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PubMed1996

Inhibition of experimentally induced cirrhosis in rats by hypothyroidism.

The coexistence of hyperkinetic circulation, hypermetabolism, and hyperactivity of the sympathetic nervous system is encountered in both cirrhosis and hyperthyroidism. Several drugs, such as propylthiouracil and propranolol, that are beneficial for treating some patients with chronic liver diseases are also prescribed for the treatment of thyrotoxicosis. We investigated the effects of experimentally induced hypo- and hyperthyroidism on the development of cirrhosis induced in rats by thioacetamide (TAA). We specifically examined whether hypothyroidism could prevent and hyperthyroidism could aggravate liver damage. Hypothyroidism induced by methimazole (MMI, 0.04%), propylthiouracil (PTU 0.05%), and by thyroidectomy was confirmed by a significant elevation of thyroid-stimulating hormone (TSH) levels. Hyperthyroidism (decreased TSH levels) was induced by eltroxin (ELT:50 micrograms/kg). Thirteen groups of 10 rats each were studied: euthyroid controls (3 groups: water, TAA 1.5 months, and TAA 3 months), hypothyroid (6 groups: MMI, PTU, surgical, MMI-TAA, PTU-TAA, surgical-TAA), and hyperthyroid (4 groups:ELT 1.5 months and 3 months, and ELT-TAA for 1.5 months and 3 months). Hepatic fibrosis (scored from 0 to 3) was significantly reduced (P < .0001) in hypothyroid rats as compared with euthyroid controls, and was aggravated in TAA-treated hyperthyroid rats (P < .0001). Quantitative microscopic analysis of liver biopsy specimens from all groups confirmed the semiquantitative histopathological scores (P < .001). Direct intrasplenic pressure measurement revealed a significant portal pressure elevation in the TAA and the ELT-treated rats (from 4.7 +/- 0.1 in the euthyroid group to 8.1 +/- 2.3 and 10.2 +/- 2.1 and 12.5 +/- 1.6 in the TAA, ELT and ELT-TAA groups, respectively). However, in the hypothyroid-TAA groups, the portal pressure was found to be within the euthyroid normal range (4.6 +/- 1.2 and 5.8 +/- 0.6 in the PTU-TAA and surgical-TAA, respectively). After 12 weeks, the mean spleen weight of rats receiving only TAA was significantly higher than the TAA-treated hypothyroid rats (P < .0001), indicating that the hypothyroid TAA-treated rats were less portal hypertensive. These results suggest that induced hypothyroidism can inhibit, whereas hyperthyroidism can aggravate, the development of cirrhosis in a rat model.

Animals↗

PubMed1998

Conserved cysteines in the type 1 deiodinase selenoprotein are not essential for catalytic activity.

The iodothyronine deiodinases are a family of oxidoreductases that catalyze the removal of iodide from thyroid hormones. Each of the three isoforms contain selenocysteine at its active site and several cysteine residues that may be important for catalytic activity. Of particular interest in the type I deiodinase (D1) is Cys124, which is vicinal to the selenocysteine at position 126, and Cys194, which has been conserved in all deiodinases identified to date. In the present studies, we have characterized the functional properties of C124A, C194A, and C124A/C194A D1 mutants, which were prepared by site-directed mutagenesis and expressed in COS-7 cells. In broken cell preparations, the sensitivity of the mutants to the selective D1 inhibitors propylthiouracil and aurothioglucose were unaltered. Mutagenesis at the Cys124 position was associated with a 7-11-fold increase in the Km of dithiothreitol, whereas Vmax values remained largely unchanged. However, both mutations resulted in marked decreases in Vmax values when glutathione or a reconstituted thioredoxin cofactor system were used in the assay. In contrast to the results of these in vitro studies, no impairment in deiodinating capability was noted in intact cells expressing equivalent levels of the mutant constructs. These studies demonstrate that Cys124 and Cys194 influence the reactivity of the D1 with thiol cofactors in in vitro assay systems but are not determinants of the sensitivity of the enzyme to propylthiouracil and aurothioglucose. Furthermore, the observation that the cysteine mutants are fully active in intact cells demonstrates that the results of commonly used broken cell assays do not accurately predict the activity of the D1 in intact cells and suggests that glutathione and thioredoxin are not the major thiols utilized in vivo to support D1 activity.

Animals↗

PubMed1989

The influence of antithyroid drugs and iodine on thyroid cell MHC class II antigen expression.

