Thyroid autoregulation: evidence for an action of iodoarachidonates and iodide at the cell membrane level.

Iodolipids are the possible mediators of excess iodide in thyroid autoregulation. Previous work from our laboratory has shown that 14-iodo-15-hydroxy-5,8,11 eicosatrienoic acid (I-HO-A) and its omega lactone (IL-w) mimic the inhibitory action of excess iodide upon several parameters of thyroid metabolism. The present experiments were performed in order to study the mechanism of the inhibitory effect of I-HO-A and IL-w on 2-deoxy-D-glucose (DOG) and aminoisobutyric acid (AIB) uptake by calf slices. I-HO-A, IL-w and KI 0.1 mM caused a 33, 31 and 25% inhibition, respectively, of AIB uptake. The presence of 0.1 mM methimazole (MMI) only reversed the effect of KI. The transport of DOG was inhibited by both compounds: I-HO-A caused a 62% decrease, while IL-w produced a 64% inhibition; and MMI failed to relieve their action. On the contrary, the 33% inhibition caused by KI disappeared when MMI was present. Taking into account that AIB and DOG transport across the membrane requires energy, supplied by Na-K-ATPase, changes in its activity were studied. TSH (10 mU/ml) produced a 74% increase in the enzyme activity which was significantly blocked by KI (82%), I-HO-A (100%) and IL-w (100%). Basal enzyme activity was impaired by IL-w (33%), but not by KI. These results were correlated with the decrease of DOG uptake produced by 1 mM ouabain. Tissue specificity effect of iodoarachidonates was demonstrated by the absence of action on DOG transport in kidney and liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

The inhibition of PB125I formation in calf thyroid caused by 14-iodo-15-hydroxy-eicosatrienoic acid is due to decreased H2O2 availability

Previous work from our laboratory has shown that 14-iodo-15-hydroxy-5,8,11-eicosatrienoic acid (I-HO-A) is a potent inhibitor of iodine organification in calf thyroid slices. The present studies were performed in order to clarify the mechanism of this action. Incubation of thyroid slices with 10(-4)M I-HO-A caused a 47 and 53% decrease in PB125I formation after 30 and 60 min incubation, respectively. In a series of experiments an inverse relationship between the degree of inhibition caused by I-HO-A and total iodine content and basal iodoprotein formation was observed. Chromatographic analysis of the labeled compounds showed a significant decrease in 125I incorporation into MIT, DIT, T3 and total iodolipid. The site of the inhibitory effect of I-HO-A was then sought. TPO was measured by three different methods. When TPO was solubilized from I-HO-A treated slices, no change in enzymatic activity was observed. Moreover, the same lack of action was found when solubilized TPO was incubated with I-HO-A. The production and release of H2O2 into the incubation medium was measured by chemiluminiscence technique. In control slices the values increased during the first 10 min and reached a plateau. Pretreatment of the slices with 10(-4)M KI caused a 51% inhibition, while the same concentration of I-HO-A produced a 59% inhibition. The possibility that I-HO-A might exert its action through a putative protein inhibitor was also explored. Incubation of slices with 10(-5)M I-HO-A caused a 46% decrease in PB125I formation and addition of actinomycin D or puromycin failed to alter this effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Exposure of dog thyroid slices to acetylcholine induces refractoriness to its subsequent stimulation of glucose oxidation

An initial incubation of dog thyroid slices with 0.1 or 1 microM acetylcholine (ACH) for at least 2 h decreases its subsequent stimulation of [1-14C]glucose oxidation. Refractoriness persists for as long as 6 h in the absence of ACH. While new protein synthesis is essential for recovery, it is not necessary for its induction. Refractoriness is prevented when 25 microM tropicamide, an atropine-like drug, is present from the beginning of the initial incubation, but not when it is added after 2 h of incubation of slices with ACH, indicating that at this time ACH is no longer necessary for refractoriness. During refractoriness induced by ACH, stimulation of glucose oxidation by thyroid-stimulating hormone, prostaglandin E1, dibutyryl cyclic AMP, and cholera toxin, but not menadiol, is also significantly diminished. Incubation of thyroid slices with ACH does not modify its stimulation of iodide organification or 32Pi incorporation into phospholipids. These results suggest that the desensitization is not due to changes in the ACH receptor but rather to intracellular metabolic effects. This phenomenon may be important in the regulation of cholinergic effects on the thyroid.     

