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PubMed2003

Thyroid hormone resistance in the heart: role of the thyroid hormone receptor beta isoform.

Several cardiac genes possess thyroid hormone (TH) response elements regulated by TH receptors. Mutation in TR-beta gene causes the human syndrome of resistance to TH, which is characterized by elevated serum concentration of T(4) and T(3) and variable degrees of insensitivity to TH. It is unclear, however, whether a mutant TR-beta could function as a dominant negative in the heart when expressed from the endogenous locus. A well-described resistance to TH (Delta337T) was either introduced into germline of mice (KI-mut) or expressed as a transgene in the heart using a cardiac-specific promoter (KS-mut). Mice were studied at baseline, after 5-propyl-2-thiouracil (PTU) or after PTU and T(3) treatment (PTU + T(3)). PTU + T(3) treatment significantly increased left ventricular mass in all groups compared with baseline measurements, although the increase in left ventricular mass was significantly less in KI-mut animals. Baseline heart rates (HRs) were similar in wild-type (WT) and KI-mut but were lower in KS-mut animals. After TH deprivation (PTU), HR decreased in WT and KI-mut animals; similarly, HR increased in WT and KI-mut after PTU + T(3). In contrast, HR in KS-mut animals did not change after either treatment. Except for cardiac hypertrophy, the presence of a germline TR-beta mutation had surprisingly little effect on cardiac function.

Animals↗
PubMed2005

Characterization of an iodothyronine 5'-deiodinase in gilthead seabream (Sparus auratus) that is inhibited by dithiothreitol.

Iodothyronine deiodinases catalyze the conversion of the thyroid prohormone T(4) to T(3) by outer ring deiodination (ORD) of the iodothyronine molecule. The catalytic cycle of deiodinases is considered to be critically dependent on a reducing thiol cosubstrate that regenerates the selenoenzyme to its native state. The endogenous cosubstrate has still not been firmly identified; in studies in vitro the sulfhydryl reagent dithiothreitol (DTT) is commonly used to activate ORD. We now have characterized an ORD activity in the teleost gilthead seabream (Sparus auratus) that is inhibited by DTT. DTT inhibited reverse T(3) (rT(3)) ORD by 70 and 100% in kidney homogenates (IC(50) 0.4 mmol/liter) and microsomes (IC(50) 0.1 mmol/liter), respectively. The omission of DTT from the incubation medium restored renal ORD Michaelis-Menten kinetics with a Michaelis constant value of 5 mumol/liter rT(3) and unmasked the inhibition by 6-n-propyl-2-thiouracil. A putative seabream deiodinase type 1 (saD1), derived from kidney mRNA, showed high homology (> or = 41% amino acid identity) with vertebrate deiodinases type 1. Features of this putative saD1 include a selenocysteine encoded by an in-frame UGA codon, consensus sequences, and a predicted secondary structure for a selenocysteine insertion sequence and an amino acid composition of the catalytic center that is identical with reported consensus sequences for deiodinase type 1. Remarkably, three of six cysteines that are present in the deduced saD1 protein occur in the predicted amino terminal hydrophobic region. We suggest that the effects of DTT on rT(3) ORD can be explained by interactions with the cysteines unique to the putative saD1 protein.

Amino Acid Sequence↗
PubMed1978

Accumulation and localization of radiocalcium in the rat thyroid gland.

To determine the rate of accumulation of radiocalcium (45Ca) in the thyroid gland, the concentration of radiocalcium was measured in the blood serum and thyroid gland at various time intervals up to 7 days after a single ip injection of 45Ca in rats. The ratio of the 45Ca concentration in thyroid gland to serum (T:S) increased with the time interval after injection. Thyroid activity was varied by hypophysectomy or by feeding a low iodine diet with or without added thiouracil. The more active the gland, the faster it equilibrated with the serum 45Ca and the less was the T:S in the steady state. The equilibration was much slower than for radioiodide, taking several days in normal control rats and hours in very active glands. Autoradiographs showed that the 45Ca concentration was higher in association with the follicular epithelium than with the follicular lumen at 1 h or less after injection. However, at 24 h, the concentration of 45Ca was higher in the lumen than elsewhere in the gland. To determine rate constants describing the equilibration, the data in the normal gland were fitted with a model that took account of the differing rates of equilibration in cell and lumen. The presence of calcium in the lumen indicates that each time the follicular epithelial cell endocytosed stored luminal colloid before secretion, it also took in an appreciable amount of calcium.