We have investigated the influence of antithyroid drugs (methimazole and propylthiouracil) and sodium iodide on the expression of major histocompatibility (MHC) class II antigen expression in human and rat thyroid cells. While methimazole and propylthiouracil significantly inhibited lectin-induced MHC class II (HLA-DR) antigen in crude human thyroid monolayer preparations these drugs had no influence on gamma-interferon induction of class II antigens in similar cell preparations. Hence, antithyroid drugs probably had a direct effect on thyroid monolayer T cells which are known to effect the lectin induction of MHC class II antigen via T-cell secretion of the cytokine, gamma-interferon (IF). Sodium iodide, similarly, had no direct influence on human thyroid cell MHC class II antigen expression induced by gamma-IF. However, iodide significantly inhibited the gamma-IF-induced expression of MHC class II antigens in a proliferating rat thyroid cell clone derived from FRTL-5 cells (clone 1B-6). In the rat thyroid cell clone this antagonistic action appeared to be exerted via inhibition of TSH-induced proliferation. These data add further support to the multifactorial nature of the profound immunosuppressive influence of antithyroid drugs in autoimmune thyroid disease and experimental thyroiditis. In contrast, iodides may have a major, and often overlooked, influence on thyroid cell proliferation and antigen expression.

Animals↗

PubMed1984

Hyperthyroidism due to inappropriate TSH secretion with associated hyperprolactinaemia--a case report and review of the literature.

A patient with inappropriate thyrotrophin (TSH) secretion is described. She initially presented with classical hyperthyroidism during pregnancy, responded to propylthiouracil and, subsequently, had a normal delivery. Hyperthyroidism persisted and 7.5 months later a subtotal thyroidectomy was performed. After a further 16 months, mild symptoms of hyperthyroidism recurred. She again responded to propylthiouracil, but developed galactorrhoea. At that stage, it was noted that she had persistently elevated circulating TSH in the presence of elevated T4 and T3 levels. Her symptomatology was mild, although objective indices of thyroid activity, including pulse rate, BMR, sex hormone binding globulin and cholesterol, were indicative of hyperthyroidism. CT scan and tomography of the sella were normal. She had a markedly exaggerated TSH response to thyrotrophin releasing hormone (TRH). Basal TSH and responsiveness to TRH was suppressed by high dose dexamethasone. The TSH response to TRH was partially suppressed by exogenous T3, but there was no effect on basal TSH levels. TSH also decreased slightly with L-dopa and bromocriptine. Circulating TSH rose markedly during methimazole administration. TSH alpha and beta subunits were elevated and appropriate for the high TSH. In addition, both subunits increased following TRH. The patient had basal hyperprolactinaemia with an impaired prolactin (PRL) response to TRH and metoclopramide. PRL suppressed with L-dopa and bromocriptine. The remaining anterior pituitary function was intact. Most of the laboratory findings argue against the presence of a TSH producing pituitary tumour and the most likely cause for inappropriate TSH secretion in this patient is selective resistance of the thyrotroph to thyroid hormones. A mild element of peripheral resistance might also be present. The hyperprolactinaemia could be related to lactotroph resistance to thyroid hormone. The complexities of treatment in this patient are stressed. Therapy was initially attempted with low dose dexamethasone, but this had no effect. T3 treatment produced an exacerbation of her symptomatology and did not influence basal TSH, thyroid hormones, or 131I uptake. Bromocriptine administration for 11 months partially suppressed basal TSH without influencing T3 and there was an increase in T4. Methimazole did decrease her T4 and T3, but TSH and PRL rose to even greater levels. Her hyperthyroidism was eventually controlled with an ablative dose of 131I. Thyroid hormone will be given in an attempt to suppress her TSH.

Adult↗

PubMed1991

Mathematical models of myosin heterodimer formation in the rat heart during thyroid hormone alterations.

To characterize the mechanisms involved in the formation of the myosin heavy chain (MHC) heterodimer V2 (alpha beta-MHC) and the homodimers V3 (beta beta-MHC) and V1 (alpha alpha-MHC), 82 5-week-old normotensive rats with homogeneous V1 were made hypothyroid with propylthiouracil (0.8%, drinking water), and the proportion of V2, V3, and V1 was determined by pyrophosphate gel electrophoresis in multicellular specimens of the left and right ventricles. After the switch from alpha-MHC to beta-MHC, the beta-MHC occurred initially in the form of the heterodimer. After 4 and 6 days, V2 was greater (p less than 0.05) than V3. At day 8, V2 was smaller than V3, and at day 10, V2 was not statistically different from V3. From day 12 onward, V2 was smaller than V3. After 21 days, the propylthiouracil treatment was stopped, and the remaining 34 hypothyroid rats were injected with a daily dose of thyroxine (average, 0.1 mg/kg body wt), resulting in a switch from beta-MHC to alpha-MHC. After 1 day, V2 still was greater than V1; however, already from day 3 onward, V2 was smaller than V1. This characteristic but unexplained heterodimeric and homodimeric organization of the thick filament was analyzed by mathematical models involving probability calculations. Two principally different models were established that assume either the exchange of MHC dimers or of single MHC in the thick filament. The parameters of the models were estimated by minimization routines using the squared discrepancies between the experimental and predicted isoenzyme populations. Based on goodness of fit and crucial model parameters, we concluded that the characteristic organization of the thick filament can be accounted for by an exchange involving predominantly MHC dimers and not single MHC. The fact that V2 was lower than expected if formation of heterodimers and homodimers were random was attributed to the preferred homodimerization of 35% of the newly synthesized MHC.

