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Division of Genetics (P.H.K.E., G.R.C., S.-L.F., M.P., S.A., W.W.C., L.E.B.), Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Department of Molecular Pharmacology (N.C.), Albert Einstein College of Medicine, Bronx, New York 10461
Address all correspondence and requests for reprints to: Peter Eng, M.D., Division of Genetics, Department of Medicine Brigham and Women’s Hospital, 75 Francis Street, Thorn 1013, Boston, Massachusetts 02115. E-mail: eng{at}rascal.med.harvard.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-Iodohexadecanal, a major iodolipid, has been shown to
inhibit nicotinamide adenine dinucleotide phosphate oxidase,
thyroid peroxidase (TPO), and TSH-induced cAMP formation in the thyroid
and thus may be a potential mediator of the effect. The so-called
escape phenomenon, however, has been less well studied. Braverman
and Ingbar suggested that adaptation to the acute Wolff-Chaikoff
effect was caused by a decrease in iodide transport into the thyroid,
which reduced the intrathyroidal iodide to concentrations that were
insufficient to sustain the decreased organification of iodide (5).
Recently, the complementary DNA (cDNA) encoding the protein responsible
for the active transport of iodide from blood to thyroid was cloned by
Dai et al. (6). This polytopic membrane protein, expressed
in thyroid follicular cells, is termed the sodium-coupled iodide
cotransporter or sodium/iodide symporter (NIS). We hypothesized that
changes in NIS expression might account for iodide autoregulation in
the thyroid. In the present study, we reexamined the mechanism
responsible for the escape from the acute Wolff-Chaikoff effect by
determining NIS messenger RNA (mRNA) and protein levels in rat thyroids
in response to acute and prolonged administration of excess iodide. |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The chronic excess iodide experiment was carried out as follows: The rats were divided into 3 groups: control and 1 and 6 days of iodide excess. Rats were given either distilled water (control group) or 0.05% iodide, as NaI, in the drinking water for 1 or 6 days. The addition of iodide to the drinking water was timed so that all the rats were killed on the same day. The thyroid gland from each rat was removed immediately, snap frozen, and stored at -70 C for measurement of specific RNA and proteins. Blood from each rat was collected, and the serum was frozen for later measurement of serum iodide, TSH, T3, and T4 concentrations. There was a total of 48 rats, with 16 rats in each group. Three separate experiments were carried out using the same protocol. The first experiment had 8 rats in each group, and the second and third experiments had 4 rats in each group. The results of the 3 experiments were pooled for the purpose of statistical analysis.
The acute excess iodide experiment was carried out as follows: Rats were divided into 5 groups: control and 1, 2, 6, and 24 h after an acute iodide load. There were 4 rats in each group. A single ip injection of 2000 µg NaI in 0.5 ml saline was administered to the 1-, 2-, 6-, and 24-h groups before being killed. Saline alone was injected into the control group. Rats were killed, and their thyroid glands and blood were obtained as described above. A similar protocol was repeated with only 3 groups of rats (6 rats in each group): control and 6 and 24 h after iodide administration. The results of the 2 experiments were pooled for statistical analysis.
This study was approved by the Harvard Area Standard Committee on Animals, and it conforms with federal and state regulations governing the use of laboratory animals.
Serum iodine and hormone measurements
Serum iodine concentrations were assayed according to the
modified Sandell-Koltoff method of Benotti and Benotti (7). Serum
thyroid hormones, T3 and T4, and TSH
concentrations were measured in duplicate by RIAs, in random order and
in the same assay for each experiment. Serum TSH was measured by RIA
using materials obtained from the National Pituitary Agency, NIH
(Bethesda, MD). Serum T3 and T4 concentrations
were determined by species-adapted RIAs using a single antibody
technique with polyethylene glycol precipitation (8).
Thyroid RNA analysis
Total thyroid RNA was extracted using a commercial kit (RNeasy;
QIAGEN, Inc., Chatsworth, CA). Northern analysis of the
RNA was carried out as follows. Total RNA (10 µg/lane) was subjected
to electrophoresis for 3 h in 1% agarose containing formaldehyde
in 1x 4-morpholinopropanesulfonic acid. The RNA was then
transferred overnight to a nylon membrane (Nytran; Schleicher & Schuell, Inc., Keene, NH) by diffusion blotting, and was UV
cross-linked. The membrane was hybridized sequentially with five rat
cDNA probes: NIS, TSH receptor (TSHr), TPO, thyroglobulin (Tg), and
cyclophilin. Cyclophilin mRNA was used for normalization of the levels
of thyroid mRNAs. The cDNA probes were labeled with
[-32P]deoxycytidine 5'-triphosphate using a random
primer protocol (Prime-It II Random Primer Labeling Kit;
Stratagene, La Jolla, CA) to a specific activity of
0.2–2 x 109 cpm/µg DNA. Purified cDNA inserts
(TSHr and TPO were provided by L. D. Kohn, National Institute of
Diabetes and Digestive and Kidney Diseases, NIH (Bethesda, MD); and Tg
by G. Vassart, Institut de Recherche Interdisciplinaire, Bruxelles,
Belgium) were used as the probes. The membrane was prehybridized 1–2 h
at 42 C, followed by an overnight hybridization at 42 C with the
radiolabeled probes. The membrane was washed twice with 6x SSPE
(NaCl, NaPO4, EDTA)/0.5% SDS at room temperature, twice
with 1x SSPE /0.5% SDS at 37 C, and (if high stringency was required)
a further wash with 0.1x SSPE/0.1% SDS at 60 C. The membrane was
exposed to a phosphorimager screen for an appropriate length of time,
and the signal intensity was analyzed by a phosphorimaging system
(Molecular Dynamics, Inc., Sunnyvale, CA).
