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Endocrinology, doi:10.1210/en.2005-1246
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Endocrinology Vol. 147, No. 6 2936-2943
Copyright © 2006 by The Endocrine Society

Amiodarone Inhibits Thyroidal Iodide Transport in Vitro by a Cyclic Adenosine 5'-Monophosphate- and Iodine-Independent Mechanism

S. Tedelind, F. Larsson, C. Johanson, H. C. van Beeren, W. M. Wiersinga, E. Nyström and M. Nilsson

Department of Medical Chemistry and Cell Biology (S.T., F.L., M.N.), Institute of Biomedicine, The Sahlgrenska Academy at Göteborg University, 405 30 Göteborg, Sweden; Departments of Radiation Physics (C.J.) and Medicine (E.N.), Sahlgrenska University Hospital, 41345 Göteborg, Sweden; and Department of Endocrinology and Metabolism (H.C.v.B., W.M.W.), Academic Medical Center, University of Amsterdam, 1100 DD Amsterdam, The Netherlands

Address all correspondence and requests for reprints to: Sofia Tedelind, Institute of Biomedicine, Department of Medical Chemistry and Cell Biology, Sahlgrenska Academy at Göteborg University, Box 420, 405 30 Göteborg, Sweden. E-mail: sofia.tedelind{at}anatcell.gu.se.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid side effects are common in patients treated for cardiac arrhythmias with amiodarone (AM). A major disturbance is inhibited thyroidal radioiodine uptake in AM-induced thyrotoxicosis, which makes 131I therapy ineffective. On the other hand, failure to escape from the Wolff-Chaikoff effect by down-regulation of the sodium/iodide symporter (NIS) is proposed to explain AM-induced hypothyroidism. However, previously no experimental studies on the possible mechanisms have been conducted. We therefore investigated the early effects of AM on thyroidal iodide transport using bicameral chamber cultures of primary pig thyrocytes that reproduce the three tissue compartments (epithelium, lumen, and extrafollicular space) of the gland. AM dose-dependently (1–50 µM) inhibited the TSH-stimulated transepithelial (basal to apical) transport of 125I– by up to 90%. The inhibitory effect was noticed already after 8 h and was further pronounced after 1–4 d, depending on the AM concentration. The intracellularly accumulated 125I– was reduced by perchlorate but not AM, and quantitative real-time RT-PCR revealed no change in the NIS expression in AM-treated cells. Blocking of cAMP degradation with 3-isobutyl-1-methylxanthine or withdrawal of AM reversed AM-induced changes in electrolyte transport but were unable to recover the suppressed 125I– transport. The iodine-free AM analog dronedarone also inhibited 125I– transport to the same extent as AM. The findings indicate that AM blocks thyroidal iodide uptake by reducing the iodide permeability of the apical plasma membrane of the thyroid epithelial cells. The effect is iodine independent and long-lasting and does not involve impaired function of NIS or the TSH receptor/cAMP signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A COMMON SIDE effect of amiodarone (AM; 2-butyl-3-(3',5'-diiodo-4'{alpha}-diethylaminoethoxybenzoyl)-benzofuran), a potent and widely used class III antiarrhythmic drug, is thyroid dysfunction (1, 2). AM-induced hypothyroidism (AIH) can affect both individuals with apparently normal thyroid glands and patients with subclinical autoimmune chronic thyroiditis. Amiodarone-induced thyrotoxicosis (AIT) also exists in two forms: type I involving excessive thyroid hormone synthesis and type II signified by an unrestricted release of preformed thyroid hormones from damaged follicles. AM shows structural resemblance to T4 and T3, and the altered peripheral metabolism of thyroid hormone regularly observed in subjects receiving the drug is partly the result of AM’s antagonistic and inhibitory effects on thyroid hormone receptors and deiodinases; this may also render peripheral tissues such as the heart and liver hypothyroid. However, because AM is an iodine-rich compound, each molecule containing two iodine atoms comprising 37.5% of the molecular weight, it is believed that the disturbed function of the thyroid gland itself is provoked by the exceedingly high iodine load (7–21 mg iodide/d) coming from the rather rapid decomposition of the drug (10% of the iodine content is estimated to be released each day) (3). In addition, iodine-independent direct cytotoxic effects of AM on the thyroid have been reported (4, 5).