Animals↗

PubMed1978

Iodothyronine metabolism in rat liver homogenates.

To investigate mechanisms of extrathyroidal thyroid hormone metabolism, conversion of thyroxine (T(4)) to 3,5,3'-triiodothyronine (T(3)) and degradation of 3,3',5'-triiodothyronine (rT(3)) were studied in rat liver homogenates. Both reactions were enzymatic. For conversion of T(4) to T(3), the K(m) of T(4) was 7.7 muM, and the V(max) was 0.13 pmol T(3)/min per mg protein. For rT(3) degradation, the K(m) of rT(3) was 7.5 nM, and the V(max) was 0.36 pmol rT(3)/min per mg protein. Production of rT(3) or degradation of T(4) or T(3) was not detected under the conditions employed. rT(3) was a potent competitive inhibitor of T(4) to T(3) conversion with a K(i) of 4.5 nM; 3,3'-diiodothyronine was a less potent inhibitor of this reaction. T(4) was a competitive inhibitor of rT(3) degradation with a K(i) of 10.2 muM. Agents which inhibited both reactions included propylthiouracil, which appeared to be an allosteric inhibitor, 2,4-dinitrophenol, and iopanoic acid. Sodium diatrizoate had a weak inhibitory effect. No inhibition was found with alpha-methylparatyrosine, Fe(+2), Fe(+3), reduced glutathione, beta-hydroxybutyrate, or oleic acid. Fasting resulted in inhibition of T(4) to T(3) conversion and of rT(3) degradation by rat liver homogenates which was reversible after refeeding. Serum T(4), T(3), and thyrotropin concentrations fell during fasting, with no decrease in serum protein binding as assessed by a T(3)-charcoal uptake. There was no consistent change in serum rT(3) concentrations. Dexamethasone had no effect in vitro. In vivo dexamethasone administration resulted in elevated serum rT(3) concentrations after 1 day, and after 5 days, in inhibition of T(4) to T(3) conversion and rT(3) degradation without altering serum T(4), T(3), or thyrotropin concentrations. Endotoxin treatment had no effect of iodothyronine metabolism in liver homogenates. In kidney homogenates the reaction rates and response to propylthiouracil in vitro were similar to those in liver. No significant T(4) to T(3) conversion or rT(3) production or degradation could be detected in other tissues. These data suggest that one iodothyronine 5'-deiodinase is responsible for both T(4) to T(3) conversion and rT(3) degradation in liver and, perhaps, in kidney. Alterations in serum T(3) and rT(3) concentrations induced by drugs and disease states may result from decreases in both T(3) production and rT(3) degradation consequent to inhibition of a single reaction in the pathways of iodothyronine metabolism.

Animals↗

PubMed1978

The influence of fasting, diabetes, and several pharmacological agents on the pathways of thyroxine metabolism in rat liver.

As judged from both paper and column chromatography, slices or homogenates of liver from rats fasted for 48 h displayed a lesser rate of generation of (125)I-labeled 3,5,3'-triiodothyronine (T(3)) from (125)I-labeled thyroxine (T(4)) added to incubation media than did preparations from normal chow-fed animals. A similar defect in the conversion of T(4) to T(3) in the livers of fasted animals was observed when preparations were incubated with substrate concentrations of T(4) so that T(3) generation could be assessed by radioimmunoassay. The effect of fasting could be prevented, wholly or in part, by administration of glucose in the drinking water to otherwise fasted animals, and the degree of prevention appeared to be proportional to the concentration of glucose employed. Diminished generation of T(3) from T(4) was similarly evident in the livers of animals with streptozotocin-induced diabetes mellitus, and this defect was overcome by the provision of insulin in vivo, but not in vitro. Decreased formation of T(3) from T(4) was also observed in preparations of liver from animals given dexamethasone, amiodarone, and propylthiouracil. In no case could these effects on the net formation of T(3) from T(4) be explained by effects of the experimental conditions on the degradation of the T(3) generated, as judged from the rate of degradation of exogenous (125)I-T(3) measured in parallel incubates. An analysis of the rate of disappearance of (125)I-T(4) from reaction mixtures in relation to the rate of appearance of (125)I-T(3) and (125)I-iodide was employed to estimate the activity of the 5-monodeiodinating pathway of T(4) metabolism that leads to the formation of 3,3',5'-triiodothyronine (reverse T(3)). Such estimates indicated that reverse T(3) formation was actively proceeding in the preparations studied, was slightly enhanced by fasting, was unaffected by dexamethasone and amiodarone, and was markedly inhibited by propylthiouracil. In view of the similarities between the effect of these experimental manipulations on the generation of T(3) from T(4) by rat liver in vitro to their effects on the production rates and serum concentrations of T(3) in man, it is concluded that the rat liver system provides a suitable model for the study of factors that influence the conversion of T(4) to T(3) in man. In addition, the findings strongly indicate that this process, at least in the liver, is closely linked to the utilization of carbohydrate.