Animals↗
PubMed1978

Mechanism of action of thioureylene antithyroid drugs: factors affecting intrathyroidal metabolism of propylthiouracil and methimazole in rats.

Experiments were performed with rats to test the physiological significance of a previously proposed mechanism of action of thioureylene antithyroid drugs, which had been derived from results obtained with a model system containing purified thyroid peroxidase. Two features of the previously proposed scheme were tested:1) the effects of drug dosage and 2) the effects of iodine deficiency. In the dosage experiments, rats were injected with graded doses of [35S]PTU (0.18-59 mumol or [35S]mmi (0.16-18 mumol). Thyroid glands were removed 1 and 6--8 h later and 35S distribution in the homogenates was determined by paper chromatography. Serum samples were also analyzed by the same procedure. From the measured 35S activity in the various components and from the known specific activity of the injected drugs, it was possible to calculate thyroidal concentrations of unchanged drug and drug metabolites. At low doses, thyroidal concentrations of unchanged 6-propyl-2-thiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MM) significantly exceeded their concentrations in serum, as reported by previous investigators. A major new finding in the present investigation was the observation that intrathyroidal metabolism of PTU and MMI is greatly affected by dosage. Marked inhibition of intrathyroidal drug metabolism was observed at 6-8 when the dosage was increased from 5.9 to 18 mumol for [35S]PTU and from 0.88 to 2.2 mumol for [35S]MMI (per 200 g rat). These findings demonstrate that with increasing dosage, PTU and MMI inhibit their own intrathyroidal metabolism. These dosage effects are similar to results previously reported for in vitro oxidation of PTU and MMI by the thyroid peroxidase system, and they offer support for the physiological significance of the previously proposed scheme. Further evidence for the physiological validity of this scheme was obtained in the experiments with iodine-deficient rats. As predicted from the in vitro findings, intrathyroidal metabolism of [35S]PTU and [35S]MMI was markedly reduced in rats on low iodine diet.

Animals↗
PubMed1979

Thiourea and cyanamide as inhibitors of thyroid peroxidase: the role of iodide.

Thiourea, methylmercaptoimidazole, propylthiouracil, and thiouracil are all potent inhibitors of thyroid peroxidase (TPO)-catalyzed iodination. Unlike the cyclic thioureylenes, thiourea at 5 mM has no effect on guaiacol oxidation. If iodide is added to guaiacol assays containing thiourea, enzyme activity is lost. The latter observation may be explained as follows. In the presence of iodide, the iodinating species [TPO.Ioxid], oxidizes thiourea to formamidine disulfide. This product decomposes to cyanamide at neutral pH. We have shown cyanamide to be an inhibitor of the peroxidative and iodinating functions of TPO. Studies in rats demonstrate that doses of thiourea which completely inhibit in vivo protein-bound iodine formation have no irreversible effect on TPO, as measured by guaiacol peroxidation after removal of the thyroids. The major in vivo action of cyanamide is similar to that of thiourea. The data suggest that the primary in vivo and in vitro mode of action of thiourea is the reversible Ioxid-trapping mechanism. The anomalous inhibition of guaiacol peroxidation seen in the presence of thiourea plus iodide derives from the formation of formamide disulfide, followed by its nonenzymic decomposition to cyanamide.

Animals↗
PubMed1980

Increase in the rough endoplasmic reticulum in capillary endothelial cells and pericytes in hyperplastic rat thyroid glands.

