The effect of iodide and chloride on transthyretin structure and stability

Transthyretin amyloid formation occurs through a process of tetramer destabilization and partial unfolding. Small molecules, including the natural ligand thyroxine, stabilize the tetrameric form of the protein, and serve as inhibitors of amyloid formation. Crucial for TTR's ligand-binding properties are its three halogen-binding sites situated at the hormone-binding channel. In this study, we have performed a structural characterization of the binding of two halides, iodide and chloride, to TTR. Chlorides are known to shield charge repulsions at the tetrameric interface of TTR, which improve tetramer stability of the protein. Our study shows that iodides, like chlorides, provide tetramer stabilization in a concentration-dependent manner and at concentrations approximately 15-fold below that of chlorides. To elucidate binding sites of the halides, we took advantage of the anomalous scattering of iodide and used the single-wavelength anomalous dispersion (SAD) method to solve the iodide-bound TTR structure at 1.8 A resolution. The structure of chloride-bound TTR was determined at 1.9 A resolution using difference Fourier techniques. The refined structures showed iodides and chlorides bound at two of the three halogen-binding sites located at the hydrophobic channel. These sites therefore also function as halide-binding sites.     

The effect of a purified preparation of lecithinase C on iodide transport

Treatment of porcine thyroid slices with highly purified lecithinase C leads to a marked reduction in the ability to accumulate iodide ion. Although this response is less sensitive as a function of lecithinase concentration than is the ability to respond to thyrotropin with an increase in glucose oxidation, phospholipid synthesis or adenyl cyclase activity, it occurs when the baseline values of these parameters are not substantially altered.     

大豆过氧化物酶－过氧化氢－碘化钾体系杀菌作用

【目的】研究豆壳过氧化物酶(SBP)-H2O2-KI三元体系杀菌作用及其可能的杀菌机制。【方法】以细菌生长的浊度(OD600)为指标检测SBP-H2O2-KI体系的抑菌作用;以活细胞计数(CFU)为指标检测SBP-H2O2-KI体系的杀菌作用;以最低抑菌浓度(MIC)为指标检测亚致死剂量的SBP-H2O2-KI体系作用下连续传代细菌的敏感性变化;以物理和化学方法检测SBP-H2O2-KI体系中活性氧基团等功能基团的生成与否,以期解释SBP-H2O2-KI体系的杀菌机制。【结果】SBP-H2O2-KI体系对多种细菌有高效、快速杀菌作用,作用时间仅为几分钟。在亚致死剂量浓度下连续培养的细菌悬液对体系的耐受能力(MIC)没有显著性变化,从中也不能分离到抗性/耐性突变株。物理和化学方法检测结果表明SBP-H2O2-KI反应过程中无羟基自由基产生;化学方法检测结果表明SBP-H2O2-KI反应过程中无超氧阴离子自由基产生;而有单线态氧和单质碘的产生。【结论】根据实验结果可以推测:SBP-H2O2-KI体系的杀菌力可能主要来源于单线态氧和碘的活性中间态,而不是羟基自由基和超氧阴离子。此外,SBP-H2O2-KI体系所具备的高效、快速的杀菌作用,以及细菌对其不易产生抗性的特点,预示此体系在医疗以及植保等方面有广泛的应用前景。     

Inhibition by iodide of iodide binding to proteins: the "Wolff-Chaikoff" effect is caused by inhibition of H2O2 generation

H2O2 generation is limiting the oxidation and binding to proteins of iodide. In dog thyroid slices thyrotropin and carbamylcholine greatly enhance protein iodination and H2O2 generation. The action of thyrotropin is mimicked by dibutyryl cyclic AMP and forskolin which suggests that it is mediated by cyclic AMP. The action of carbamylcholine was mimicked by ionomycin and by phorbol myristate ester. This suggests that the effect of carbamylcholine is mediated by the two intracellular signals generated by the Ca++ phosphatidylinositol cascade: Ca++ and diacylglycerol. The Wolff-Chaikoff effect is the inhibition by iodide of its own organification. In dog thyroid slices, iodide greatly inhibited H2O2 generation stimulated by thyrotropin and by carbamylcholine. Iodide decreased the production of intracellular signals induced by TSH and carbamylcholine but it also inhibited the action of probes of these intracellular signals (dibutyryl cAMP, forskolin, ionomycin, phorbol-myristate ester) on the H2O2 generating system itself. These effects were suppressed by methimazole an inhibitor of iodide oxidation.     