Animals↗

PubMed1979

Regulation of the conversion of thyroxine to triiodothyronine in the perfused rat liver.

This study was undertaken to determine what factors control the conversion of thyroxine (T(4)) to triiodothyronine (T(3)) in rat liver under conditions approximating those found in vivo. Conversion of T(4) to T(3) was studied in the isolated perfused rat liver, a preparation in which the cellular and structural integrity is maintained and that can perform most of the physiologic functions of the liver. The perfused liver readily extracted T(4) from perfusion medium and converted it to T(3). Production of T(3) by the perfused liver was a function of the size of the liver, the uptake of T(4) by the liver, and the presence of T(4)-5'-deiodinase activity. Production of T(3) was increased by increasing the uptake of T(4) by liver, which could be accomplished by increasing the liver size, by increasing the perfusate T(4) concentration, or by decreasing the perfusate albumin concentration. These changes occurred without altering the conversion of T(4) to T(3). The liver had a large capacity for extracting T(4) and for T(4)-5'-deiodination to T(3), which was not saturated at a T(4) concentration of 60 mug/dl. Production of T(3) was decreased by inhibiting hepatic T(4)-5'-deiodinase with propylthiouracil, which decreased T(3) production by decreasing the conversion of T(4) to T(3). Propylthiouracil did not alter hepatic T(4) uptake. Fasting resulted in a progressive decrease in hepatic T(4) uptake to 42% of control levels by the 3rd d of fasting; this was accompanied by a proportionate decrease in T(3) production. The rate of conversion of T(4) to T(3) did not change during fasting. When T(4) uptake in 2-d-fasted rat livers was raised to levels found in fed rats by increasing the perfusate T(4) concentration from 10 to 30 mug/dl, T(3) production returned to normal. Again, no change in the rate of conversion of T(4) to T(3) was observed. These results indicate that the decreased hepatic T(3) production during fasting primarily results from decreased hepatic uptake of T(4), rather than from changes in T(4)-5'-deiodinase activity. Thus, these studies have delineated a new mechanism that functions independently of enzyme quantity or activity whereby production of T(3) from T(4) is regulated.

Animals↗

PubMed2003

Thyroid hormone enhances aggrecanase-2/ADAM-TS5 expression and proteoglycan degradation in growth plate cartilage.

Effects of thyroid hormone on proteoglycan degradation in various regions of cartilage were investigated. In propylthiouracil-treated rats with hypothyroidism, proteoglycan degradation in epiphyseal cartilage during endochondral ossification was markedly suppressed. However, injections of T(4) reversed this effect of propylthiouracil on proteoglycan degradation. In pig growth plate explants, T(3) also induced breakdown of proteoglycan. T(3) increased the release of aggrecan monomer and core protein from the explants into the medium. Accordingly, the level of aggrecan monomer remaining in the tissue decreased after T(3) treatment, and the monomer lost hyaluronic acid-binding capacity, suggesting that the cleavage site is in the interglobular domain. The aggrecan fragment released from the T(3)-exposed explants underwent cleavage at Glu(373)-Ala(374), the major aggrecanase-cleavage site. The stimulation of proteoglycan degradation by T(3) was less prominent in resting cartilage explants than in growth plate explants and was barely detectable in articular cartilage explants. Using rabbit growth plate chondrocyte cultures, we explored proteases that may be involved in T(3)-induced aggrecan degradation and found that T(3) enhanced the expression of aggrecanase-2/ADAM-TS5 (a disintegrin and a metalloproteinase domain with thrombospondin type I domains) mRNA, whereas we could not detect any enhancement of stromelysin, gelatinase, or collagenase activities or any aggrecanase-1/ADAM-TS4 mRNA expression. We also found that the aggrecanse-2 mRNA level, but not aggrecanase-1, increased at the hypertrophic stage during endochondral ossification. These findings suggest that aggrecanse-2/ADAM-TS5 is involved in aggrecan breakdown during endochondral ossification, and that thyroid hormone stimulates the aggrecan breakdown partly via the enhancement of aggrecanase-2/ADAM-TS5.

ADAM Proteins↗

PubMed1982

Effects of neonatal hyperthyroidism on the development of the hypothalamic-pituitary-thyroid axis in the rat.