Observatios were made on the rough endoplasmic reticulum (RER) of the blood capillary endothelium, pericytes, and lymphatic endothelium in the course of studies of changes occurring in the thryoid gland during the development of thyroid hyperplasia. To induce the hyperplasia, Fischer rats were fed a goitrogen (thiouracil) an a low iodine diet for various time intervals from 3-100 days. Thyroid glands were fixed by perfusion with glutaryldehyde and embedded in Epon. A noticeable increase in the RER in the capillary endothelium was seen by 3 days. This increase was very pronounced by 7 days and persisted in many cells at 100 days. The RER response was also pronounced in pericytes. In the lymphatic endothelium, however, the increase in RER was barely perceptible. These results suggest that capillary endothelium and pericytes have protein synthetic and secretory functions of an unknown nature that become strongly activated in stimulated thyroid glands.

Animals↗
PubMed1981

Relationship of polycystic ovary induction to prolactin secretion: prevention of cyst formation by bromocriptine in the rat.

PRL levels were measured over 30 days of cyst induction in the rat. Polycystic ovaries (PCO) were induced by dietary incorporation of thiouracil (Thio) for 30 days and injection of 10 IU hCG sc for the last 20 days of the experimental period. Animals were killed at specific intervals 24 hours after the last injection. Ovaries and thyroids were removed and weighed. Sera were analyzed for PRL by RIA. PRL levels increased in animals receiving Thio plus hCG by day 30 compared to animals receiving Thio alone. The increase in PRL in the Thio, hCG-stimulated group coincides with formation of follicular cysts. Administration of bromocriptine (1 mg/day) concurrent with hCG during the last 20 days of Thio-feeding significantly suppressed ovarian weight gain and PCO formation. Serum PRL levels in bromocriptine-treated rats were significantly reduced compared to the PCO group yet were also significantly greater than all control groups at day 30. These findings suggest that PRL is important to the etiology of ovarian cysts in the rat.

Animals↗
PubMed1981

The influence of thyroidal status on rat hepatic mitochondrial NADH duroquinone reductase.

Hepatic mitochondrial NADH duroquinone reductase and alpha-glycerophosphate dehydrogenase activities were measured in rats with altered thyroidal status. Whereas alpha-glycerophosphate dehydrogenase activity was decreased in hypothyroid rats, NADH duroquinone reductase was increased approximately 3-fold in both thiouracil-fed and thyroidectomized rats. In hyperthyroid animals, NADH duroquinone reductase activity was decreased, whereas there was the expected elevation in mitochondrial alpha-glycerophosphate dehydrogenase. Maximum velocity measurements of NADH duroquinone reductase demonstrated that the increase in enzyme activity associated with hypothyroidism occurred without an alteration in Michaelis constants for the reaction. Rats bearing mammary carcinomas induced by N-nitrosomethylurea also showed an increase in hepatic NADH duroquinone reductase when rendered hypothyroid, but the enzyme was unaffected by thyroxine administration.

Animals↗
PubMed1982

Preferential inhibition of thyroxine and 3,5,3'-triiodothyronine formation by propylthiouracil and methylmercaptoimidazole in thyroid peroxidase-catalyzed iodination of thyroglobulin.

