growth restriction (IUGR). J Clin Endocrinol Metab 83:2964–2971[Abstract/Free Full Text]
  45. Santini F, Chiovato L, Ghirri P, Lapi P, Mammoli C, Montanelli L, Scartabelli G, Ceccarini G, Coccoli L, Chopra IJ, Boldrini A, Pinchera A 1999 Serum iodothyronines in the human fetus and the newborn: evidence for an important role of placenta in fetal thyroid hormone homeostasis. J Clin Endocrinol Metab 84:493–498[Abstract/Free Full Text]
  46. Darras VM, Hume R, Visser TJ 1999 Regulation of thyroid hormone metabolism during fetal development. Mol Cell Endocrinol 151:37–47[CrossRef][Medline]
  47. Muller A, Zuidwijk MJ, Simonides WS, van Hardeveld C 1997 Modulation of SERCA2 expression by thyroid hormone and norepinephrine in cardiocytes: role of contractility. Am J Physiol 272:H1876–H1885
  48. Tagami T, Nakamura H, Sasaki S, Mori T, Yoshioka H, Yoshida H, Imura H 1990 Immunohistochemical localization of nuclear 3,5,3'-triiodothyronine receptor proteins in rat tissues studied with antiserum against C-ERB A/T3 receptor. Endocrinology 127:1727–1734[Abstract]
  49. Barlet C, Ben Abdelkhalek M, Doucet A 1985 Sites of thyroid hormone action on Na-K-ATPase along the rabbit nephron. Pflugers Arch 405:52–57[CrossRef][Medline]
  50. Kohrle J 1997 [Transfer and metabolism of thyroid gland hormones in the placenta]. Acta Med Austriaca 24:138–143[Medline]
  51. Izumo S, Nadal-Ginard B, Mahdavi V 1986 All members of the MHC multigene family respond to thyroid hormone in a highly tissue-specific manner. Science 231:597–600[Abstract/Free Full Text]
  52. Orlowski J, Lingrel JB 1990 Thyroid and glucocorticoid hormones regulate the expression of multiple Na,K-ATPase genes in cultured neonatal rat cardiac myocytes. J Biol Chem 265:3462–3470[Abstract/Free Full Text]
  53. Huang CJ, Geller HM, Green WL, Craelius W 1999 Acute effects of thyroid hormone analogs on sodium currents in neonatal rat myocytes. J Mol Cell Cardiol 31:881–893[CrossRef][Medline]
  54. Hartong R, Wang N, Kurokawa R, Lazar MA, Glass CK, Apriletti JW, Dillmann WH 1994 Delineation of three different thyroid hormone-response elements in promoter of rat sarcoplasmic reticulum Ca2+ATPase gene. Demonstration that retinoid X receptor binds 5' to thyroid hormone receptor in response element 1. J Biol Chem 269:13021–13029[Abstract/Free Full Text]
  55. Wu J, LaPointe MC, West BL, Gardner DG 1989 Tissue-specific determinants of human atrial natriuretic factor gene expression in cardiac tissue. J Biol Chem 264:6472–6479[Abstract/Free Full Text]
  56. Duncan KG, Haidar MA, Baxter JD, Reudelhuber TL 1990 Regulation of human renin expression in chorion cell primary cultures. Proc Natl Acad Sci USA 87:7588–7592[Abstract/Free Full Text]
  57. Cao L, Chen SC, Cheng T, Humphreys MH, Gardner DG 1998 Ligand-dependent regulation of NPR-A gene expression in inner medullary collecting duct cells. Am J Physiol 275:F119–F125
  58. Docter R, Krenning EP, Bernard HF, Visser TJ, Hennemann G 1988 Inhibition of uptake of thyroid hormone into rat hepatocytes by preincubation with N-bromoacetyl-3,3',5-triiodothyronine. Endocrinology 123:1520–1525[Abstract]
  59. Gottesman MM, Pastan I 1993 Biochemistry of multidrug resistance mediated by the multidrug transporter. Annu Rev Biochem 62:385–427[CrossRef][Medline]
  60. Obregon MJ, Mallol J, Escobar del Rey F, Morreale de Escobar G 1981 Presence of L-thyroxine and 3,5,3'-triiodo-L-thyronine in tissues from thyroidectomized rats. Endocrinology 109:908–913[Abstract]
  61. Escobar-Morreale HF, Obregon MJ, Escobar del Rey F, Morreale de Escobar G 1995 Replacement therapy for hypothyroidism with thyroxine alone does not ensure euthyroidism in all tissues, as studied in thyroidectomized rats. J Clin Invest 96:2828–2838
  62. Weiss RE, Hayashi Y, Nagaya T, Petty KJ, Murata Y, Tunca H, Seo H, Refetoff S 1996 Dominant inheritance of resistance to thyroid hormone not linked to defects in the thyroid hormone receptor {alpha} or ß genes may be due to a defective cofactor. J Clin Endocrinol Metab 81:4196–4203[Abstract]
  63. Weiss RE, Refetoff S 1999 Treatment of resistance to thyroid hormone–primum non nocere [Editorial; Comment]. J Clin Endocrinol Metab 84:401–404[Free Full Text]
  64. Reutrakul S, Sadow PM, Pannain S, Pohlenz J, Carvalho GA, Macchia PE, Weiss RE, Refetoff S 2000 Search for abnormalities of nuclear corepressors, coactivators, and a coregulator in families with resistance to thyroid hormone without mutations in thyroid hormone receptor ß or {alpha} genes. J Clin Endocrinol Metab 85:3609–3617[Abstract/Free Full Text]
  65. Beck-Peccoz P, Chatterjee VK 1994 The variable clinical phenotype in thyroid hormone resistance syndrome. Thyroid 4:225–232[Medline]
  66. Muller B, Zulewski H, Huber P, Ratcliffe JG, Staub JJ 1995 Impaired action of thyroid hormone associated with smoking in women with hypothyroidism. N Engl J Med 333:964–969[Abstract/Free Full Text]
  67. Refetoff S, Weiss RE, Usala SJ 1993 The syndromes of resistance to thyroid hormone. Endocr Rev 14:348–399[CrossRef][Medline]
  68. Everts ME, Visser TJ, Moerings EP, Docter R, van Toor H, Tempelaars AM, de Jong M, Krenning EP, Hennemann G 1994 Uptake of triiodothyroacetic acid and its effect on thyrotropin secretion in cultured anterior pituitary cells. Endocrinology 135:2700–2707[Abstract]
  69. Everts ME, Verhoeven FA, Bezstarosti K, Moerings EP, Hennemann G, Visser TJ, Lamers JM 1996 Uptake of thyroid hormones in neonatal rat cardiac myocytes. Endocrinology 137:4235–4242[Abstract]



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