The pathogenesis of AIT and AIH has not been elucidated. Clearly the type of thyroid dysfunction caused by AM is influenced by the iodine status in the population, with AIH prevailing in areas with sufficient iodine supply and AIT being more common in iodine-deficient regions (6, 7). The excessive iodine exposure accompanying AM treatment might therefore trigger different responses, depending on the gland’s previous long-term adaptation to normal or low circulating and intrathyroidal iodide levels. However, why this results in diametrically opposed outcomes, either stimulated or suppressed thyroid hormone synthesis and secretion, in different subjects is not understood. Apart from being envisaged a causal role in the development of destructive thyroiditis characterizing type II AIT, potential pathogenetic effects of AM itself (iodine excluded) on the emergence of thyroid dysfunction have not been studied, neither clinically nor experimentally. Nevertheless, the circulating AM levels during treatment is in the micromolar range (8, 9), and both AM and desethylamiodarone (DEA), the main active metabolite of the drug, accumulate in the thyroid in vivo (10) and in cultured thyrocytes (11). Because of its amphiphilic nature, AM is a membrane-active drug whose antiarrhythmic properties comprising prolongation of the action potential duration are mainly achieved by a direct inhibition of sodium, potassium, and calcium channels in the sarcolemma of cardiomyocytes (12). Although no previous studies have addressed the issue, it can be assumed that electrolyte transport in thyroid cells is also affected by AM.

Patients with AIT often show a poor radioiodide uptake that makes diagnostic imaging difficult or impossible, and therapy with 131I is therefore not optional in most cases (1, 2). No feasible explanation to this phenomenon has been offered, although it is often suggested to be the result of AM-induced thyroid tissue destruction in which the disrupted and leaky follicle lack colloid content including iodide. Alternatively, a diminished ability of the AM-treated thyroid gland to concentrate radioiodide might be related to inhibited iodide transport. This possibility is, however, not supported by earlier observations that subjects with AIH show uninhibited thyroidal uptake of radioiodine (13). In fact, the pathogenesis of AIH is generally believed to be caused by the inhibitory action of high iodine levels on thyroid hormone synthesis, the so-called Wolff-Chaikoff effect (14), and failure to escape from this (1, 2). From the clinical point of view, it thus appears that AM exerts multiple effects on thyroidal iodine metabolism that cannot be explained by a single mechanism, and it is also reasonable to assume that some features may be secondary to long-term AM treatment, leaving possible primary actions of the drug on, in general, iodide transport so far unknown. In the present study, we therefore investigated whether AM and dronedarone (Dron), an iodine-free analog, and their active metabolites influence iodide transport mechanisms in vitro early after drug exposure. For this purpose, we used polarized pig thyrocytes cultured in bicameral chambers that allow basolateral iodide uptake via the sodium/iodide symporter (NIS) and iodide efflux across the apical plasma membrane to be simultaneously monitored.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Isolation and culture of pig thyrocytes
Follicle isolation from pig thyroid glands was performed as earlier described (15). Briefly, minced tissue was digested with 0.25 mg/ml collagenase (Worthington, Freehold, NJ) in the presence of 0.1 mg/ml trypsin inhibitor type I-S and 2 µg/ml DNase I (both purchased from Sigma Chemical Co., St. Louis, MO). By filtering the cell suspension through nylon filters with diminishing pore size and repeated washings and centrifugations at 600 rpm for 5 min, connective tissue remnants and blood cells to a major extent were excluded from the follicle preparation. The follicles consisting mainly of ruptured segments with no remaining colloid were either frozen in fetal calf serum (PAA Laboratories GmbH, Linz, Austria) containing 10% dimethylsulfoxide (Sigma) and stored at –80 C for later use or directly seeded (density: 1200 follicle fragments per square millimeter) on permeable filter of Transwell inserts (no. 3413; filter pore size 0.4 µm; Corning Costar Co., Acton, MA) precoated with 0.3 mg/ml collagen S type I (Roche Diagnostics Co., Indianapolis, IN). The cells were cultured in 5% CO2 at 37 C in Eagle’s MEM supplemented with 5% fetal calf serum, amphotericin B (2.5 µg/ml), penicillin (200 U/ml), and streptomycin (200 U/ml) (all cell culture reagents from PAA). Confluent thyrocytes forming a tight monolayer after culture for 7 or more days were stimulated with bovine TSH (1 mU/ml) (Sigma) for 24 h followed by exposure to AM (1–50 µM; Sigma), Dron (5–20 µM; gift from Sanofi-Synthelabo Inc., Montpellier, France), or the metabolites DEA or desbutyldronedarone (DBDron) (both 10 µM; Sanofi-Synthelabo) for an additional 24–96 h in the continuous presence of TSH unless otherwise indicated. All reagents were added to the basal compartment of the bicameral chamber.