The present study was undertaken to determine whether the thioureylene antithyroid drugs propylthiouracil [6-propyl-2-thiouracil (PTU)] and methylmercaptoimidazole [1-methyl-2-mercaptoimidazole (MMI)] have a specific inhibitory effect on the thyroid peroxidase (TPO)-catalyzed conversion of diiodotyrosine to T4 (coupling reaction) independent of their well known inhibitory effect on peroxidase-catalyzed iodination. We have employed model incubation systems containing highly purified TPO to examine this question. Most experiments were performed with a model iodination system containing TPO, low iodine thyroglobulin, [131]iodide, and glucose-glucose oxidase. Both PTU and MMI are effective inhibitors of iodination of this system at physiological concentrations, and the system is well suited for studying the simultaneous action of these drugs on iodination and coupling. The addition of graded doses of the drugs to the iodination system demonstrated a relatively greater inhibitory effect on iodothyronine than on iodotyrosine formation. However, this observation in itself does not establish a specific inhibitory effect on coupling, since the formation of T4 involves a reaction between two molecules of 3,5-diiodotyrosine (DIT). The rate of this reaction, therefore, is second order with respect to DIT concentration, and the inhibition of DIT formation by thioureylene drugs would be expected to result in a disproportionately greater reduction in T4 formation even id there were no selective inhibitory effect of the drugs on the coupling reaction. Under certain conditions of incubation, however, it was possible to demonstrate a significant inhibitory effect on T4 and T3 formation without any decrease (in fact, a slight increase) in diiodotyrosine formation. These observations indicate that, at least under some conditions, PTU and MMI can exert a specific inhibitory effect on the coupling reaction. In the case of PTU, a specific inhibitory effect on coupling was also demonstrated with an incubation system in which TPO-catalyzed coupling was measured in the absence of iodination.

Animals↗
PubMed1983

Mechanism of action of thioureylene antithyroid drugs in the rat: possible inactivation of thyroid peroxidase by propylthiouracil.

We have previously shown that the thioureylene antithyroid drugs 6-propyl-2-thiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI) can inactivate thyroid peroxidase (TPO) in a model iodination system containing relatively high concentrations of iodide. The purpose of the present study was to determine whether these drugs may also inactivate TPO in vivo in rats. Assays for total TPO activity after injection of PTU or MMI did not prove to be a valid approach. As TPO inactivation might be expected to result in a relatively prolonged inhibition of enzyme activity, most of our experiments involved measurement of the duration of the inhibitory effect of a single injection of drug. Young rats were injected with low doses of PTU or MMI, and the effect on thyroidal organic iodine formation was determined at intervals after injection, either by 1-h pulse labeling with 131I- in vivo or by incubation of excised thyroid lobes in a medium containing 131I-. Results of both types of experiment demonstrated that the inhibitory effect of a small dose of PTU (1 mumol/100 g BW) was still very marked 17-18 h after injection. Moreover, an inhibitory effect of this small dose of PTU on the metabolism of [35S]MMI could also be demonstrated. Administration of MMI to rats, on the other hand, did not show the prolonged inhibitory effect observed with PTU. This is most likely attributable to the much lower thyroidal uptake of MMI than of PTU in rats. Intrathyroidal metabolism of [35S]PTU and [35S]MMI was also investigated. In contrast to the rapid disappearance of 35S from plasma, both drugs showed accumulation and retention of 35S in the thyroid. However, we obtained no evidence that thyroidal accumulation of PTU or one of its metabolites could explain the prolonged inhibitory effect of this drug. It seemed more likely that this was attributable to TPO inactivation. The clinical implications of our findings are discussed with relation to the dosage schedule commonly employed in the treatment of Graves' disease with antithyroid drugs.

Animals↗
PubMed1984

Metabolism of 3,3'-diiodothyronine in rat hepatocytes: interaction of sulfation with deiodination.