The acute and latent effects of neonatal hyperthyroidism (NH) on the hypothalamic-pituitary-thyroid axis were studied in the rat after treatment of newborn animals with L-T4 (0.4 microgram/g BW, daily) for a period of 12 days. NH was associated with a permanent reduction in body weight in both male and female rats, in addition to a delay in the attainment of peak concentrations of hypothalamic TRH and pituitary and serum TSH. Serum TSH, T4, and T3 concentrations also were significantly and permanently reduced in NH animals (P less than 0.01) after cessation of L-T4 treatment. The serum TSH secretory response to 1 microgram synthetic TRH also was evaluated in 120-day-old control and NH rats, before and after the administration of L-T4 (0.6 microgram/100 g BW for 7 days) or propylthiouracil (0.05% in the drinking water for 14 days). In the baseline state, adult NH rats had a net secretory response similar to that of controls (189.0 +/- 31.3 vs. 227.0 +/- 29.3 microgram/ml . min). Administration of T4 significantly decreased while propylthiouracil treatment significantly increased the net TSH secretory response of NH rats compared to similarly treated control rats. These data are compatible with the hypothesis that NH leads to a permanent resetting of the regulatory set-point for pituitary TSH secretion and to increased sensitivity to the feedback inhibitory effects of thyroid hormones.

Animals↗

PubMed1984

Iodothyronine deiodination reaction types in several rat tissues: effects of age, thyroid status, and glucocorticoid treatment.

Two types of iodothyronine 5'-deiodination have been characterized previously in rat tissues. They can be distinguished by inhibition of type I but not type II by 6-n-propylthiouracil, by the relative suitability of T4 and rT3 as substrates, rT3 much better than T4 for type I and T4 as good as, or better than, rT3 for type II, and by the concentration of T4 required to inhibit deiodination of rT3, 1-10 microM for type I and 1-10 nM for type II. Type I activity (6-n-propylthiouracil sensitive) is most abundant in liver and kidney. Type II activity has, to date, been identified only in the pituitary, central nervous system, and brown adipose tissue. Iodothyronine tyrosyl deiodination has also been identified in homogenates of rat brain, liver, and placenta. It is not clear how many different enzymes carry out this latter reaction. In the present studies, we have extended previous work by determining maturational patterns of the deiodinating pathways in several thyroid hormone-responsive rat tissues, possible modulation of those patterns by glucocorticoids, and the age of onset of responsivity of the deiodinases to hypothyroidism. Iodothyronine 5'-deiodinating activity was found in rat lung and eye, and the reaction was all or nearly all type I in both. Activity in the eye was virtually absent from the lens and vitreous humor. In immature rat cerebrum, pituitary, lung, and eye, between gestational day 17 and postnatal day 21, there was a uniform pattern of an increase in type I 5'-deiodination activity over time, until adult levels were attained. The ages at which adult activity levels were reached varied from tissue to tissue, however. Type II activity was present at the earliest ages tested in the cerebrum (gestational day 17), pituitary, and brown adipose tissue (day of birth). In cerebral cortex, type II activity was highest at day 21 postnatally and equal at birth and in adulthood, and in pituitary and brown adipose tissue it was higher in adulthood than at birth. T3 tyrosyl ring deiodinating activity was several times greater in homogenates of eye and placenta than in cerebral homogenates. In all three tissues, there was similar, dose-dependent inhibition of [125I]T3 tyrosyl deiodination by 5 nM and 20 nM nonradioactive T3. In the eye and brain, T3 tyrosyl deiodination rates decreased progressively with age from gestational day 17 to postnatal day 7.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue, Brown↗

PubMed1987

Thyroid angiogenesis: endotheliotropic chemoattractant activity from rat thyroid cells in culture.

Thyroid enlargement in response to chronic hypersecretion of TSH reflects the coordinated growth of both parenchyma and stroma. Because Wollman et al. observed in propylthiouracil-fed rats that enlargement and remodeling of thyroid capillaries were strictly localized around follicles, they hypothesized that growth of perifollicular blood vessels is stimulated by angiogenic factors secreted by neighboring follicular epithelial cells. In support of this hypothesis, we report that media conditioned by rat thyroid cells were very active in an in vitro angiogenesis bioassay that measures stimulation of endothelial cell migration through chemotaxis membranes in microwell Boyden chamber assemblies. Primary cultures of thyroid cells from collagenase-dispersed glands from male or female Holtzman rats fed 0.01% propylthiouracil in the drinking water released activity that produced up to 5-fold increases in endothelial cell migration rates relative to those in identical unconditioned medium. Thyroid-derived activity was primarily chemotactic (i.e. only weakly chemokinetic) to both rabbit aortic and microvascular endothelial cells. That endotheliotropic activity is derived from thyroid parenchyma is indicated by the finding that media conditioned by FRTL cells, a clonally derived thyroid follicular epithelial cell line, produced parallel chemoattractant responses. Thyroid-conditioned media were also chemoattractant to mouse BALB/c-3T3 cells, which have endothelial cell characteristics. In contrast, thyroid-conditioned media did not increase the high spontaneous migration rate of Walker rat sarcoma (WR256) cells. T4, T3, thyroglobulin, bovine fibroblast growth factor (alpha and beta), and media conditioned by rabbit endothelial cells were inactive. Chemoattractant activity in serum containing conditioned media was retained by both 10,000 and 30,000 mol wt cut-off (MWCO) ultrafilters. Activity in serum-free thyroid-conditioned media was largely retained by 10,000 MWCO filters, but only partially retained by 30,000 MWCO filters; activity in the 30,000 filtrate was recoverable in a 10,000 MWCO retentate. These findings support the hypothesis that capillary growth during thyroid enlargement occurs, at least in part, as a result of a parenchymal-stromal (epithelial-mesenchymal) paracrine interaction mediated by specific endotheliotropic (angiogenic) factors released by follicular epithelial cells and distinct from T3, T4, and thyroglobulin.