Thyroid protein analysis
Total protein from the thyroid glands was extracted as follows.
Frozen thyroid glands were thawed in RIPA buffer (1x PBS, 1% Nonidet
P-40, 0.5% sodium deoxycholate, 0.1% SDS) and protease inhibitors
(aprotinin, sodium orthovanadate, and phenylmethylsulfonylflouride)
were added. Homogenization was performed using a mechanical device
(Polytron; Kinematica, Luzern, Switzerland). The homogenate was
centrifuged at 15,000 x g, and the supernatant (which
contained the whole cell lysate) was quantified spectrophotometrically
using a modified Lowry method (Dc Protein Assay; Bio-Rad Laboratories, Inc., Hercules, CA). Western blot analysis was
carried out as follows: Twenty-five micrograms of protein per lane were
loaded on an 8% SDS polyacrylamide gel and subjected to electophoresis
at a constant voltage (150 V). Electroblotting to a nitrocellulose
membrane (Protran; Schleicher & Schuell, Inc.) was
performed for 2 h at 90 mA using a semidry electroblotting system
(MultiphorII Electrophoresis System; Pharmacia LKB
Biotechnology, Uppsala, Switzerland). Blocking was done overnight using
TTBS/milk (TBS, 1% Tween 20 and 5% milk). The membrane was incubated
with a 1:5000 dilution of affinity-purified anti-NIS antibody in
TTBS/milk. Two 5-min and two 15-min washes in TTBS were then performed.
The membrane was incubated with a 1:25,000 dilution of a horseradish
peroxidase conjugated antirabbit antibody (Pierce Chemical Co., Rockford, IL) in TTBS/milk. Two 5-min and two 15-min washes
were again performed. The membrane was then incubated with an enhanced
chemiluminescent substrate (Supersignal Substrate Western Blotting,
Pierce Chemical Co.) and exposed to film. Quantitation of
the signal intensity was performed by densitometry (Molecular Dynamics, Inc.).
Statistical analysis
Statistical analysis was performed using a statistical analysis
program (Instat v 2.02; GraphPad Software, Inc., San
Diego, CA). Comparison between groups was by ANOVA, followed by
Tukey-Kramer test for intergroup comparison. Results are expressed as
mean ± SE.
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Results |
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Chronic iodide ingestion
Serum iodine and hormone levels. Serum iodine levels, after 1
and 6 days of iodide administration, were markedly elevated (more than
60-fold), compared with values in the control group (Table 1
). There was no statistical difference
in the serum iodine levels between the 1-day and 6-day groups. Serum
T3 and T4 concentrations were significantly
decreased in the rats treated with iodide for 1 day, compared with
values in the control rats. Serum T3 concentrations, after
6 days of iodide administration, were similar to those in the control
rats; but T4 concentrations, after 6 days of iodide
administration, were significantly higher than 1-day and control rats.
TSH concentrations were not significantly different from control values
at 1 or 6 days after iodide administration, although values were
slightly higher at 1 day (Fig. 1).
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). TPO mRNA was decreased significantly
6 days after iodide administration but not after 1 day (Fig. 3). In contrast, TSHr mRNA and Tg mRNA
were unchanged during iodide administration (data not shown).
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Acute iodide administration
Serum iodine and hormone levels. Serum iodine concentrations
were markedly increased 1 h after iodide administration and
progressively decreased at 2, 4, 6, and 24 h, but they were still
greater than 100 µg/dl at 24 h (Table 1
). There were no
significant changes in serum T3, T4, or TSH
concentrations at any time point after iodide administration (data not
shown).