Quantitative real-time RT-PCR
Total RNA from pig thyrocytes grown on filters was isolated using the RNeasy microkit (QIAGEN GmbH, Hilden, Germany) according to the manufacturer’s instructions and thereafter quantified by UV spectrophotometry at 260 nm. RNA quality was verified by measuring the OD absorption ratio at 260 and 280 nm. cDNA was synthesized from 2 µg RNA using random hexamers and TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA) according to the manufacturer’s instructions. Oligonucleotide primers for the pig NIS (pNIS) gene and for the reference gene (18S) were designed using the Primer Express software (Applied Biosystems) and were purchased from TAG Copenhagen A/S (Copenhagen, Denmark). The sequence of the primers were as follows: pNIS forward primer, 5'-ctctcctggcagggcatatct-3', reverse primer, 5'-gctgagggtgccgctgta-3'; 18S forward primer, 5'-gtaacccgttgaaccccatt-3', reverse primer, 5'-ccatccaatcggtagtagcg-3'. The relative quantitative measurement of pNIS cDNA was performed with the QuantiTect SYBR Green PCR kit using the ABI PRISM 7900HT sequence detection system (Applied Biosystems). The real-time PCR was preincubated at 50 C for 2 min and initiated at 95 C for 10 min; the mixtures were then subjected to 40 cycles of a two-step PCR, comprising 15 sec of denaturation at 95 C and a 1-min annealing/elongation phase at 60 C.

The crossing point values were used for calculation of the relative expression ratios between negative control and treated cells by the formula described by Pfaffl (16), and the statistical significance of the data were analyzed by using the relative expression software tool (REST) (17).

Radioiodide transport assay
Experiments were carried out in a 37 C water bath. The culture medium was changed to Tyrode's salt solution (pH 7.2), containing methimazole (1 mM; Sigma) to prevent iodide organification and potassium iodide (10–7 M; added basally only) as carrier for radioiodide. Trace amounts of 125I– (125 kBq/ml; Nycomed Amersham Plc., Little Chalfont, UK) was added to the basal culture chamber. When indicated, potassium perchlorate (1 mM) was added along with radioiodide to prevent NIS-mediated uptake. After 30 min, samples of the apical medium were taken for measurement of the amount of 125I– transported across the epithelium from the basal to the apical culture compartment. Thereafter the filters with attached cells were rapidly but carefully washed three times to diminish extracellular radioactivity before being cut out. Radioactivity present in medium and cell samples was measured with a {gamma}-counter (B5002; Packard Instrument International, Zurich, Switzerland).

Transepithelial electrical resistance (TER)
The transepithelial electrolyte transport was estimated by measuring the TER and the potential difference (PD) with a Millicell ERS ohmmeter (Millipore, Bedford, MA). Provided that the cells are confluent with intact tight junctions, these parameters reflect the conductivity of the apical and basolateral plasma membranes and the overall transcellular transfer of ions across the cell monolayer mediated by ion pumps and channels. TSH is known to stimulate a cAMP-mediated flux of Na+ in the basal direction by activating an amiloride-sensitive Na+ channel in the apical plasma membrane (18); this is monitored as a decrease in TER and an augmented PD that persist as long as TSH is present. TER data presented in graphs were corrected for the background resistance measured across filters without cells.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Amiodarone inhibits transepithelial iodide transport without affecting NIS mRNA expression
125I– was used as tracer to monitor transepithelial iodide transport in the filter-cultured pig thyroid cell monolayers after exposure to AM. This showed that AM decreased the TSH-stimulated basal-to-apical transport of 125I– in a dose-dependent manner (Fig. 1AGo); 10 µM AM caused an approximately 30% inhibition, whereas 50 µM of the drug reduced the 125I– transport by 80% or more after treatment for 24 h. The highest AM concentration (50 µM) inhibited 125I– transport by 15–30% already 8 h after addition (data not shown). Also, prolonged incubation for 96 h with lower AM concentrations (1–10 µM) caused a further decrease in 125I– transport (Fig. 1BGo).