Animals↗

PubMed1989

A study of hepatic low Km iodothyronine 5'-monodeiodinase.

To document the presence of a low Km rT3 and T4-5'-monodeiodinase (5'MDL; Km in nanomolar concentrations) in the liver and to study its characteristics in comparison with the high Km 5'MD (5'MDH; Km in micromolar concentrations), we incubated rat liver microsomal protein (20 micrograms for rT3 substrate and 200 micrograms for T4 substrate) with 125I-labeled rT3 or T4 and dithiothreitol (DTT; up to 20 mM) for 5 min (for rT3) or 30-120 min (for T4) and determined the amount of 125I liberated during incubation. Pilot studies had shown that the activity of rT3 5'MDH is markedly (greater than or equal to 85%) inhibited in the presence of 2 M NaCl, while the rT3 5'MDL is essentially unaffected, and both low and high Km T4 5'MD are minimally (approximately 20%) inhibited. The representative kinetics of various substrates studied were: Km, 13 nM for rT3 5'MDL, 640 for rT3 5'MDH, 26 for T4 5'MDL, and 3620 for T4 5'MDH; maximum velocity, 0.28 nmol/h.mg protein for rT3 5'MDL, 46 for rT3 5'MDH, 0.002 for T4 5'MDL, and 0.46 for T4 5'MDH. Propylthiouracil and iopanoate inhibited all enzymic activities studied. The relative Ki values (micromolar concentrations) for propylthiouracil were: 7.1 for rT3 5'MDL, 1.5 for rT3 5'MDH, 24 for T4 5'MDL, and 40 for T4 5'MDH; those for iopanoate were 0.4 for rT3 5'MDL, 18 for rT3 5'MDH, 7.0 for T4 5'MDL, and 4.0 for T4 5'MDH. DTT was a potent stimulator of enzyme activities studied; its dose (millimolar concentrations) that caused a 50% maximal stimulation was 0.04 for rT3 5'MDL, 1.0 for rT3 5'MDH, 0.025 for T4 5'MDL, and 0.035 for T4 5'MDH. T4 inhibited rT3 5'-monodeiodination and vice versa. The Ki of T4 was 1.3 microM for rT3 5'MDL and 2.0 for rT3 5'MDH, while that of rT3 was 0.4 for T4 5'MDL and 0.6 for T4 5'MDH. We examined the activity of the hepatic 5'MDL (rT3, 0.5 nM; DTT, 0.06 nM; 2 M NaCl) and 5'MDH (rT3, 0.5 microM; DTT, 20 mM; no NaCl) in groups (six animals per group) of rats that were saline treated (control), thyroidectomized, or hyperthyroid (given T3, 20 micrograms/day for 5 days). The relative values for 5'MDL were (mean +/- SD) 17 +/- 3.0, 4.0 +/- 2.0 (P less than 0.01), and 24 +/- 2.0 (P less than 0.01) pmol/h.mg protein, respectively, whereas those for 5'MDH were 13 +/- 4.0, 3.0 +/- 1.6 (P less than 0.01), and 25 +/- 1.0 (P less than 0.01) nmol/h.mg protein, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1990

Stimulation of rat atrial natriuretic peptide (rANP) synthesis by triiodothyronine and thyroxine (T4): T4 as a prohormone in synthesizing rANP.

Thyroid hormones have been reported to increase the secretion and synthesis of atrial natriuretic peptide (ANP) in vitro. In this study, we focused on the role of type I T4 5'-deiodinase to investigate the stimulating effects of T4 on ANP synthesis and secretion by measuring cellular content and secretion into the medium of immunoreactive rat ANP (IR-rANP) and rANP mRNA levels in cultured rat neonatal atrial myocytes. Both T3 (10(-9)-10(-7) M) and T4 (10(-8)-10(-6) M) increased cellular content and secretion into the medium of IR-rANP in a dose-dependent manner. However, these effects of T4 were completely inhibited by the addition of propylthiouracil, which selectively suppressed type I T4 5'-deiodinase activity. Methimazole, which did not alter T4 5'-deiodinase activity, had no effects on T4-induced IR-rANP increase. In the measurements of rANP mRNA levels by dot blot analysis, T3 (10(-8) and 10(-7) M) and T4 (10(-7) and 10(-6) M) increased rANP mRNA levels in the same way as they increased IR-rANP content. Although T4-increased rANP mRNA levels were also inhibited by propylthiouracil, methimazole did not alter the effect of T4. Moreover, T4-induced rANP mRNA accumulation in atrial myocytes was further stimulated by the addition of dithiothreitol, suggesting that the deiodinating activity was thiol sensitive. These data suggest that the stimulating effect of T4 on cellular IR-rANP content and rANP mRNA levels is entirely induced after it is converted to T3 by type I T4 5'-deiodinase in atrial myocytes and that T4 serves as a prohormone for T3 in synthesizing rANP.