Effect of iodoarachidonates on thyroid FRTL-5 cells growth.

Excess iodide inhibits several thyroid parameters, by a putative organic iodocompound. Different iodolipids, including iodinated derivatives of arachidonic acid (IAs), are produced by rat, calf and pig thyroid. The action of two iodolactones, one bearing the iodine atom at the position 6 (IL-d) and the other at position 14 (IL-w) on growth of FRTL-5 cells was studied. KI, IL-w and IL-d exert a dose-related inhibition on FRTL-5 cell proliferation. The first two compounds caused inhibition at 1 microM while IL-d was effective at 10 microM. This inhibitory action of iodolactones (ILs) was not altered by 1 mM methyl-mercaptoimidazol (MMI), indicating that they exert their effect per se. The action of ILw on cell growth was reversible. The growth-stimulating effect of 10 microM forskolin was inhibited by IAs, showing that one possible site of action lies at the cAMP pathway. The present results give further support to our hypothesis about the role of IAs in thyroid growth autoregulation.     

The subcellular distribution of 32P-labelled phospholipids, 32P-labelled ribonucleic acid and 125I-labelled iodoprotein in pig thyroid slices. Effect in vitro of thyrotrophic hormone and dibutyryl-3′,5′-(cyclic)-adenosine monophosphate

1. The incorporation in vitro of [(32)P]phosphate into phospholipids and RNA and of [(125)I]iodide into protein-bound iodine by pig thyroid slices incubated for up to 6hr. was studied. The subcellular distribution of the labelled products formed after incubation with radioactive precursor in the nuclear, mitochondrial, smooth-microsomal, rough-microsomal and cell-sap fractions was also studied. 2. Pig thyroid slices actively took up [(32)P]phosphate from the medium during 6hr. of incubation; the rate of incorporation of (32)P into phospholipids was two to five times that into RNA. 3. The uptake of [(125)I]iodide by the slices from the medium was rapid for 4hr. of incubation, 6-10% of the label being incorporated into iodoprotein. 4. Much of the (32)P-labelled phospholipid accumulated in mitochondria and microsomes, whereas the nuclear fraction contained most of the (32)P-labelled RNA. After 2hr. of incubation most of the (32)P-labelled cytoplasmic RNA accumulated in the rough-microsomal fraction. The major site of localization of proteinbound (125)I was the smooth-microsomal fraction, and gradually increasing amounts appeared in the soluble cytoplasm fraction, suggesting a vectorial discharge of [(125)I]iodoprotein (presumably thyroglobulin) from smooth vesicles into the colloid. 5. The addition of 0.1-0.4 unit of thyrotrophic hormone/ml. of incubation medium markedly enhanced the accumulation of (32)P-labelled phospholipids in the microsomal fractions and to a much smaller extent that of (32)P-labelled RNA without any increase in the total uptake of the label. Almost simultaneously the hormone increased the uptake of [(125)I]iodide by the slices and enhanced the accumulation of protein-bound (125)I in the smooth-microsomal fraction. 6. As a function of time of incubation, thyrotrophic hormone had a biphasic effect on [(125)I]iodide uptake and protein-bound (125)I formation, the stimulatory effect being reversed after 4hr. of incubation. 7. 6-N-2'-O-Dibutyryl-3',5'-(cyclic)-AMP, but not 3',5'-(cyclic)-AMP or 5'-AMP, mimicked the action of thyrotrophic hormone on iodine uptake as well as on iodination of protein. On the other hand, the mimicry by 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP of the stimulatory effect of thyrotrophic hormone on the formation of labelled thyroid phospholipids and RNA was only an apparent one resulting from an enhanced uptake of [(32)P]phosphate. 8. It is concluded that thyrotrophic hormone causes a co-ordinated increase in the formation or accumulation of phospholipids, RNA and iodoprotein associated with the endoplasmic reticulum, and that 6-N-2'-O-dibutyryl-3',5'-(cyclic)-AMP mimics the more rapid effects of thyrotrophic hormone on transport and metabolic functions of thyroid cells, but does not influence their slower biosynthetic responses to the hormone.     