Figure 1
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  		FIG. 1. Effect of AM on TSH-stimulated iodide transport in polarized pig thyroid cells. A, Dose-dependent inhibition of basal-to-apical 125I– transport after exposure to AM for 24 h. B, Time-dependent inhibition of the same transport by low AM concentrations. Filter-cultured cells were prestimulated with 1 mU/ml TSH for 24 h before AM was added, and TSH stimulation continued during AM treatment. Data are expressed as relative change of 125I– transport in percentage of that monitored in cultures stimulated with only TSH. Means ± SD (n = 4).

 
To determine whether the AM-induced inhibition of iodide transport was caused by a reduced NIS expression, analogous to that characterizing escape of the Wolff-Chaikoff effect after exposure to excess iodide (19), pNIS mRNA was quantified by real-time RT-PCR. This showed that TSH stimulation for 48 h enhanced the NIS mRNA level almost 60-fold, compared with control cells (Table 1Go). However, AM (50 µM) present during the last 24 h of TSH stimulation, corresponding to experiments shown in Fig. 1AGo, had no effect on the ability of TSH to up-regulate NIS (Table 1Go). If NIS is targeted by AM explaining the effect on 125I– transport it therefore must be by a posttranscriptional mechanism. Alternatively, the inhibited 125I– transport might be unrelated to NIS function. A clue to this came from analysis of cellular 125I– uptake and retention. In AM-treated cultures in which the transport from the basal to the apical medium was markedly reduced (Fig. 2Go, upper panel), the amount of 125I– accumulated in the cell layer did not differ from or was even higher than that monitored in cultures given only TSH (Fig. 2Go, lower panel). For comparison, both the intracellular accumulation and apical transfer of 125I– were almost abolished by perchlorate added from the start of transport study (Fig. 2Go). Collectively, these findings indicate that apical efflux rather than basolateral uptake of 125I– was affected by AM.


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  		TABLE 1. Quantification of NIS mRNA by real-time PCR in primary cultured pig thyroid cells

 

Figure 2
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  		FIG. 2. Effects of AM and perchlorate on the TSH-stimulated transepithelial transport (upper panel) and the corresponding cellular accumulation (lower panel) of 125I–. TSH-prestimulated cultures were exposed to 50 µM AM for 24 h. Perchlorate (ClO4–; 1 mM) was added to the basal medium at the start of 125I– transport monitoring. The activity in the apical medium and cells was measured in the same cultures. Data are expressed as changes relative to the uptake values obtained in untreated cultures set to 1. Means ± SD (n = 4).

 
Amiodarone inhibits TSH-stimulated electrolyte transport by cAMP-dependent and independent mechanisms
In view of the fact that AM is a membrane-perturbing compound with profound effects on membrane functions including gating-properties of ion channels, we wanted to examine whether AM influenced the thyroidal transport of other ions besides iodide. For this purpose, we adopted a previously reported model of evaluating TSH-stimulated electrolyte transport by monitoring changes in TER and PD (20). As shown in Fig. 3Go, in cultures prestimulated with TSH for 24 h, the steady-state levels of TER and PD were 0.6–0.8 k{Omega} x cm2 and 45–55 mV, respectively. After addition of AM (50 µM) with ongoing TSH stimulation, the TER was gradually increased to 2.3 k{Omega} x cm2 between 2–6 h and further to 3.5 k{Omega} x cm2, accompanied by a decrease of the PD to nearly 20 mV after 24 h (Fig. 3Go). Also lower AM concentrations (≤10 µM) had similar but less pronounced and more delayed effects on TER and PD (data not shown). Overall, it is likely that these changes reflect an altered conductance of the major electrolytes (sodium, chloride, and potassium), and the pattern resembles that seen when TSH stimulation is abrogated (20).