Animals↗

PubMed2002

Substitution of cysteine for a conserved alanine residue in the catalytic center of type II iodothyronine deiodinase alters interaction with reducing cofactor.

UNLABELLED: Human type II iodothyronine deiodinase (D2) catalyzes the activation of T(4) to T(3). The D2 enzyme, like the type I (D1) and type III (D3) deiodinases, contains a selenocysteine (SeC) residue (residue 133 in D2) in the highly conserved catalytic center. Remarkably, all of the D2 proteins cloned so far have an alanine two residue-amino terminal to the SeC, whereas all D1 and D3 proteins contain a cysteine at this position. A cysteine residue in the catalytic center could assist in enzymatic action by providing a nucleophilic sulfide or by participating in redox reactions with a cofactor or enzyme residues. We have investigated whether D2 mutants with a cysteine (A131C) or serine (A131S) two-residue amino terminal to the SeC are enzymatically active and have characterized these mutants with regard to substrate affinity, reducing cofactor interaction and inhibitor profile. COS cells were transfected with expression vectors encoding wild-type (wt) D2, D2 A131C, or D2 A131S proteins. Kinetic analysis was performed on homogenates with dithiothreitol (DTT) as reducing cofactor. The D2 A131C and A131S mutants displayed similar Michaelis-Menten constant values for T(4) (5 nM) and reverse T(3) (9 nM) as the wt D2 enzyme. The limiting Michaelis-Menten constant for DTT of the D2 A131C enzyme was 3-fold lower than that of the wt D2 enzyme. The wt and mutant D2 enzymes are essentially insensitive to propylthiouracil [concentration inhibiting 50% of activity (IC(50)) > 2 mM] in the presence of 20 mM DTT, but when tested in the presence of 0.2 mM DTT the IC(50) value for propylthiouracil is reduced to about 0.1 mM. During incubations of intact COS cells expressing wt D2, D2 A131C, or D2 A131S, addition of increasing amounts of unlabeled T(4) resulted in the saturation of [(125)I]T(4) deiodination, as reflected in a decrease of [(125)I]T(3) release into the medium. Saturation first appeared at medium T(4) concentrations between 1 and 10 nM. IN CONCLUSION: substitution of cysteine for a conserved alanine residue in the catalytic center of the D2 protein does not inactivate the enzyme in vitro and in situ, but rather improves the interaction with the reducing cofactor DTT in vitro.

Alanine↗

PubMed1992

Cloning and in vitro expression of the human selenoprotein, type I iodothyronine deiodinase.

The type I 5' iodothyronine deiodinase (5' DI) catalyzes the deiodination of T4 to the biologically active hormone T3 and accounts for a significant fraction of its production. We have recently cloned the complementary DNA (cDNA) for the rat 5' DI, which contains the rare amino acid selenocysteine, and used this to screen human liver and kidney cDNA libraries to identify a human 5' DI cDNA clone. From these, we constructed a cDNA encoding a functional 5' DI. The 2222 base pair human 5' DI cDNA is approximately 200 nucleotides shorter than the 2.4-kilobase hybridizing band in Northern blots of human liver, kidney, and thyroid, because of missing 5' untranslated sequence and the poly A tail. The deduced amino acid sequence codes for a protein of 28.7 kilodaltons assuming the UGA codon at position 382 encodes selenocysteine, and is highly homologous (88% similarity) to the rat. We transiently expressed the 5' DI in COS-7 cells to establish that it encodes a functional enzyme and to study its kinetics. These show saturable deiodination of rT3 (Ka 0.52 +/- 0.04 mumol/L and Vmax 63.2 +/- 16.4 pmol min-1 mg-1). T4 and gold thioglucose are competitive inhibitors of rT3 deiodination. 6-n-Propylthiouracil (PTU) is an uncompetitive inhibitor (with rT3) and competitive inhibitor (with dithiothreitol) of rT3 deiodination. 6-n-Propylthiouracil inhibits T4 to T3 conversion. Labeling of COS-7 cells transiently transfected with the human 5' DI cDNA with bromoacetyl-125I-T3 demonstrates a 28-kilodalton protein. This indicates that in the human, as well as in the rat messenger RNA, the UGA encodes selenocysteine and translation terminates at the UAA codon at nucleotides 754 to 756. Reverse T3 and gold thioglucose (100 nmol/L) block bromoacetyl-125I-T3 labeling of the transiently expressed human and rat 5' DI proteins. These results demonstrate that the human 5' DI is a selenoprotein, analogous to the rat enzyme. Given the previously demonstrated critical role of the selenium atom in catalyzing deiodination by this protein, we conclude that this trace element is essential for normal thyroid hormone action in man.