Role of calcium in acetylcholine-induced desensitization in dog thyroid slices

Incubation of dog thyroid slices with 1 microM acetylcholine (ACH) for 3 h followed by a second 2-h incubation without it induces a diminution of stimulation of glucose oxidation by ACH during a third incubation of 45 min. Using a calcium-free medium during all incubations prevents the desensitization and reduces, but does not abolish, ACH stimulation of glucose oxidation. EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid] (2 mM) added to the calcium-free medium in all incubations prevents both refractoriness and stimulation of glucose oxidation induced by ACH. Calcium depletion during the first incubation only, achieved by using EGTA and a calcium-free medium, also prevents refractoriness but not the augmentation of glucose oxidation caused by ACH. Incubation of thyroid slices with 1 microM ionophore A23817 during the 3-h first incubation decreases the stimulation of glucose oxidation induced by its readdition or by 1 microM ACH added for the first time in the third incubation. Ionophore-induced desensitization is not related to a cholinergic muscarinic receptor effect. Initial incubation of dog thyroid slices with 1 microM ACH diminishes the subsequent stimulation of glucose oxidation by 0.5 microM ionophore. However, the ACH-induced desensitization to ionophore can be overcome by a 10-fold increase in the amount of ionophore in the third incubation. Ionophore (1 microM) in the first incubation also induces refractoriness to thyroid-stimulating hormone (TSH) (10 mU/ml)-stimulated glucose oxidation in the third incubation. In contrast, initial incubation of thyroid slices with TSH (25 mU/ml) does not affect the stimulation of glucose oxidation by 0.5 microM ionophore added during the third incubation. These results suggest that increased intracellular calcium plays a major role in, or even mediates, ACH-induced desensitization in the thyroid gland.     

From the molecular characterization of iodide transporters to the prevention of radioactive iodide exposure

In the event of a nuclear reactor accident, the major public health risk will likely result from the release and dispersion of volatile radio-iodines. Upon body exposure and food ingestion, these radio-iodines are concentrated in the thyroid, resulting in substantial thyroidal irradiation and accordingly causing thyroid cancers. Stable potassium iodide (KI) effectively blocks thyroid iodine uptake and is thus used in iodide prophylaxis for reactor accidents. The efficiency of KI is directly related to the physiological inhibition of the thyroid function in the presence of high plasma iodide concentrations. This regulation is called the Wolff-Chaikoff effect. However, to be fully effective, KI should be administered shortly before or immediately after radioiodine exposure. If KI is provided only several hours after exposure, it will elicit the opposite effect e.g. lead to an increase in the thyroid irradiation dose. To date, clear evaluation of the benefit and the potential toxicity of KI administration remain difficult, and additional data are needed. We outline in this review the molecular characterization of KI-induced regulation of the thyroid function. Significant advances in the knowledge of the iodide transport mechanisms and thyroid physiology have been made. Recently developed molecular tools should help clarify iodide metabolism and the Wolff-Chaikoff effect. The major goals are clarifying the factors which increase thyroid cancer risk after a reactor accident and improving the KI administration protocol. These will ultimately lead to the development of novel strategies to decrease thyroid irradiation after radio-iodine exposure.     

Stimulation by iodide of H(2)O(2) generation in thyroid slices from several species

The regulation of thyroid metabolism by iodide involves numerous inhibitory effects. However, in unstimulated dog thyroid slices, a small inconstant stimulatory effect of iodide on H(2)O(2) generation is observed. The only other stimulatory effect reported with iodide is on [1-(14)C]glucose oxidation, i.e., on the pentose phosphate pathway. Because we have recently demonstrated that the pentose phosphate pathway is controlled by H(2)O(2) generation, we study here the effect of iodide on basal H(2)O(2) generation in thyroid slices from several species. Our data show that in sheep, pig, bovine, and to a lesser extent dog thyroid, iodide had a stimulatory effect on H(2)O(2) generation. In horse and human thyroid, an inconstant effect was observed. We demonstrate in dogs that the stimulatory effect of iodide is greater in thyroids deprived of iodide, raising the possibility that differences in thyroid iodide pool may account, at least in part, for the differences between the different species studied. This represents the first demonstration of an activation by iodide of a specialized thyroid function. In comparison with conditions in which an inhibitory effect of iodide on H(2)O(2) generation is observed, the stimulating effect was observed for lower concentrations and for a shorter incubation time with iodide. Such a dual control of H(2)O(2) generation by iodide has the physiological interest of promoting an efficient oxidation of iodide when the substrate is provided to a deficient gland and of avoiding excessive oxidation of iodide and thus synthesis of thyroid hormones when it is in excess. The activation of H(2)O(2) generation may also explain the well described toxic effect of acute administration of iodide on iodine-depleted thyroids.     

