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PubMed1978

Effects of chronic treatment of intact and hypophysectomized rats with thyroid-stimulating hormone on the metabolism of [35S]methimazole and [35S]propylthiouracil.

Chronic treatment of intact rats with various doses of TSH increased the thyroidal 35S accumulation after single doses of [35S]methimazole (MMI) and [35S]propylthiouracil (PTU). However, no effect on the intrathyroidal breakdown of the drugs was observed. Thus absolute thyroidal levels of unmetabolized MMI and PTU were increased by factors of up to 2 and 3, respectively, compared to the control groups. Simultaneous decreases in the levels of thyroidal total iodine were observed. Hypophysectomized rats showed a marked inhibition of both thyroidal accumulation and oxidation of [35S]-MMI but TSH treatment of hypophysectomized rats restored the accumulation and oxidation to sham-operated and control group levels. The results show that in rats TSH has an important role in the control of thyroidal levels of antithyroid drugs currently used in the treatment of hyperthyroidism.

Animals↗

PubMed1978

Thyroxine 5'-deiodinase activity of rat kidney: observations on activation by thiols and inhibition by propylthiouracil.

Enzymatic 5'-deiodination of T4 in various tissues is known to be stimulated by thiols and inhibited by propylthiouracil (PTU). Dithiothreitol (DTT) stimulation of rat kidney T4 5'-deiodinase followed saturation kinetics. Inhibition of the enzyme by PTU (10 (-5) M) was overcome in a concentration-dependent manner by DTT, methimazole (MMI), and thiourea. Pretreatment of catalytically active kidney microsomal preparations with PTS caused persistent inhibition of enzyme activity, assayed in the absence of PTU. Similarly, injection of PTU in rats in a dosage of 5 microgram/100 g BW or greater significantly impaired T4 5'-deiodination in subsequently isolated kidney microsomal preparations. Reagents that cleave disulfide bonds (DTT and KCN), as well as the reductant, Na2S2O4, and the thioureylene, MII, partially restored activity to PTU-inactivated enzyme in a time- and concentration-dependent way. These observations suggest that PTU inhibition of T4 5'-deiodinase results from an interaction of PTU with the enzym, possibly via a PTU-enzyme disulfide which can be prevented or reversed by thiols or thioureylene agents, and raise the possibility that protein sulfhydryl groups may undergo oxidation during the reductive 5'-deiodination of L-T4.

Animals↗

PubMed1979

Oxidative metabolites of [2-14C]propylthiouracil in rat thyroid.

The identities and relative amounts of the major metabolites in rat thyroids 6 h after the administration of [14C]propylthiouracil ([14C]PTU) have been investigated. Rat thyroid extracts were chromatographed on columns of Bio-Gel P-2 and DEAE-Sephadex and in various thin layer chromatography systems. The extracts contained protein-bound PTU metabolites; an unknown peak 3; peak 1, which was chromatographically similar to PTU--SO2H; peak 2, which was similar to PTU--SO3H; PTU; and small amounts of 6-n-propyluracil (PU). The major metabolites were isolated and purified by column chromatograpy. On the basis of chromatographic properties identical to cochromatographed standards in seven different systems and the products formed after treatment with various reagents, peak 1 was identified as PTU-SO2H and peak 2 as PTU--SO3H. Peak 3 was seen only on Bio-Gel P-2 columns, was very unstable, and was not similar to any known PTU standard. The properties of this compound suggest that it may be a thiolsulfonic ester (formula:see text), but the data are insufficient for positive identification. Approximately 85% of the radioactivity in the protein peak was bound to thyroglobulin. HCl converted 86.5% of the protein-bound radioactivity to PU, and H2S converted 91% to PTU, indicating that an oxidized S was involved in the linkage to protein. Dithiothreitol released 23.6% of the protein-bound radioactivity as PTU, and mercaptoethanol released 32.5%, indicating that 25-35% of the PTU is bound in disulfide linkage. Approximately 50% of the radioactivity released by mercaptoethanol was S-ethanol PTU, which suggests a PTU-protein bond similar to a thiolsulfonic ester. Quantitation of the metabolites revealed that protein-bound metabolites accounted for 21-29% of the total radioactivity, unknown peak 3 accounted for 7.1%, PTU--SO2H for 48-50%, PTU--SO3H for 8-10%, and PTU for 10.7-16.5%. Only traces of PU were observed. The data demonstrate that PTU--SO2H is the major PTU metabolite in rat thyroid and must be the compound X observed by other investigators and that all metabolites identified are oxidative products of PTU. These findings support the earlier conclusion of Taurog and Riesco that protein binding of PTU occurs as a consequence of oxidation.