Figure 3
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  		FIG. 3. Effect of AM on TER and PD in TSH-stimulated thyroid cells. AM (50 µM) was added to cultures prestimulated with TSH (1 mU/ml) for 24 h at 0 time, after which TER (squares) and PD (circles) were repeatedly measured as indicated. Open symbols, TSH only; filled symbols, TSH and AM. Means ± SD (n = 4).

 
Considering previous notions that AM can interfere with the TSH receptor signaling pathway by decreasing the cAMP level (21), it was of interest to elucidate whether AM affected the bioelectrical parameters by blocking the signal transduction via cAMP. Indeed, the AM-induced increase of TER was inhibited by the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) added either from the start of experiment or after AM exposure for 24 h (Fig. 4AGo, upper panel). However, the major increase in TER was not preventable by IBMX, which also did not appear to notably influence the AM-induced decrease in PD (Fig. 4AGo, lower panel). It is likely therefore that AM affects the transepithelial electrolyte transport in thyroid cells partly by interfering with the cAMP signaling downstream of TSH receptor activation but that a cAMP-independent or -insensitive mechanism predominates. To further corroborate this, we investigated whether AM influenced the change in TER that appears soon after TSH withdrawal and reflects a gradual deactivation of the TSH receptor signaling (20). In comparison with cultures not exposed to AM, removal of TSH from AM-treated cells caused a much faster increase in TER, which also leveled off at a higher level (Fig. 4BGo). Importantly, readdition of TSH provoked a sudden drop in TER (Fig. 4BGo), indicating that the TSH receptor was still present at the cell surface and susceptible to stimulation by the ligand. Together, this suggests that AM accelerates the degradation of cAMP but that the TSH receptor signaling pathway targeting membrane-permeating mechanisms otherwise is fully working in AM-treated thyroid cells.


Figure 4
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  		FIG. 4. Effects of phosphodiesterase inhibitor IBMX and TSH withdrawal on the AM-induced changes in TER and PD. A, Cells were prestimulated with TSH (1 mU/ml) for 24 h and then exposed to 50 µM AM. IBMX (0.5 mM) was added simultaneously with AM (first arrow) or after AM treatment for 24 h (second arrow). TER (upper panel) and PD (lower panel) were measured at the indicated times. See upper panel for keys to the different treatments. B, TSH-stimulated cultures were incubated with (filled symbols) or without (empty symbols) AM (50 µM) for 24 h and then subjected to TSH withdrawal (first arrow) followed by TSH stimulation again (second arrow). TER was monitored every 15 min. For clarity reasons, due to the fact that the TER levels in AM-exposed and AM-free cultures were much different (illustrated at 24 h in A), the data in B are given as changes relative to TER recorded in the same treatment group immediately before TSH washout. Means ± SD (n = 4) in both A and B.

 
Inhibition of iodide transport by amiodarone is cAMP-independent and persists after drug removal
Because TSH-stimulated transepithelial iodide transport in the present experimental model is cAMP dependent (22), it was of interest to investigate whether the inhibitory effect of AM was sensitive to IBMX similar to the partial blocking of electrolyte transport. TSH-prestimulated cultures were therefore treated with 50 µM AM together with IBMX for 24 h after which 125I– transport was studied. This showed that the 125I– transport was reduced to the same extent by AM independently of the presence or absence of phosphodiesterase inhibitor (Fig. 5AGo). It was also found that the inhibited 125I– transport persisted or was further pronounced, although AM was removed from the medium and culture continued for an additional 3 d (Fig. 5BGo). In contrast, the suppressive effect of AM on TSH-regulated TER changes was shown to be reversible already 24 h after the drug was omitted (Fig. 5CGo).


Figure 5
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  		FIG. 5. Effects of IBMX and withdrawal of AM on AM-induced changes in iodide transport and TER. A, Cells were stimulated with TSH (1 mU/ml) for 24 h and then exposed to AM (50 µM) in the presence or absence of IBMX (0.5 mM) for an additional 24 h (with ongoing TSH stimulation), after which 125I– transport was measured as previously indicated. B, TSH-prestimulated cultures were treated with 10 or 50 µM AM for 24 h and then directly taken for 125I– transport study or, with continued TSH stimulation, switched to AM-free medium and further cultured for 72 h before 125I– transport was measured. C, Cultures were treated as outlined in B and TER was monitored daily (first arrow: addition of AM, second arrow: AM withdrawal). Data in A–C are expressed as relative changes in percentage of, respectively, the 125I– transport and TER values monitored in cultures stimulated with only TSH. Means ± SD (n = 4) in all experiments.