Effects of forskolin on adenylate cyclase,cyclic AMP,protein kinase and intermediary metabolism of the thyroid gland

Forskolin (40 μM) stimulated adenylate cyclase activities of bovine thyroid plasma membranes without pthe addition of guanine nucleotides. GDP had little effect on the forskolin-stimulated adenylate cyclase activity while Gpp[NH]p (0.1–1.0 μM) decreased it. In the presence of TSH (10 mU/0.11), Gpp[NH]p no longer caused inhibition. Forskolin did not affect phosphodiesterase activities of thyroid homogenates. Forskolin (10 μM) rapidly increased cAMP levels in bovine thyroid slices both in the absence and presence of a phosphodiesterase inhibitor. The effect of TSH (50 mU/ml) on cAMP levels was additive or greater than additive to that of forskolin. An initial 2-h incubation of slices with forskolin did not decrease their subsequent cAMP responses to either forskolin and/or TSH while similar treatment of slices with TSH induced desensitization of the cAMP response to TSH, but not to forskolin. Forskolin (10 μM) as well as TSH (50 mU/ml) activated cAMP-dependent protein kinase of slices in the absence of a phosphodiesterase inhibitor. Although forskolin activated the adenylate cyclase cAMP system, it did not stimulate iodide organification or glucose oxidation, effects which have been attributed to cAMP. In fact, forskolin inhibited these parameters and ³²P incorporation into phospholipids as well as their stimulation by TSH. These results indicate that an increase in cAMP levels and cAMP-dependent protein kinase activity in thyroid slices may not necessarily reproduce the effects of TSH on the thyroid.     

The effect of long-term external ionizing radiation on the functional activity of rat thyroid under enhanced potassium iodide consumption

The study was devoted to the effect of long-term (20 days) external ionizing radiation at a dose of 0.5 Gy on the iodide metabolism in the rat thyroid under supplementation of high iodine doses (10 daily KI doses). It was found that the potassium iodide administration partially prevented the effects of a post radiation decrease of serum thyroid hormone levels (the level of T4 was normal and that of T3 was 77.4% of the controls). After the supplementation of 10 daily iodide doses, the rat thyroid tissue showed the most pronounced increase in the levels of total, free and protein-bound iodide compared to the groups of animals consuming normal and elevated KI doses. Pronounced inhibition of thyroid peroxidase activity (3.1-fold) was noted in the same group. The data obtained indicate a radiation-induced activation of iodide uptake during its enhanced supplementation and disturbed iodide enzymatic oxidation and organification.     

Incubation of bovine thyroid slices with thyrotropin is associated with a decrease in the ability of pertussis toxin to adenosine diphosphate-ribosylate guanine nucleotide regulatory component(s)

Pretreatment of bovine thyroid slices with TSH resulted in desensitization of TSH-sensitive adenylyl cyclase activity but no change in stimulatory nucleotide binding regulatory component of adenylyl cyclase (Gs) activity assessed by reconstitution of the Gs-defective cyc-S49 adenylyl cyclase system. Possible changes in substrates for pertussis toxin (PT)-induced ADP ribosylation due to TSH treatment and/or in endogenous ADP ribosylation of membrane proteins were explored. Using 10 microM [32P]NAD+ as substrate, endogenous ADP ribosylation was not observed in membranes from control or TSH-treated slices. ADP ribosylation of alpha-subunits of Gs by cholera toxin was also unaffected by incubation of thyroid slices with TSH. In contrast, ADP ribosylation of 40 kilodalton (kDa) substrates for PT was decreased between 40% and 60% by TSH treatment. This effect of TSH was dependent on its concentration and the time of incubation of the slices and was specific for labeling of the 40 kDa PT substrate. Prostaglandin E1 treatment of thyroid slices, which results in a much smaller homologous desensitizing effect, did not result in changes in ADP ribosylation by PT. The effect of incubation of slices with TSH was abolished by pretreatment of the membranes with 0.3-1.0% Lubrol PX, which increased the labeling of the 40 kDa polypeptides. The data suggests that TSH induces in thyroid tissue a redistribution of 40 kDa polypeptides changing their availability to PT.     