Production of 3,3'-diiodothyronine (3,3'-T2) is an important step in the peripheral metabolism of thyroid hormone in man. The rapid clearance of 3,3'-T2 is accomplished to a large extent in the liver. We have studied in detail the mechanisms of this process using monolayers of freshly isolated rat hepatocytes. After incubation with 3,[3'-125I]T2, chromatographic analysis of the medium revealed two major metabolic routes: outer ring deiodination and sulfation. We recently demonstrated that sulfate conjugation precedes and in effect accelerates deiodination of 3,3'-T2. In media containing different serum concentrations the cellular clearance rate was determined by the nonprotein-bound fraction of 3,3'-T2. At substrate concentrations below 10(-8) M 125I- was the main product observed. At higher concentrations deiodination became saturated, and 3,3'-T2 sulfate (T2S) accumulated in the medium. Saturation of 3,3'-T2 clearance was found to occur only at very high (greater than 10(-6)M) substrate concentrations. The sulfating capacity of the cells exceeded that of deiodination by at least 20-fold. Deiodination was completely inhibited by 10(-4) M propylthiouracil or thiouracil, resulting in the accumulation of T2S while clearance of 3,3'-T2 was little affected. No effect was seen with methimazole. Hepatocytes from 72-h fasted rats showed a significant reduction of deiodination but unimpaired sulfation. Other iodothyronines interfered with 3,3'-T2 metabolism. Deiodination was strongly inhibited by 2 microM T4 and rT3 (80%) but little by T3 (15%), whereas the clearance of 3,3'-T2 was reduced by 27% (T4 and rT3) and 12% (T3). It is concluded that the rapid hepatic clearance of 3,3'-T2 is determined by the sulfate-transferring capacity of the liver cells. Subsequent outer ring deiodination of the intermediate T2S is inhibited by propylthiouracil and by fasting, essentially without an effect on overall 3,3'-T2 clearance.

Animals↗
PubMed1985

Thyroxine 5'-deiodinase activity in anterior pituitary glands transplanted under the renal capsule in the rat.

The conversion of T4 to T3 by the anterior pituitary gland appears to be of considerable physiological importance in the control of pituitary function. To determine a possible role of hypothalamic factors in controlling this enzymatic process, iodothyronine 5'-deiodinase (I5'D) activity was studied in rats 6 weeks after homologous transplantation of pituitary (implanted animals) or muscle tissue (sham animals) under the renal capsule. Intrasellar pituitaries remained intact, and serum T3, T4, and TSH levels were similar in both groups. I5'D activity was determined by quantifying T3 production rates in tissue homogenates at T4 concentrations of 0.002-4 microM, and with 20 mM dithiothreitol. Sellar pituitaries from sham and implanted animals displayed similar nonlinear reaction kinetics, suggesting the presence of two enzymatic processes having approximate Michaelis-Menten constant (Km) values of 2 nM and 0.3 microM. Maximum velocity (Vmax) was 51.3 +/- (SE) 4.0 fmol T3/min X mg protein (units) and 40 +/- 6 U for the low and high Km components, respectively. In transplanted pituitary tissue, I5'D activity was markedly altered; the low Km activity was significantly decreased (Km, 6 nM; Vmax, 13.0 +/- 1.1 U; P less than 0.001 compared to sellar pituitaries), whereas the high Km activity was increased 15-fold (Km, 5 microM; Vmax, 620 U). The in vitro addition of 6-n-propyl-2-thiouracil (0.1 mM) inhibited high Km I5'D activity in homogenates of both transplanted pituitary and renal tissue by approximately 50% (P less than 0.001), but had no effect on low Km I5'D activity in either sellar or transplanted pituitaries. In sham and implanted animals rendered hypothyroid by the inclusion of 1 g/dl NaClO4 in their drinking water for 6 weeks, low Km I5'D activity was increased approximately 3-fold in sellar and transplanted pituitary tissue. The levels of activity reached in transplanted tissue, however, were only 20-30% of those noted in sellar pituitary homogenates (P less than 0.001). High Km I5'D activity was estimated to be decreased 55% in transplanted tissue from hypothyroid animals. These studies demonstrate that transplantation of the anterior pituitary gland under the renal capsule in the rat results in marked alterations in two distinct components of pituitary I5'D activity. This suggests that neuroendocrine factors are important in the control of pituitary T4 to T3 conversion. Furthermore, it provides evidence for a unique mechanism whereby the hypothalamus, by modulating local thyroid hormone metabolism, may influence pituitary function.

Animals↗
PubMed1985

Activity of the hypothalamo-pituitary axis and testicular development in prepubertal ram lambs with induced hypothyroidism or hyperthyroidism.