Animals↗

PubMed1980

Intrathyroidally generated iodide: the role of propylthiouracil-sensitive processes in its production.

Thyroid radioiodide to serum radioiodide concentration (*T/*S) ratios 1 h after 131I administration were 3 times higher in rats pretreated with propylthiouracil (PTU) than in rats with binding thyroids. Although indications of a preponderance of transported iodide in the thyroid early after 131I injection were found, a reduction of the 1-h *T/*S ratio in TSH-treated rats by the inhibitor of iodotyrosine dehalogenation, mononitrotyrosine, points to an early contribution of intrathyroidally generated (internal) iodide to total thyroid iodide. In rat thyroids labeled 72 h earlier, there was no rise in electrophoretically separated radioiodide 2 h after the administration of PTU alone, but the elevation of thyroid iodide due to TSH was augmented by concurrently given PTU. On the assumption that PTU affects transported components of thyroid iodide identically whether they are labeled 1 or 72 h earlier, we conclude that PTU must severely depress the generation of internal iodide. This effect is no longer evident if TSH is given simultaneously. Further, our results are consistent with the hypothesis that internal iodide passes through a stage in which it is not available for organic binding before it mixes with transported iodide. TSH appears to facilitate the transfer of internal iodide into the pool which it shares with external iodide.

Animals↗

PubMed1981

Homologous radioimmunoassay for canine thyrotropin: response of normal and x-irradiated dogs to propylthiouracil.

A RIA for canine TSH (cTSH) has been developed using a double antibody method. Rabbit anti-cTSH antisera was slightly cross-reactive with ovine TSH. No evidence of cross-reactivity was noted with human, bovine, rat RSH, or ovine LH. Serum cTSH in euthyroid dogs (9-10 yr of age; 7-14 kg) was 7.0 +/- 0.9 ng/ml (mean +/- SE; range, 2.7-7.9). Normal and x-irradiated (localized to the thyroid or cranium) dogs received 334 mg propylthiouracil (PTU) im in oil daily for 1 week as a goitrogenic challenge. A significant increase (P less than 0.05) in cTSH was noted in normal dogs and in two of four dogs that received 1000 Rads to the thyroid. No significant increase was observed in cTDH in any of the dogs previously given either 100 or 1000 Rads to the head. Although serum T3 and T4 were only slightly decreased (0.10 greater than P less than 0.15 and 0.15 greater than P less than 0.20, respectively) in thyroid-irradiated dogs, a significant decrease (P less than 0.01) in T3 was observed in both normal and x-irradiated dogs maintained on PTU for 1 week. The inability of head x-irradiated cTSH, suggests that moderate to high doses of x-irradiation can impair the ability of the hypothalamic-hypophysial-thyroid axis to respond to a goitrogenic challenge.

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↗

PubMed1982

Immunoreactive somatostatin and beta-endorphin content in the brain of mature rats after neonatal exposure to propylthiouracil.