 
Dron, an iodine-free amiodarone analog, also inhibits iodide transport
Dron, an AM analog, mimics some AM actions (23) but is believed to exert fewer thyroid side effects due to the fact that it is completely devoid of iodine. To test whether this drug yet might reproduce the blocking effect of AM on 125I– transport, Dron and its active metabolite DBDron were investigated. Thyroid cell toxicity was apparently higher with Dron than AM, and 50 µM rapidly caused a loss of TER and detachment of cells with condensed nuclei designating apoptosis (data not shown). However, 5–20 µM Dron did not compromise cell viability and the epithelial barrier function remained intact throughout experiments. As shown in Fig. 6AGo, Dron dose-dependently inhibited the transepithelial 125I– transport (Fig. 6AGo, upper panel) and caused a slight increase in the cellular 125I– content (Fig. 6AGo, lower panel) to about the same extent as did AM. In further similarity with the response to AM, the inhibited transport of 125I– persisted 3 d after washout of Dron (Fig. 6BGo). Moreover, nonlethal concentrations of DBDron and DEA, the major AM metabolite, were found to inhibit iodide transport as well (Fig. 6CGo).


Figure 6
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  		FIG. 6. Effects of Dron, DBDron, and DEA on iodide transport. A, Cultures prestimulated with TSH (1 mU/ml) for 24 h were exposed to Dron (5–20 µM) for an additional 24 h in the continuous presence of TSH, after which basal-to-apical transport of 125I– (upper panel) and accumulation of 125I– in cells (lower panel) were measured in the same cultures. B, TSH-prestimulated cells were treated with Dron (10 µM) for 24 or 96 h, and for 24 h followed by incubation in Dron-free medium for 72 h, in the continued presence of TSH. 125I– transport was measured at the indicated times. C, TSH-prestimulated cultures were exposed to DEA and DBDron (10 µM) for 24 h followed by 125I– transport. Data in A–C are expressed as relative changes in percentage of the 125I– content in the apical medium or cell layer of matching cultures stimulated with only TSH. Means ± SD (n = 4 in A and B and n = 3 in C).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Thyroidal iodine metabolism is often affected in patients treated with AM for severe cardiac arrhythmias, but the involving mechanisms have not been experimentally elucidated. In this study, carried out in primary cultures of pig thyroid cells with preserved epithelial characteristics, we show that TSH-stimulated transepithelial iodide transport is dose-dependently inhibited by AM and that the blocking effect mainly involves the apical efflux of iodide, without simultaneous down-regulation of NIS mRNA expression. Because Dron, an iodine-free AM analog, similarly inhibits iodide transport, it is likely that the benzofuranic compound itself rather than the iodine content of the drug exerts the effect. The AM concentrations found to suppress iodide transport in vitro are slightly higher than the plasma levels in AM-treated patients (24) but correspond well with those measured in the thyroid gland after long-term drug exposure (25, 26).

The working in vitro three-compartment model of thyroidal iodide transport used in this study is well established. For example, it has been used previously to identify a TSH-regulated iodide efflux mechanism in the apical domain of the thyrocyte’s plasma membrane (15) and characterize the dysregulated iodide transport appearing after growth factor stimulation (27, 28) and 131I irradiation (29). Also, active cellular uptake of radioiodine occurs only from the basal chamber medium (15), confirming that NIS is selectively expressed in the basolateral membrane that in vivo faces the extrafollicular space (30). So by measuring the amount of radioiodide transported across the cell layer in apical direction and the radioactivity amount retained in the cell layer (at steady-state transport), it can be determined with high accuracy whether a transport blocking effect involves basolateral uptake or apical efflux or both. The present experiments unequivocally showed that AM markedly reduced the transepithelial iodide transport without a corresponding suppression of the cellular iodide content. Rather, more iodide accumulated in the AM-treated cells. In contrast, no radioiodide was found to be transported apically or retained in the cells when NIS-mediated uptake of iodide was blocked by perchlorate. Together, this indicates that AM targets mainly the apical efflux mechanism resulting in a diminished delivery of iodide to the apical medium corresponding to the follicular lumen in vivo.