Prepubertal (16 weeks old) ram lambs were used to investigate the effects of hyperthyroidism and hypothyroidism on development of reproductive endocrine function. Over a period of 8 weeks, ram lambs were made hypothyroid (serum T4 less than or equal to 3 ng/ml compared with controls congruent to 30 ng/ml) by daily oral administration of methyl thiouracil or hyperthyroid (serum T4 congruent to 135 ng/ml) by daily sc injection of T4. Hyperthyroidism was associated with decreases in LH pulse frequency (2.25 +/- 0.75/12 h compared with controls 5.75 +/- 0.48/12 h), basal LH, and mean LH concentrations, together with arrested testicular growth and aspermatogenesis. Hypothyroid rams showed normal pubertal development. After iv injection of LHRH, hyperthyroid ram lambs showed similar LH responses to control and hypothyroid rams. Basal testosterone production (5.6 +/- 1.0 ng/min) and plasma testosterone concentrations after human CG (2.0 +/- 0.7 ng/ml) in hyperthyroid rams were significantly lower than in controls (67.5 +/- 24.0 ng/min and 5.2 +/- 1.0 ng/ml, respectively). It is concluded that retarded testicular development in hyperthyroid ram lambs results from changes in hypothalamo-pituitary activity manifested in a decreased LH pulse frequency.

Animals↗
PubMed1986

Hormonal control of a low Km (type II) iodothyronine 5'-deiodinase in cultured NB41A3 mouse neuroblastoma cells.

The central nervous system manifests complex homeostatic mechanisms for the maintenance of thyroid hormone economy. The present studies used the NB41A3 mouse neuroblastoma cell line as a model system to study the hormonal regulation of the enzymatic conversion of T4 to T3 in neural tissue. NB41A3 cells manifested a thiol-dependent 6-n-propyl-2-thiouracil-insensitive iodothyronine 5'-deiodinase (I5'D) with a Km for T4 of approximately 10 nM. I5'D activity was increased 2- to 4-fold in cells grown in thyroid hormone-depleted medium. Exposure of cells in situ to various thyroid hormones resulted in a rapid dose-dependent inhibition of enzyme activity with the following order of potency: rT3 = T4 greater than T3. The potent inhibitory effect of rT3 on I5'D activity could not be attributed to substrate competition with T4 in the reaction assay. The addition of dexamethasone (2 X 10(-7) M) to the culture medium also inhibited I5'D activity by 46 +/- 6% (+/- SE; n = 4 experiments; P less than 0.02), whereas insulin and epinephrine were without effect. In other experiments, saturation analysis using a purified preparation of isolated nuclei from NB41A3 cells demonstrated the presence of saturable, high affinity nuclear binding sites which had a Kd value for T3 of 0.13 +/- 0.05 nM and a maximum binding capacity of 0.13 +/- 0.01 pmol T3/mg DNA. These studies demonstrate that NB41A3 cells have a low Km (type II) I5'D process and nuclear T3-binding sites very similar to those previously described in the rat central nervous system. I5'D activity in this cell line appears to be regulated by multiple serum factors, including thyroid hormones and glucocorticoids. The potent regulatory effect of rT3 and T4 suggests that T3 formation by thyroid hormones in neural tissue is controlled by a unique cellular mechanism independent of the nuclear T3 receptor. Since tissue and plasma concentrations of T4 are considerably higher than those of rT3, the former hormone is likely to be the principal thyroid hormone regulating this enzymatic process.

Animals↗
PubMed1987

Comparison of kidney and brown adipose tissue iodothyronine 5'-deiodinases.