Affinity Labels↗

PubMedDate not supplied

Diagnosis and treatment of Graves disease.

OBJECTIVE: To review the etiology, diagnosis, and clinical presentation of Graves disease and provide an overview of the standard and adjunctive treatments. Specifically, antithyroid drugs, beta-blockers, inorganic iodide, lithium, and radioactive iodine are discussed, focusing on current controversies. DATA SOURCES: Primary articles were identified through a MEDLINE search (1966-July 2000). Key word searches included beta-blockers, Graves disease, inorganic iodide, lithium, methimazole, and propylthiouracil. Additional articles from these sources and endocrinology textbooks were also identified. We agreed to include articles that would highlight the most relevant points, as well as current areas of controversy. DATA SYNTHESIS: Graves disease is the most common cause of hyperthyroidism. The 3 main treatment options for patients with Graves hyperthyroidism include antithyroid drugs, radioactive iodine, and surgery. Although the antithyroid drugs propylthiouracil (PTU) and methimazole (MMI) have similar efficacy, there are situations when 1 agent is preferred. MMI has a longer half-life than PTU, allowing once-daily dosing that can improve patient adherence to treatment. PTU has historically been the drug of choice for treating pregnant and breast-feeding women because of its limited transfer into the placenta and breast milk. Adjuvant therapies for Graves disease include beta-blockers, inorganic iodide, and lithium. beta-Blockers are used to decrease the symptoms of hyperthyroidism. Inorganic iodide is primarily used to prepare patients for thyroid surgery because of its ability to decrease the vascularity of the thyroid gland. Lithium, which acts in a manner similar to iodine, is not routinely used due to its transient effect and the risk of potentially serious adverse effects. In the US, radioiodine therapy has become the preferred treatment for adults with Graves disease. It is easy to administer, safe, effective, and more affordable than long-term treatment with antithyroid drugs. Hypothyroidism is an inevitable consequence of radioiodine therapy. Radioiodine is contraindicated in pregnant women because it can damage the fetal thyroid gland, resulting in fetal hypothyroidism. Bilateral subtotal thyroidectomy, which was once the only treatment available, is now performed only in special circumstances. In addition to the normal risks associated with surgery, laryngeal nerve damage, hypoparathyroidism, and hypothyroidism can occur following that procedure. CONCLUSIONS: Despite extensive experience with medical management, controversy prevails regarding choosing among the various drugs for treatment of Graves disease. None of the treatment options, including antithyroid drugs, radioiodine, and surgery, is ideal. Each has risks and benefits, and selection should be tailored to the individual patient.

Antithyroid Agents↗

PubMedDate not supplied

Clinical aspects of hyperthyroidism in hospitalised patients in Albania.

The purpose of this study was to evaluate certain clinical aspects of hyperthyroidism in Albania, which is an iodine deficient country, as it is known that iodine intake may influence the type of thyroid hyperfunction. The files of sixty-six patients with thyrotoxicosis who were hospitalised for their disease were retrospectively analysed. 59.1% of these patients suffered from toxic multinodular goiter, 27.3% from Grave's disease (toxic diffuse goiter), 10.6% from toxic adenoma, 1.5% from iodide-induced hyperthyroidism and 1.5% from transient hyperthyroidism due to subacute thyroiditis. There was an increased female to male ratio (83.3% vs 16.7%, respectively, p<0.001). 83.9% of all hyperthyroid patients lived in cities, while 16.1% lived in villages. Ophthalmopathy was found in 11.1% of patients with Graves' disease, and thyrotoxic heart disease was found in 14% of patients with thyrotoxicosis. 71.9% of all patients with hyperthyroidism were treated with propylthiouracil (PTU), while 28.1% of them were treated with methimazole; 67.2% of all these patients also received propranolol hydrochloride, while 32.8% were prescribed atenolol. Compliance was lower than that reported in other studies as only 41% of all patients received their treatment regularly. Side effects from treatment with antithyroid drugs were as follows: 4.1% (2/48) of patients treated with propylthiouracil presented leukopenia with agranulocytosis, and 6.1% of them toxic hepatitis, while 11.1% (2/18) of patients treated with methimazole presented agranulocytosis. In conclusion, the mode of presentation and side effects of hyperthyroidism appears to be different in Albania when compared with other countries, probably as a result of iodine deficiency and/or possibly nutritional status. Compliance with treatment is lower than that reported in other series, while antithyroid drug side effects seem to be more frequent. The latter observation may be due to the fact that only hospitalised patients were analysed in this study.

Journal Article↗