There are several possible mechanisms by which AM might inhibit apical iodide efflux. It has previously been shown that high concentrations of AM counteract TSH-stimulated cAMP production in FRTL-5 cells (21) and human thyrocytes (31), and because the iodide permeability of the apical plasma membrane in filter-cultured pig thyroid cells is positively regulated by cAMP (22), impaired function of the TSH receptor signaling pathway could be a cause. However, although inhibition of phosphodiesterase by IBMX in the present study was found to partly reverse the AM-induced increase in TER, the same treatment was unable to recover iodide transport. Moreover, the electrophysiological measurements showed that TSH readily activated its receptor and that the signal was transduced to the effectors in the plasma membrane that mediates an increased ion conductance designated by a lowered TER. It is therefore likely that AM acts downstream of cAMP and possibly at the plasma membrane level to directly block the apical efflux of iodide. The affected efflux mechanism may yet be TSH and cAMP regulated as it is known to be in normal thyroid cells (22) but made insensitive to stimulus by the drug action.

Due to its amphiphilic properties, AM is known to be incorporated into the lipid bilayer of biological membranes in which the intercalated drug decreases the membrane fluidity and hence the mobility and function of membrane components (32, 33). The molecule(s) mediating TSH-stimulated apical iodide efflux in thyroid cells has not been unequivocally determined, although the apical plasma membrane houses several putative candidates, e.g. pendrin (34) and a newly identified protein, the apical iodide transporter (35). An important issue for future studies will be to investigate whether the residency in the apical membrane and iodide-permeating properties of these membrane proteins are affected by AM.

The AM-induced inhibition of TSH-stimulated iodide transport was not accompanied by a reduced NIS expression at the transcriptional level. However, it cannot be excluded that NIS turnover is altered posttranscriptionally, as has been reported for the ß-adrenoceptor after AM exposure (36). Yet the cellular retention of radioiodide was the same as or even higher in the AM-treated thyroid cells in comparison with those stimulated with only TSH, indicating that the NIS-mediated iodide uptake continued also when the apical iodide efflux at the same time was inhibited. If AM unspecifically perturbs membrane function, it can be questioned why NIS located basolaterally is not at all or much less affected than the apical iodide efflux mechanism(s). The reason for this is unknown, but it can be speculated that different lipid composition of the basolateral and apical plasma membranes might play a role. Recent progress in the understanding of epithelial cell polarization highlights the importance of cholesterol-rich lipid rafts in the formation and intracellular sorting of transport carriers of cargo that are segregated and routed mainly to the apical cell surface (37). Once inserted in the membrane, the turnover of cholesterol also seems to differ between the apical and basolateral membranes of epithelial cells (38). Notably, the cholesterol content of both synthetic and native membranes has been shown to modulate the membrane activity of AM (39). Whether AM alters the properties of lipid rafts or whether iodide transporters in thyroid cells at all depend on such membrane microdomains for their function have not been investigated.

We also found that prolonged AM treatment for up to 4 d further suppressed the transepithelial transport of iodide and that there were no signs of recovery, even though the exposure to drug was limited to 24 h. In contrast, the AM-induced increase in TER was fully reversed already 24 h after drug washout. This suggests either that iodide transport is more susceptible than electrolyte transport to AM or that the renewal rate of damaged transporters are much different. It is previously known that the half-life of NIS is very long, but information of the turnover of iodide efflux mediator(s) is lacking. Irrespective of this, it is possible that AM that has accumulated intracellularly, e.g. bound to ingestible lipids deposited in lysosomes (40, 41), exerts long-lasting effects, even though the drug is removed from the culture medium; a slow release of drug from such deposits might enter the regular vesicular transport machinery that recycles membrane to the exocytic pathway, which in turn could influence the properties of the apical plasma membrane. Although not equivalent due to the great differences in drug exposure times, it is yet of interest to compare these in vitro data with the clinical signs of patients on AM treatment that develop thyroid dysfunction and particularly AIT. In this situation AM often impairs radioiodine uptake for a long time even after drug withdrawal. The mechanism is unknown, but because of the very long half-life of the drug (24), it is suggested that iodide continues to be released from AM accumulated in adipose tissue and other organs and that the thyroid gland suffers from the high iodine load. However, in cultured thyrocytes exposed to Dron (lacking iodine) for only 24 h the same prolonged inhibitory effect on iodide transport as with AM was found, indicating that this results from a direct action of the drug and that the mechanism therefore primarily is iodine independent. From this, it is also suggested that the prevailing view on the cause of a poor thyroidal iodide uptake in AIT might be reconsidered. AIT type II patients are believed to become thyrotoxic on the basis of a drug- or iodine-induced destructive thyroiditis, and any iodide that may be taken up by NIS would leak out of the damaged follicles. As an alternative explanation, we propose that failure of AM-treated glands to concentrate radioiodide can be caused by impaired apical iodide efflux without the necessity of a broken thyroid epithelial barrier.