mRNA expression. The level of NIS mRNA was unchanged at 1 and
2 h after the acute administration of 2000 µg NaI ip but was
significantly decreased at 6 h (60% of control) and further
decreased at 24 h (40% of control) (Fig. 5). TPO mRNA was unchanged at 1, 2, and
6 h but was decreased at 24 h (Fig. 6). There were no statistical differences
among the levels of TSHr or Tg mRNA at 1, 2, 6, and 24 h after the
acute administration of iodide (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It had been postulated in 1963 that the adaptation to or escape from the acute Wolff-Chaikoff effect was caused by a decrease in the active transport of iodide from the plasma into the thyroid, thereby decreasing the high concentrations of intrathyroidal iodide that inhibit hormone synthesis. We have now reinvestigated this postulate by determining the level of NIS mRNA and protein in the thyroids of iodide-exposed rats. The level of NIS mRNA significantly decreased after 1 and 6 days of iodide ingestion. The decrease in NIS protein was even more dramatic, decreasing to approximately 23% of the control values at both time points. The decrease in NIS occurred in the absence of a significant increase in TSH, which would have increased NIS or reduced the iodide induced-decrease in NIS, because TSH has been shown to increase NIS expression (11, 12). The decrease in NIS expression, after 1 day of excess iodide exposure, suggested the possibility that the active transport of iodide into the thyroid, which induces the acute Wolff-Chaikoff effect, might occur rapidly (<24 h) after iodide administration. To evaluate this hypothesis, rats were killed from 1–24 h after the acute administration of excess iodide ip. No change in NIS mRNA occurred at 1 or 2 h after iodide administration, but a significant decrease was observed at 6 and 24 h. Also, no change in NIS protein was observed at 6 h, when NIS mRNA was already decreased; however, a marked decrease in NIS protein was found at 24 h. These findings suggest that the decrease in active iodide transport into the thyroid occurred between 6 and 24 h after excess iodide exposure. Thus, it is likely that the excess iodide transported into the thyroid, before the decrease in NIS protein, was responsible for the acute inhibition of hormone synthesis previously described (2, 13). Uyttersprot et al. (14) recently reported that the acute administration of a single small dose of iodide to the iodine-deficient, propylthiouracil-, and perchlorate-treated dog, with elevated serum TSH concentrations, decreased NIS mRNA in the thyroid at 48 h. They did not measure NIS protein. Also, they did not determine serum iodine concentrations. Our observations are also likely more physiological, because the rats were on a normal iodine intake, were not receiving any antithyroid drugs, and did not have markedly elevated serum TSH values.
The present results also suggest that the half-life of NIS protein in the thyroid, in the presence of excess iodine, is less than 24 h, which is far shorter than the half-life of 4 days, observed in FRTL5 cells in vitro (15, 16). It is possible that the degradation of NIS protein is higher in the presence of excess iodide, and further studies of the half-life of NIS protein, in the presence and absence of excess iodide, are indicated. Because both the NIS mRNA and NIS protein are decreased, it seems likely that NIS regulation by iodine is (at least partly) transcriptional, although a posttranscriptional mechanism cannot be ruled out. Pulse-chase experiments are planned to examine the translational or posttranslational effects of iodine on NIS protein turnover.
Although excess iodide administration did not affect Tg or TSHr mRNAs, TPO mRNA levels were decreased after 6 days of chronic iodide ingestion and 1 day after the acute administration of a large dose of iodide. These findings are similar to those observed in the iodine-deficient hypothyroid dog, 1 day after the administration of an acute dose of iodine (14). The decrease in TPO mRNA, after iodine administration, would tend to negate the escape phenomenon. Indeed, if the decrease in TPO mRNA occurred alone, organification of iodine and subsequent hormone synthesis would continue to be impaired. In spite of this possible decrease in TPO, the reduction in iodide transport and subsequent decrease in thyroidal iodine content would permit iodine organification and hormone synthesis to resume.
We have recently reported that chronic iodine administration to the diabetes and lymphocytic thyroiditis (LT) prone BB/Wor rat increases the incidence of LT and decreases TPO mRNA in the follicular cells in contact with infiltrating lymphocytes (17). Although spontaneous LT or iodine-induced LT does not occur in iodine-sufficient or -deficient Sprague Dawley rats (18), it is possible that a decrease in TPO mRNA might be one of the mechanisms responsible for iodine-induced hypothyroidism, so common in Hashimoto’s thyroiditis (19).
In summary, we have shown that excess iodide, given to rats, chronically or acutely decreases both thyroid NIS mRNA and protein. Our findings are consistent with the hypothesis that the escape from the Wolff-Chaikoff effect is caused by a down-regulation of the NIS, resulting in decreased iodide transport into the thyroid. This would then lower the intrathyroidal iodine below a critical threshold and would allow organification to resume. The decrease in NIS is likely to be, at least in part, transcriptional. In addition, we have also found that excess iodide decreases TPO mRNA and that this decrease may contribute to iodide-induced hypothyroidism commonly seen in patients with Hashimotos’s thyroiditis.
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Footnotes |
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Received January 19, 1999.
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(TNF ), and
decreased expression of thyroglobulin and thyroid peroxidase mRNA in
the thyroids of iodide treated BB/Wor rats. Eur J Endocrinol 139:539–545[Abstract]
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