The contents of immunoreactive somatostatin (IR-SRIF) and beta-endorphin (IR-beta-EP) in 12 brain regions were examined in rats exposed neonatally to propylthiouracil (PTU) through the mother's milk. Since the dose of PTU used in this study is lower than the usual dose employed to induce hypothyroidism, a milder form of neonatal hypothyroidism resulted. This conclusion is supported by the only mild subnormal growth of rats to adulthood and serum T4 and T3 concentrations in the normal range. Adult rats treated with PTU neonatally had significantly higher IR-SRIF contents in several brain regions compared to controls, whereas IR-beta-EP levels were not significantly different (significant increase only in the thalamus) in most regions. The results indicate that even mild hypothyroidism during early postnatal development causes permanent impairment of brain function, which manifests itself in part by an altered brain content of IR-SRIF.

Animals↗

PubMed1983

Propylthiouracil (PTU) pharmacology in the rat. I. Serum and thyroid PTU measurements by radioimmunoassay.

We have developed a highly sensitive and specific RIA for propylthiouracil (PTU) which uses 125I-labeled PTU as the radioactive ligand. At a final antibody dilution of 1:10,000, the detection limit for PTU was 100 pg; cross-reactivity with circulating, urinary, and intrathyroid PTU metabolites was negligible. Using this assay, serum and thyroid PTU levels were determined after short term (1 week) and long term (1 month) PTU treatment at doses of 0.0001-0.05%. Serum PTU was a linear function of the PTU dose (r = 0.99; P less than 0.001), whereas thyroid PTU was a linear function of the logarithm of the PTU dose (r = 0.99; P less than 0.001). Serum PTU levels were higher after 1 month of treatment than after administration for 1 week, probably because steady state conditions were not achieved after 1 week. At several doses, thyroid PTU levels were also higher after 1 month of treatment, but the differences were not as striking as those seen in the serum levels. The pharmacokinetic data are consistent with a multicompartmental model for PTU distribution. The logarithmic relationship between thyroid PTU and PTU dose suggests a saturable uptake mechanism for PTU by the thyroid; inhibition of thyroid PTU uptake by PTU itself could also explain these observations.

Animals↗

PubMed1983

Propylthiouracil (PTU) pharmacology in the rat. II. Effects of PTU on thyroid function.

Using a sensitive and specific RIA for propylthiouracil (PTU), we examined the effects of short term (1 week) and long term (1 month) PTU treatment on thyroid function in the rat, and correlated changes in thyroid function with serum and thyroid PTU levels. After 1 week, dose-dependent decreases in thyroid PBI, serum T4, and serum T3 were observed, with concomitant elevations in the serum rT3 to T4 ratio and serum TSH. Fifty percent suppression of thyroid PBI occurred at a PTU concentration in the drinking water of 0.0005% (ED50), with concomitant serum and thyroid PTU levels of 0.3 micrograms/ml and 300 ng/thyroid, respectively. After 1 month of PTU, serum T4 values were lower than after 1 week of treatment for all PTU concentrations, but values for the other thyroid functional variables were similar to those in the 1 week group at comparable PTU dosage. The PTU dose-response curve for thyroid PBI was similar to that seen after 1 week of treatment, with an ED50 of 0.0004%. After discontinuation of PTU treatment, PTU disappeared from serum in a biexponential fashion, with an early rapid distribution phase (t 1/2 = approximately 4 h) and a second slower elimination phase (t 1/2 = approximately 2.6 days). In the thyroid, an initial increase in PTU content was seen up to 18 h after PTU withdrawal; thereafter, thyroid PTU declined linearly, with a t 1/2 of 1.4 days in both groups. After PTU withdrawal, thyroid PBI recovered with a t 1/2 of 1.09 days after 1 week on PTU, but recovery was prolonged (t 1/2 = 2.8 days) after 1 month of treatment. Log thyroid PTU and log thyroid PBI were linearly related after PTU withdrawal (r = 0.97; P less than 0.001) after 1 week but not after 1 month. Serum T4 and serum T3 remained below control values for 2 days, but then rapidly normalized, with T3 values rising transiently above the control value. This rebound occurred at a time when PTU was still present within the thyroid, before thyroid PBI had returned to baseline. These data indicate a close inverse relationship between PTU dose and both thyroid hormone biosynthesis and peripheral T4 deiodination. In addition, short and long term PTU treatments have quantitatively similar effects on thyroid function, although recovery of thyroid function is prolonged after long term treatment. The biexponential disappearance of PTU from the serum is compatible with a two-compartment model of PTU distribution. The early increase in thyroid PTU after drug withdrawal is suggestive of an inhibitory effect of PTU upon its own uptake by the thyroid, whereas the faster disappearance of PTU from the thyroid than from serum is consistent with intrathyroid drug metabolism.