We have examined the influence of assay conditions on the 6-n-propyl-2-thiouracil (PTU) sensitivity of the iodothyronine 5'-deiodinase in brown adipose tissue (BAT) from hypothyroid rats. These results were compared with similar studies of 5'-deiodinase activity in kidney microsomes from euthyroid animals. Even though BAT microsomes contain largely type II (PTU-insensitive) deiodinase activity, the 5'-deiodination of T4 can be inhibited by PTU if the dithiothreitol (DTT) concentration in the assay is reduced to 5 mM or less. The apparent Ki for PTU of BAT microsomes was 4.3 mM at 5.0 mM DTT and 0.41 mM at 0.5 mM DTT. The kinetics of inhibition were noncompetitive. With kidney microsomes, PTU inhibition of rT3 5'-deiodination was both time and enzyme/substrate ratio dependent. For example, using 1 microgram microsomal protein, 2 nM rT3, and 5 mM DTT, the inhibitory effect of PTU was not maximal until 12 min after PTU addition. At stable reaction velocities PTU inhibition was uncompetitive, and the Ki was about 1 microM. Deiodination by kidney microsomes was completely inhibited by 50 microM PTU. Even though it is possible to inhibit the type II 5'-deiodinase activity with high concentrations of PTU (in the presence of low DTT concentrations), the deiodinase in kidney is about 1000-fold more sensitive to PTU. By these criteria the kidney microsome 5'-deiodinase is type I.

Adipose Tissue, Brown↗
PubMed1989

Metabolism of triiodothyroacetic acid (TA3) in rat liver. II. Deiodination and conjugation of TA3 by rat hepatocytes and in rats in vivo.

The hepatic metabolism of 3,3',5-triiodothyroacetic acid (TA3), a naturally occurring side-chain analog of T3, was studied in vitro and in vivo. Metabolites were quantified by HPLC after Sephadex LH-20 prepurification of samples obtained after incubation of [125I]TA3 or 3,[3'-125I]diiodothyroacetic acid (3,[3'-125I]TA2) with isolated rat hepatocytes under various conditions or after iv administration of [125I]TA3 to normal or 6-propyl-2-thiouracil (PTU)-treated rats. In protein-free incubations with hepatocytes, TA3 glucuronide (TA3G) and I- were normally the main TA3 products, i.e. 44% and 49%, respectively. In the presence of the type I deiodinase inhibitor PTU, the I- production from added TA3 decreased to 3%, and TA3 sulfate (TA3S) increased from 2-14%. Normally, 3,3'-TA2 was converted to I-, but in the presence of PTU 3,3'-TA2S was produced. In SO4(2-)-depleted cultures incubated with TA3 or 3,3'-TA2, production of I- was diminished, and the glucoronides of the substrates and the deiodinated products were generated. If both sulfation and deiodination were inhibited, TA3 and 3,3'-TA2 were cleared completely via glucuronidation. The metabolism of TA3 and especially 3,3'-TA2 was greatly retarded in cultures with 0.1% BSA. PTU treatment of TA3-injected rats reduced plasma I- levels 6-fold, increased plasma sulfates 2.6-fold, but did not affect plasma TA3 clearance. Biliary excretion of radioactivity until 4 h after [125I]TA3 injection amounted to 55% of the dose in controls vs. 85% in PTU-treated rats. In both groups, an unknown metabolite X was detected in serum and its sulfate conjugate XS in bile. The mean percent distribution of TA3G/TA3S/XS in bile amounted to 70:8:13 in control and 57:22:12 in PTU rats. In conclusion, TA3 is effectively metabolized in rat liver by glucuronidation and subsequent biliary excretion of TA3G, which may explain its rapid in vivo clearance relative to T3. Furthermore, a significant proportion of TA3 is deiodinated by the type I deiodinase, either directly or after prior sulfation.

Animals↗
PubMed1989

Effects of propylthiouracil on the biliary clearance of thyroxine (T4) in rats: decreased excretion of 3,5,3'-triiodothyronine glucuronide and increased excretion of 3,3',5'-triiodothyronine glucuronide and T4 sulfate.