Intracellular and extracellular sites of iodination in dispersed hog thyroid cells.

Iodination and hormone synthesis has been studied in isolated hog thyroid cells in suspension. We characterized three iodination processes by use of pharmacological agents. (1) Intracellular iodination dependent on active iodide transport, which was inhibited by NaClO4 or ouabain, but not by catalase. This iodination was linear for 6h with no apparent Km for iodide of 1.5 muM, was stimulated by thyrotropin or N6O2'-dibutyryladenosine 3':5'-cyclic monophosphate, yielded mostly iodinated thyroglobulin and was efficient for tetraiodothyronine synthesis. (2) Extracellular iodination, which was sensitive to catalase, but not to NaClO4 or ouabain. This iodination plateaued after 2h and the apparent Km was 16.5 muM. This process was insensitive to thyrotropin and dibutyryl cyclic AMP. The major products were iodoprotein other then thyroglobulin and iodolipid and the yield of tetraiodothyronine was low. (3) Intracellular iodination from passively diffused iodide, which was not sensitive to inhibitors. Other characteristics of passive intracellular iodination were intermediate between active intracellular iodination and extracellular iodination. The fact that the three processes are inhibited by similar concentrations of methimazole, and their apparent Km values, when corrected for the concentrating effect of iodide trapping, are all of the same order as the Km of purified thyroid peroxidases, suggest that although their locations are different, the enzymic systems involved are identical. These results show that, besides an extracellular site of iodination, dispersed thyroid cells process an intracellular site of iodination with biochemical characteristics of physiological relevance.     

Thyroid-stimulating hormone increases active transport of perchlorate into thyroid cells

Perchlorate blocks thyroidal iodide transport in a dose-dependent manner. The human sodium/iodide symporter (NIS) has a 30-fold higher affinity for perchlorate than for iodide. However, active transport of perchlorate into thyroid cells has not previously been demonstrated by direct measurement techniques. To demonstrate intracellular perchlorate accumulation, we incubated NIS-expressing FRTL-5 rat thyroid cells in various concentrations of perchlorate, and we used a sensitive ion chromatography tandem mass spectrometry method to measure perchlorate accumulation in the cells. Perchlorate caused a dose-related inhibition of 125-iodide uptake at 1-10 microM. The perchlorate content from cell lysate was analyzed, showing a higher amount of perchlorate in cells that were incubated in medium with higher perchlorate concentration. Thyroid-stimulating hormone increased perchlorate uptake in a dose-related manner, thus supporting the hypothesis that perchlorate is actively transported into thyroid cells. Incubation with nonradiolabeled iodide led to a dose-related reduction of intracellular accumulation of perchlorate. To determine potential toxicity of perchlorate, the cells were incubated in 1 nM to 100 microM perchlorate and cell proliferation was measured. Even the highest concentration of perchlorate (100 microM) did not inhibit cell proliferation after 72 h of incubation. In conclusion, perchlorate is actively transported into thyroid cells and does not inhibit cell proliferation.     