Animals↗

PubMed1986

A new class of propylthiouracil analogs: comparison of 5'-deiodinase inhibition and antithyroid activity.

After in vivo administration, propylthiouracil (PTU) inhibits not only thyroid iodide uptake and organification, but also T4 5'-deiodinase activity in most peripheral organs. The present report describes the effects of some previously untested 6-substituted 2-thiouracil derivatives on in vivo and in vitro iodide uptake and organification, and on T4 5'-deiodinase activity in liver and pituitary homogenates. When added to homogenates, many analogs were as potent or more potent than PTU in inhibiting hepatic T4 5'-deiodinase activity. Three derivatives, 6-anilino-2-thiouracil (A compound), 6-(p-ethylanilino)2-thiouracil (B compound), and 6-(p-n-butylanilino) 2-thiouracil (C compound), which were among the most potent inhibitors of hepatic T4 5'-deiodinase, when added in vitro inhibited T4 5'-deiodinase activity in liver homogenates after in vivo administration. When added to pituitary homogenates prepared from hypothyroid rats, these compounds also significantly inhibited pituitary T4 5'-deiodinase activity. In a concentration of 1 mM in the presence of 20 mM dithiothreitol, the percent inhibition of pituitary T4 5'-deiodinase activity was 19.7 +/- 7.4 (mean +/- SE), 34.0 +/- 3.2, 47.3 +/- 3.1, and 89.0 +/- 1.0 for PTU and the A, B, and C compounds, respectively (P less than 0.05 for all groups vs. one another and vehicle). Despite their ability to inhibit hepatic T4 5'-deiodinase activity, none of the 13 analogs tested altered thyroid iodide uptake or organification after administration of 0.1 mg/rat. PTU, in the same dose, inhibited thyroid iodide uptake by 78.2 +/- 2.4% (P less than 0.001) and thyroid iodide organification by 36.4 +/- 7.3% (P less than 0.01). Furthermore, the A, B, and C compounds did not inhibit thyroid iodide uptake or iodide organification when administered in higher doses of 5, 5, and 1 mg/rat, respectively. In contrast to these in vivo results, the A, B, and C compounds were more potent than PTU in inhibiting iodide organification in a purified thyroid peroxidase system and in porcine thyroid slices. The concentrations causing 50% inhibition of iodide organification in the purified thyroid peroxidase system were 30, 7, 8, and 14 microM for PTU and the A, B, and C compounds, respectively. However, PTU was far more potent in inhibiting iodide organification in intact incubated thyroid lobes compared to the A, B, and C compounds.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1986

Beta-endorphin and its congeners in rat pituitary and thyroid: effects of propylthiouracil and thyroid hormone administration.

Immunoreactive beta-endorphin (ir-beta EP) and immunoreactive N-acetyl-endorphin (ir-NacEP) have been demonstrated in the rat thyroid by specific RIA and characterized by reverse phase HPLC. In addition, pituitary and thyroid ir-beta EP and ir-NacEP levels have been determined after manipulation of the pituitary-thyroid axis by chronic (21 days) treatment with 1) propylthiouracil (PTU), 2) L-T4, 3) L-T3, or 4) T3 plus PTU. No difference in anterior pituitary or neurointermediate lobe ir-beta EP was seen between controls and treated groups (n = 8/group). In contrast, levels of ir-NacEP were markedly lower (P less than 0.01) in both hypo- and hyperthyroid groups than in controls, in both anterior pituitary and neurointermediate lobe. In the thyroid, levels of both ir-beta EP and ir-NacEP were profoundly depressed (P less than 0.01) in all treated groups, with no change in calcitonin levels, suggesting that the thyroid effect of PTU and T3/T4 may be specific for the synthesis, processing, and/or release of pro-opiomelanocortin derived peptides. The findings in this study suggest 1) that acetylation of pituitary and thyroid beta EP is similarly sensitive to PTU and thyroid hormone administration and 2) that in the thyroid, but not in the pituitary, both PTU and thyroid hormones markedly lower levels of pro-opiomelanocortin-derived peptides.