The liver metabolizes T4 by deiodination and conjugation to T4 glucuronide (T4G), but little information exists about the formation of T4 sulfate (T4S) in vivo. We have examined the excretion of T4G, T4S, T3 and rT3 glucuronide (T3G and rT3G) in bile, collected under pentobarbital anesthesia 0-8 h or 17-18 h after iv [125I]T4 injection to control and 6-propyl-2-thiouracil (PTU)-treated rats. Radioactivity in bile, plasma, feces, and urine was analyzed by Sephadex LH-20 chromatography and HPLC. PTU induced a 2-fold increase in the biliary excretion of total radioactivity (26.6% vs. 15.0% dose between 0-8 h; 2.0% vs. 1.0% dose between 17-18 h). Biliary metabolites, 17-18 h after T4 injection, in control vs. PTU rats amounted to (percent dose): T4G, 0.44 vs. 0.75; T3G, 0.19 vs. 0.07; rT3G, 0.02 vs. 0.15; and T4S, 0.06 vs. 0.32. Similar results were obtained for control rats when bile was collected between 7-8 h after iv T4. The excretion rate of T3G was lower and that of rT3G higher when bile was continuously collected for 8 h immediately after T4 administration, probably due to prolonged experimental stress. However, regardless of the period of bile collection, PTU induced a more than 24-fold decrease in the T3G/rT3G ratio and a 5-fold increase in T4S excretion. In the animals killed 18 h after T4 injection, PTU treatment increased plasma T4 retention by 50%, reduced urinary I- excretion by 74%, and increased fecal radioactivity by 47%. No conjugates were detected in feces, and the distribution of fecal T4:T3:rT3 was 70:18:2 in control and 68:7:6 in PTU-treated rats. The results indicate that 1) the glucuronidative clearance of T4 is not affected by PTU; 2) the T3G/rT3G ratio in bile is a sensitive indicator of type I deiodinase inhibition; 3) T4 undergoes significant sulfation in rats in vivo, and 4) biliary excretion of T4S is enhanced if its type I deiodination is inhibited.

Animals↗
PubMed1989

Ligand-induced inactivation of type I iodothyronine 5'-deiodinase: protection by propylthiouracil in vivo and reversibility in vitro.

The inactivation of the phenolic ring deiodinases by natural substrates and iodinated contrast agents such as iopanoic acid (IOP) is a principal mechanism regulating the peripheral metabolism of thyroid hormones. The present studies were designed to investigate the cellular and biochemical mechanisms mediating this effect on type I 5'-deiodinase (5'DI) in rat liver and kidney. We reasoned that if inactivation requires the direct interaction of ligand (e.g. IOP) with 5'DI, then this effect should be blocked by a prior injection of 6n-propyl-2-thiouracil (PTU), which binds to an essential sulfhydryl group at the enzyme's active site and inhibits catalytic cycling. Experiments were performed using liver and kidney microsomes prepared from male rats injected 2.5 or 6 h earlier with various combinations of PTU, IOP, and/or vehicle. In initial studies, conditions were defined whereby active 5'DI could be regenerated in vitro from PTU-injected animals. This involved the treatment of microsomes with 50 mM dithiothreitol, pH 7.0, before assay. After this reductive treatment, 5'DI activity in both liver and kidney microsomes from rats previously injected with PTU, or the combination of PTU followed 30 min later by IOP, was equivalent to that in vehicle-injected control animals, whereas activity was decreased 60% in microsomes from animals injected with only IOP. This demonstrates that PTU protects 5'DI against ligand-induced inactivation, suggesting that this process is initiated by substrate binding to the enzyme's active site. In other experiments, liver microsomes from IOP-injected animals were pretreated with 100 mM dithiothreitol, pH 9.0, then washed, and 5'DI activity was determined. Under these stringent reducing conditions, 5'DI activity was completely recovered in microsomes from rats injected with IOP 2.5 h earlier, but only partially recovered 6 h after the IOP injection. Thus, ligand-induced inactivation of 5'DI may involve initially a reversible oxidation of the enzyme, followed by its irreversible inactivation.

Animals↗