Negative Effect of Iodide On the Survival of Newly Divided Epithelial Cells In Chronically Stimulated Rat Thyroid

Abstract. The aim of this work was to investigate some aspects of the thyroid epithelial cell kinetics during the iodide-induced involution of a hyperplastic goitre in the rat. Rats were made iodine-deficient for 6 months, and propylthiouracil (PTU) (0.15%) was added to the diet during the last 2 months. Thereafter, rats were refed with iodide and PTU was removed (day 0).
Forty-eight hours previously, all the rats were injected with tritiated thymidine ([³H]TdR) (1 μCi/g). Some animals were killed 1 hr or 24 hr after [³H]TdR injection (i.e. on day -2 and -1, day O corresponding to the restoration of a normal iodine diet); the other animals were killed after different delay periods and following L³H]TdR injection. Autoradiography of thyroid sections, iodine determination of plasma iodide and protein-bound iodine (PBI), and RIA of plasma thyroid stimulatory hormone (TSH) were performed. Plasma TSH concentration was very high on day O of iodide refeeding (3000 ± 330 ng/ml) and remained at this level until day 8. Plasma PBI was very low on day O, remained so until day 4 and greatly increased on day 8. Plasma iodide was also very low on day O, but markedly increased on day 1, then did not vary significantly until day 43 of iodine refeeding. Thyroid weight, elevated on day O, decreased relatively quickly until day 30, then more slowly until day 73.     

Spectral studies with lactoperoxidase and thyroid peroxidase: interconversions between native enzyme, compound II, and compound III

Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide.     

Stimulatory effects of potassium iodide on auxin action in the coleoptile segments of maize (Zea mays)

Potassium iodide (KI) was found to stimulate IAA-induced elongation of coleoptile segments in maize (Zea mays L.). The promoting effects of KI on coleoptile elongation, which were optimal at 1 mM in the presence of IAA, did not occur as a result of better conservation of IAA in the incubation medium. In addition, KI did not affect fusicoccin- or epibrassinolide-induced elongation. Additionally, sodium iodide (NaI) induced similar stimulatory effects on IAA-induced elongation, however, potassium chloride (KCl) showed no effect, suggesting that iodide is the active component. KI also enhanced IAA-induced ethylene biosynthesis in maize coleoptile segments. Taken together, these results suggest the involvement of KI-sensitive step(s) in auxin action before effectors of the signal transduction pathway split to elongation growth and ethylene biosynthesis. In-yong Hwang and Soo Chul Chang contributed equally to this work.     

Fluorescence quantitation of thyrocyte iodide accumulation with the yellow fluorescent protein variant YFP-H148Q/I152L

The thyroid gland accumulates iodide for the synthesis of thyroid hormones. The aim of the current study was to quantify iodide accumulation in cultured thyroid cells by live cell imaging using the halide-sensitive yellow fluorescent protein (YFP) variant YFP-H148Q/I152L. In vivo calibrations were performed in FRTL-5 thyrocytes to determine the sensitivity of YFP-H148Q/I152L to iodide. In the presence of ion-selective ionophores, YFP-H148Q/I152L fluorescence was suppressed by halides in a pH-dependent manner with 20-fold selectivity for iodide versus chloride and competition between the two halides. At a physiological pH of 7 and a chloride concentration of 15mM, the affinity constant of YFP-H148Q/I152L for iodide was 3.5mM. In intact FRTL-5 cells, iodide induced a reversible decrease in YFP-H148Q/I152L fluorescence. FRTL-5 cells concentrated iodide to 60 times the extracellular concentration. Iodide influx exhibited saturation kinetics with respect to extracellular iodide with a K(m) of 35 microM and a V(max) of 55 microM/s. Iodide efflux exhibited saturation kinetics with respect to intracellular iodide concentration with a K(m) of 2.2mM and a V(max) of 43 microM/s. The results of this study demonstrate the utility of YFP-H148Q/I152L as a sensitive and selective biosensor for the quantification of iodide accumulation in thyroid cells.     

Studies on doses of methimazole (MMI) and its administration regimen on broiler metabolism