Acetylation↗

PubMed1986

Iodothyronine 5'-deiodinase in brown adipose tissue: thiol activation and propylthiouracil inhibition.

Brown adipose tissue (BAT) of hypothyroid rats contains a low Km (type II) iodothyronine 5'-deiodinase (I-5'D) that has been characterized as being insensitive to inhibition by propylthiouracil (PTU), based mainly on observations with homogenates prepared in a medium containing 10 mM dithiothreitol (DTT) and enzymatic assays in the presence of 20 mM DTT in vitro. In the studies reported herein, BAT homogenates from hypothyroid rats prepared in a DTT-free medium were found to contain I-5'D activity at 20 mM DTT, comparable to that in homogenates prepared in a DTT-containing medium, and were activated by submillimolar concentrations of DTT with an EC50 of approximately 0.5 mM. Almost all of the homogenate activities could be accounted for in microsomal preparations. The activity was substantially inhibited by 1 mM PTU. The PTU inhibition was progressively alleviated with increasing concentrations of added DTT and was not seen at DTT concentrations higher than 10 mM. At 250 microM DTT, the Km and maximum velocity values for rT3 and T4 were 2.9 and 1 nM and 70 and 200 fmol/mg protein X h, respectively, with a Ki for PTU of approximately 200 microM. On administration of PTU in vivo (2 mg/100 g BW; 1 h before killing) and subsequent assay at 250 microM DTT, the I-5'D in the homogenates was about 50% inhibited, and the microsomes showed a state of persistent inhibition, with activity levels about 70% of the control value. The data show that BAT type II I-5'D can be substantially activated at submillimolar concentrations of DTT, and this activation is sensitive to inhibition by PTU administered both in vitro and in vivo.

Adipose Tissue, Brown↗

PubMed1986

Increase in the number of type II insulin-like growth factor receptors during propylthiouracil-induced hyperplasia in the rat thyroid.

We have observed that membranes isolated from rat thyroids contain receptors for the insulin-like growth factors (IGF). As IGFs are known to be important mediators of tissue growth, we conducted this study to determine whether modulation of thyroid IGF receptors might be involved in TSH-stimulated hyperplasia. A substantial increase in both the weight of the thyroid and its DNA content was observed within 2 days of exposing adult male rats to 0.1% propylthiouracil (PTU) in their drinking water. Serum T4 reached unmeasurable levels and serum TSH rose 3-fold over control by the tenth day of treatment. [125I]Iodo-human(h)IGF-II binding to membranes isolated from hyperplastic glands was significantly higher than control beginning at 2 days. A maximum was reached after 5 days (13.3 +/- 0.8%/25 micrograms protein vs. a control level of 6.7 +/- 0.7%, mean +/- SEM). The increase had disappeared by 15 days of PTU exposure, paralleling the drastic fall in the growth rate of the glands. This increase in binding was specific for the thyroid, as it was not seen in other organs. In both treated and control animals, the receptor involved was shown to be type II by preferential binding to IGF-II, lack of interaction with insulin, and molecular sizing. The observed increase in binding could be accounted for by an increase in receptor site number, the affinity remaining essentially the same. We conclude that the TSH-stimulated hyperplasia of the rat thyroid, induced by PTU, is associated with an increase in the binding sites of the type II IGF receptor. This observation raises the possibility that modulation of this receptor may play a role in the mediation of the mitogenic effect of TSH on the thyroid gland.