碘甲烷对大鼠肝脏合成功能的影响

目的研究碘甲烷对大鼠肝脏合成功能的影响。方法将24只雄性sD大鼠随机分成4组,放入静式染毒罐中,实验组分别给予浓度为650mg／m3、260mg／m3、130mg／m3的碘甲烷,每天吸入染毒4h,连续染毒一周,对照组给予相同环境条件不染毒。染毒结束后对血清总蛋白（TP）、血清白蛋白（ALB）、血清球蛋白（GLO）、血清胆碱酯酶（CHE）、血清谷丙转氨酶（ALT）以及血清谷草转氨酶（AST）进行测定。结果随着碘甲烷浓度的升高,大鼠TP含量逐渐降低,高、中、低剂量组与对照组存在统计学差异,且高剂量组与中、低剂量组也存在统计学差异。ALB浓度随着碘甲烷浓度的升高逐渐降低,高、中、低剂量组与对照组存在统计学差异,且高剂量组与中、低剂量组也存在统计学差异。GLO含量随着碘甲烷浓度升高变化不大,各组间差异无统计学意义。CHE活力随着碘甲烷浓度升高逐渐降低,各组之间差异均有统计学意义。ALT含量随着碘甲烷浓度的升高逐渐升高,且实验组与对照组相比存在统计学差异,高剂量组与低剂量组比较差异有统计学意义。AST含量随着碘甲烷浓度的升高逐渐升高,实验组与对照组之间比较存在统计学差异,高、中剂量组与低剂量组比较,存在统计学差异。结论碘甲烷暴露对大鼠的肝脏合成功能有损害作用。     

A nonradioactive iodide uptake assay for sodium iodide symporter function

The standard assay for sodium iodide symporter (NIS) function is based on the measurement of radioiodide uptake (¹²⁵I) in NIS-expressing cells. However, cost and safety issues have limited the method from being used widely. Here we describe a simple spectrophotometric assay for the determination of iodide accumulation in rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast and highly reproducible with a Z′ factor of 0.70. This procedure allows the screening of more than 800 samples per day and can easily be adapted to robotic systems for high-throughput screening of NIS function modulators. Using this method, the potency of several known inhibitors of NIS function was evaluated in a single day with high accuracy and reliability. Measured IC₅₀ values were essentially identical to those determined using Na¹²⁵I.     

Acetylcholine receptor-controlled ion translocation caused by phenyltrimethylammonium and nereistoxin: simple estimation of equilibrium constants of the minimal model

The rate of slow Li+ influx and the fraction of active form of acetylcholine receptor (AChR) of Electrophorus electricus membrane vesicles at equilibrium between the active and desensitized forms of the receptor were measured in the presence of various concentrations of phenyltrimethylammonium (PTA) and nereistoxin (NTX), by a simple filtration assay and flame emission spectroscopy. The equilibrium constants of these ligands in the minimal model, which accounts for the AChR-mediated ion flux, were estimated simply from these two measurements, since the equilibrium constants for acetylcholine (ACh) and carbamylcholine (Carb) estimated from two kinetic measurements agreed well with those estimated from five sophisticated kinetic measurements of AChR-mediated ion fluxes. PTA showed high potency but not high efficacy, and showed inhibition when large doses were applied. NTX showed both low potency and low efficacy and acted as an inhibitor when it was added with Carb. The apparent dissociation constants of these three agonists evaluated from the minimal model and the equilibrium constants agreed with those obtained by assay of inhibition of radiolabeled ligand binding.     

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.     

Dynamic iodide trapping by tumor cells expressing the thyroidal sodium iodide symporter

The thyroidal sodium iodide symporter (NIS) in combination with various radioactive isotopes has shown promise as a therapeutic gene in various tumor models. Therapy depends on adequate retention of the isotope in the tumor. We hypothesized that in the absence of iodide organification, isotope trapping is a dynamic process either due to slow efflux or re-uptake of the isotope by cells expressing NIS. Iodide efflux is slower in ARH-77 and K-562 cells expressing NIS compared to a thyroid cell line. Isotope retention half times varied linearly with the number of cells expressing NIS. With sufficient NIS expression, iodide efflux is a zero-order process. Efflux kinetics in the presence or absence of perchlorate also supports the hypothesis that iodide re-uptake occurs and contributes to the retention of the isotope in tumor cells. Iodide organification was insignificant. In vivo studies in tumors composed of mixed cell populations confirmed these observations.     