It is assumed that AIH depends on subtle defects in the organification of iodine and, particularly in patients with preexisting autoimmune thyroiditis, an increased susceptibility to the inhibitory effect of iodine on thyroid hormone synthesis (1, 2). This can be regarded as a variant of the classical Wolff-Chaikoff effect induced experimentally by excess iodide (14); hence, the long-lasting iodide load prevailing in AM-treated patients is held responsible. It is also believed that thyroid cells in patients with AIH fail to escape from the Wolff-Chaikoff effect (1, 2), meaning that the mechanism of thyroid autoregulation that relieves the gland from the high iodine load to eventually resume normal function is not working. It is now established that escape from the Wolff-Chaikoff effect relies on a rapid down-regulation of the NIS expression (19). Although yet incompletely studied with regard to prolonged exposure times and possible synergistic effects with excessive iodide, the present quantitative real-time RT-PCR analysis of pNIS supports the notion that NIS might be excluded from the thyroid-suppressive effects of AM. Indeed, clinical studies show that there may be even an increased radioiodide uptake and a positive perchlorate discharge test, indicating incomplete organification of iodide, in AIH patients (13). However, a possible contribution of AM acting directly on the apical plasma membrane in which iodination normally takes place is plausible and warrants further investigation. It also appears important to distinguish early (primary) and chronic (secondary) responses to AM and iodide and the possibility of additive or synergistic effects between them. In further attempts to elucidate these issues the experimental model used here will be instrumental.

Finally, it is noteworthy that AM (and Dron; data not shown) markedly influenced the electrophysiological properties of the thyroid epithelium. This is probably reminiscent of the well-known effects of the drug on cardiac ion channels that are intimately involved in the antiarrhythmic action [reviewed by Kodama et al. (42)]. The fact that AM targets electrolyte transporters in cells other than cardiomyocytes may not be surprising, taking into account the unspecific nature of the mode of action, but this is actually a neglected field of investigation. It is conceivable to assume that the membrane potential of nonexcitable cells is modulated by AM, so also in thyroid cells. With regard to epithelia, as observed in the present study, the transepithelial transport of electrolytes seems to be a major site of interference. The functional significance of this finding remains to be elucidated. A pertinent question to answer would be whether AM-induced changes in electrolyte fluxes might alter the physicochemical properties of the colloid and the metabolism of thyroglobulin and iodine stored in the follicular lumen.


   Acknowledgments
 
We are grateful to Madeleine Nordén for valuable discussions and Therése Carlsson for excellent technical assistance.


   Footnotes
 
This study was supported by Grant 537 from the Swedish Medical Research Council.

S.T., F.L., C.J., H.C.v.B., E.N., and M.N. have nothing to declare. W.M.W. has consulted for Genzyme and has received lecture fees from Merck. This is not in conflict of interest with the material being published.

First Published Online March 9, 2006

Abbreviations: AIH, Amiodarone-induced hypothyroidism; AIT, Amiodarone-induced thyrotoxicosis; AM, amiodarone; DBDron, desbutyldronedarone; DEA, desethylamiodarone; Dron, dronedarone; IBMX, 3-isobutyl-1-methylxanthine; NIS, sodium/iodide symporter; PD, potential difference; pNIS, pig NIS; TER, transepithelial electrical resistance.

Received September 30, 2005.

Accepted for publication March 2, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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