We designed three experiments to determine both the optimal dose of and time on experiment for methimazole (MMI; 1-methyl-2-mercaptimidazole). Our goals were to determine if chicken growth was related to thyroid hormone levels and if intermediary metabolism changed along with changes in thyroid hormone levels. Initiating MMI at one week of age decreased (P<0.01) plasma thyroid levels and growth in four-week old birds. In contrast, initiating MMI at two and three weeks of age decreased (P<0.05) hormone levels without affecting growth as severely. Although initiating MMI at two weeks of age depressed (P<0.05) plasma thyroid hormones at four weeks, there was little change in vitro lipogenesis at four weeks. Again, initiating MMI at one week of age decreased body weight, plasma thyroid hormones and in vitro lipogenesis at four weeks of age. In addition, this treatment also decreased (P<0.05) malic enzyme activity at this same age period. The second experiment showed that MMI, initiated at 14 days, had no significant effect on 28-day body weight and again decreased both plasma T(3) and T(4) but T(3) replacement increased plasma T(3) in both 14-28-day treatment groups. All body weights were similar at 30 days, however. Lastly, diets containing graded levels of MMI decreased thyroid hormones and body weight (0>0.25>0.5>1 g MMI/kg). In contrast, only the two higher levels (0.5 and 1 g MMI/kg) decreased in vitro lipogenesis. Growth depression, caused by MMI feeding, can occur without changes in lipid metabolism. The length of MMI administration may be as important as dose level in obtaining effects (growth, thyroid hormone depression and inhibition of lipogenesis).     

Effect of antioxidants (vitamin C,E and turmeric extract) on methimazole induced hypothyroidism in rats

The study was to investigate the protective effect of antioxidants against methimazole (MMI) induced hypothyroidism in rats. Male Wistar rats were fed MMI, MMI plus vitamin C, MMI plus vitamin E and MMI plus turmeric extract (TE) supplemented diet. At the end of the experiments, thyroid weights, thyroxine (T4), triiodothyronine (T3) and cholesterol levels were determined. It was observed that MMI treated rats showed increase in thyroid weights, very low levels of circulating T4, T3 and increased levels of total cholesterol as compared to controls (P< 0.001). However, rats which received Vit. C, Vit. E or TE along with MMI showed reduced weights (38-55% less) in thyroid glands (P < 0.01), less suppressed T4 and T3 levels (2-6% and 7-35% respectively) and less increase in total cholesterol levels (19-52%) which are statistically significant. The data suggest the positive effect of antioxidants on thyroid gland which could be due to direct involvement of antioxidants on thyroid gland.     

Iodide-induced inhibition of adenylate cyclase activity in horse and dog thyroid

The characteristics of the iodide-induced inhibition of cyclic AMP accumulation in dog thyroid slices have been previously described [Van Sande, J., Cochaux, P. and Dumont, J. E. (1985) Mol. Cell. Endocrinol. 40, 181-192]. In the present study we investigated the characteristics of the iodide-induced inhibition of adenylate cyclase activity in dog and horse thyroid. The inhibition of cyclic AMP accumulation by iodide in stimulated horse thyroid slices was similar to that observed in dog thyroid slices. The inhibition was observed in slices stimulated by thyroid-stimulating hormone, cholera toxin and forskolin. Increasing the concentration of the stimulators did not overcome the iodide-induced inhibition. Adenylate cyclase activity, assayed in crude homogenates or in plasma-membrane-containing particulates (100,000 x g pellets), was lower in homogenates or in particulates prepared from iodide-treated slices than from control slices. This inhibition was observed on the cyclase activity stimulated by forskolin, fluoride or guanosine 5'-[beta, gamma-imino]triphosphate, but also on the basal activity. It was relieved when the homogenate was prepared from slices incubated with iodide and methimazole. Similar results were obtained with dog thyroid. The inhibition persisted when the particulate fraction was washed three times during 1 h at 100,000 x g, in the presence of bovine serum albumin or increasing concentration of KCl. It was similar whatever the duration of the cyclase assay, in a large range of protein concentration. These results indicate that a stable modification of adenylate cyclase activity, closely related to the plasma membrane, was induced when slices were incubated with iodide. Iodide inhibition did not modify the affinity of adenylate cyclase for its substrate (MgATP), but induced a decrease of the maximal velocity of the enzyme. The percentage inhibition was slightly decreased when Mg2+ concentration increased, and markedly decreased when Mn2+ concentration increased. A detectable adenylate cyclase activity was demonstrated when intact slices were incubated in the presence of [alpha-32P]ATP, probably because of the presence of broken cells produced during the slicing. Iodide had no direct effect on this cyclase system, which confirms that iodide needs the integrity of the cell to induce the inhibition and suggests that the inhibition is not transmitted between cells.