EDTA inhibits peroxidase-catalyzed iodide oxidation through interaction at the iodide binding site

EDTA inhibits the formation of I3- from iodide catalysed by various pure peroxidases. The inhibition is concentration-dependent and chloroperoxidase (CPO) is more sensitive than horseradish peroxidase (HRP) and lactoperoxidase (LPO). EDTA is more active than EGTA or other biological chelators tested. Zn2+, Mn2+ and Co2+ are equally active in reversing the effect of EDTA on both CPO and HRP almost completely, but ineffective in the case of LPO. The effect of EDTA on HRP can be reversed by a higher concentration of iodide but not by H2O2. EDTA causes a hypsochromic change in the absorption of the Soret band of HRP at 402 nm, and iodide can reverse this effect. EDTA can effectively displace radioiodide specifically bound to HRP. It is suggested that EDTA inhibits iodide oxidation by interacting at the iodide binding site of the HRP.     

侧脑室注射Orexin-1受体拮抗剂对大鼠心血管效应的影响

目的:用侧脑室微量注射和免疫组化方法研究食欲素-1受体(OX1R)拮抗剂SB408124对麻醉大鼠的心血管效应及其作用机制。方法:雄性SD大鼠,侧脑室微量注射SB408124,及甲硝阿托品、六甲溴铵静脉注射预处理后侧脑室注射SB408124,观测动脉血压(MAP)和心率(HR)的变化。然后,用免疫组化方法检测侧脑室注射SB408124对大鼠脑延髓头端腹外侧区(RVLM)内酪氨酸羟化酶(TH)阳性神经元的影响。结果:侧脑室注射SB408124可显著降低麻醉大鼠的MAP和HR,但是HR变化不如MAP变化明显。应用六甲溴铵可完全阻断SB408124的心血管效应,但甲硝阿托品不能阻断SB408124的心血管效应。侧脑室注射SB408124可使鼠脑延髓头端腹外侧区内酪氨酸羟化酶阳性神经元的数量显著降低。结论:侧脑室注射OX1R拮抗剂SB408124可显著降低麻醉大鼠的MAP和HR,其作用主要是通过抑制交感神经系统的活性而实现的。     

Effect of internally loaded iodide, thiocyanate, and perchlorate on sodium-dependent iodide uptake by phospholipid vesicles reconstituted with thyroid plasma membranes: iodide counterflow mediated by the iodide transport carrier

Na+-dependent I- transport and I- counterflow were studied using phospholipid vesicles (P-vesicles) made of porcine thyroid plasma membranes and soybean phospholipid by sonication. 1) I- uptake by P-vesicles incubated in the presence of external Na+ was higher than that by P-vesicles incubated in choline+ instead of Na+. The vesicles exhibited Na+-dependent I- uptake. When P-vesicles were internally loaded with I- prior to incubation in Na+, a further increase in Na+-dependent I- uptake was observed, although the concentration of internal I- was very much higher than that outside. In the absence of external Na+, I- uptake by P-vesicles preloaded with I- was comparable to baseline uptake. 2) Na+-dependent I- uptake by P-vesicles not loaded with I- and enhanced Na+-dependent I- uptake by P-vesicles preloaded with I- were both inhibited by either of SCN- and ClO4- added outside the vesicles. 3) When P-vesicles were loaded with SCN- instead of I- and incubated in Na+, I- uptake by these vesicles was also higher than baseline Na+-dependent I- uptake. However, a ClO4- load did not result in an increase in I- uptake. These results indicate that Na+-dependent I- transport including Na+-dependent I- counterflow is specifically mediated by the thyroid I- carrier. SCN- - I- counterflow in addition to I- - I- counterflow occurs dependently on Na+, but ClO4- - I- counterflow does not.     

Acute and chronic leptin effect upon in vivo and in vitro rat thyroid iodide uptake

Leptin has stimulatory effects on the hypothalamic-pituitary-thyroid axis and on deiodinases activities. Here, we evaluated the effect of leptin injection upon in vivo and in vitro thyroid 125I uptake (RAIU). We designed two experiments: acute leptin (LepA) with a single dose of leptin (8 microg/100 g BW/sc), and chronic leptin (LepC), injected with the same dose of LepA, once a day, for 6 days. In parallel, control groups were saline-injected. For in vivo study, part of the animals were injected with 125I (3700 Bq) and killed after 15 or 120 min. In vivo thyroid RAIU was not changed in LepA animals. However, LepC animals showed higher in vivo thyroid RAIU (15 min:+130% and 120 min:+72%; p<0.05). For in vitro study, the other animals were killed and their thyroids were incubated with 125I. Thyroids explants from LepA and LepC groups presented lower thyroid 125I content (-32% and -29% p<0.05, respectively). The amount of our data suggest that, in vitro, leptin causes a direct inhibition of the rat thyroid RAIU, but in vivo, the effect of leptin was different according to the treatment period, which indicates that other indirect mechanisms are involved in the in vivo leptin chronic stimulation of the thyroid gland.     

Efflux of preloaded iodide from the thyroid induced by externally added iodide. A study using a biological model of the thyroid iodide transport system

Efflux of preloaded I- from the thyroid induced by externally added I- was studied using a biological model of the thyroid I- transport system. Phospholipid vesicles (P-vesicles) made from thyroid plasma membranes and soybean phospholipids were capable of accumulating I- in the presence of external Na+. P-vesicles incubated in 136 mM Na+ containing 0.9 microM I- with 125I- for 2 min accumulated I- so that the I- concentration in the vesicles became about 2 microM. Addition of 5-20 microM stable I- to the incubation mixture at 2 min incubation resulted in a dose-dependent decrease in previously loaded 125I- in the vesicles. In other words, a dose-dependent increase in efflux of preloaded 125I- was observed. While the efflux occurred, Na+-dependent I- influx into P-vesicles was preserved. When 2 mM ClO4-, a specific inhibitor of Na+-dependent I- influx, was added together with 10 microM I-, the external I- failed to diminish preloaded 125I- in P-vesicles. The 125I- efflux did not occur when a large amount of stable I- entered P-vesicles independently of Na+ in the presence of ClO4-. Similar 125I- efflux induced by externally added 5 microM SCN- was also blocked by simultaneously added ClO4-. These observations suggest that such I- efflux from the thyroid is a certain type of uphill I- transport which is closely related to Na+-dependent I- transport and that ClO4- and SCN- act on a common site of the I- transport system.     

Negative effect of iodide on the survival of newly divided epithelial cells in chronically stimulated rat thyroid

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 microCi/g). Some animals were killed 1 hr or 24 hr after [3H]TdR injection (i.e. on day -2 and -1, day 0 corresponding to the restoration of a normal iodine diet); the other animals were killed after different delay periods and following [3H]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 0 of iodide refeeding (3000 +/- 330 ng/ml) and remained at this level until day 8. Plasma PBI was very low on day 0, remained so until day 4 and greatly increased on day 8. Plasma iodide was also very low on day 0, but markedly increased on day 1, then did not vary significantly until day 43 of iodine refeeding. Thyroid weight, elevated on day 0, decreased relatively quickly until day 30, then more slowly until day 73. The [3H]TdR labelling index (LI) of the thyroid epithelial cells (TEC) was high on day 0 (56 +/- 3 labelled cells/10,000 cells), and 24 hr thereafter increased to 104 +/- 3, by division of the labelled cells. On day 1 of iodine refeeding, the LI had abruptly decreased to about half this value and then remained stable for 3 more days. Between day 4 and day 16, a progressive decline in the LI, (by about 3-4 per day), was observed. The LI showed no further modification, up to day 73, the longest period investigated. The decrease in LI occurred without any significant changes in the labelling intensity (grain count) of the remaining labelled cells between day 1 and 16, this indicates that no cell division took place during this period. The data are therefore interpreted as showing a biphasic elimination after iodide refeeding, of cells that were actively proliferating during the goitrous state.(ABSTRACT TRUNCATED AT 400 WORDS)