Animals↗

PubMed1988

Propylthiouracil and methimazole display contrasting pathways of peripheral metabolism in both rat and human.

We have developed HPLC procedures for analyzing the metabolites of [35S]methylmercaptoimidazole [( 35S] MMI) and [35S]propylthiouracil [( 35S]PTU) in bile, urine, serum, and liver of rats. We also studied urinary metabolites of [35S] MMI and [35S]PTU in one human subject. In bile collected from [35S]MMI-injected rats, two major metabolites accounted for 80-90% of the total 35S. Incubation of these metabolites either with or without beta-glucuronidase led to the appearance of a 35S-labeled compound less polar than MMI. In contrast, the major [35S]PTU metabolite in bile (greater than 50% of total 35S) was completely converted to [35S]PTU on incubation with beta-glucuronidase and showed no conversion in a control incubation. From these results we conclude that the major biliary metabolites of MMI in rats are not glucuronides. They appear to be labile conjugates of a metabolite of MMI. After [35S]MMI injection into rats, two major and at least four minor metabolites were observed in urine. In one human who received [35S]MMI orally, the HPLC profile of 35S in urine was similar to that of the rat. Incubation of human urine or of its isolated major component with beta-glucuronidase had no significant effect on the HPLC profile. On the other hand, the major urinary metabolite of [35S]PTU in human and rat urine was completely converted to [35S]PTU on incubation of whole urine with beta-glucuronidase. These results indicate that glucuronides comprise at most only a minor fraction of MMI metabolites in urine of rats or humans. Based on similarities in elution time, the metabolites of [35S]PTU in urine closely resembled those in bile of rats. In contrast, the metabolites of [35S]MMI in urine were strikingly different from those in bile. PTU displays noncovalent binding to serum protein to a much greater extent than does MMI. However, after injection of [35S]MMI into rats, a significant fraction of the 35S was firmly bound to protein in both serum and liver. This binding appeared to be covalent and involved metabolism of [35S]MMI. This type of binding was much less detectable after the injection of [35S]PTU into rats.

Animals↗

PubMed1988

Iodothyronine 5'-deiodination in rat kidney microsomes: sensitivity to propylthiouracil.

When activated by dithiothreitol, iodothyronine 5'-deiodinase (I-5'D) activity in kidney microsomes is less sensitive to inhibition by propylthiouracil (PTU) and iopanoate (IOP) at nanomolar, compared to micromolar, substrate concentrations. The enzymatic activities at nanomolar substrate concentrations are, however, completely eliminated in the presence of a combination of 10 microM IOP and 100 microM PTU. In this report we present evidence that 1) the relative PTU insensitivity results from the residual activities of the high Km enzyme which, while being very sensitive to PTU inhibition at micromolar substrate concentrations, becomes progressively less PTU sensitive as substrate concentrations decline relative to its Km; and 2) the relative IOP insensitivity is due to the presence in kidney microsomes of a low Km enzyme which is relatively insensitive to IOP, but highly sensitive to inhibition by PTU. Classifying the deiodinases on the basis of PTU sensitivity, therefore, requires that not only the thiol concentrations, but, as in the case of the type I enzyme, also the substrate concentrations be specified. The PTU resistance of the type I enzyme at nanomolar substrate concentrations suggests a role of this enzyme in T3 neogenesis in PTU-treated animals.

Animals↗

PubMed1989

Enhancement of cytosolic tyrosine kinase activity by propylthiouracil-induced hyperplasia in the rat thyroid.

Hyperplasia of the thyroid gland induced by propylthiouracil (PTU) is a well established model of rapid cell proliferation in vivo. Recent evidence indicates that tyrosine kinase activity is associated with growth factor receptors and oncogene protein products and may have an important regulatory action in the control of cell growth. Thus, we examined tyrosine kinase activity in rat thyroid membrane and cytosol preparations at rest and during PTU-induced hyperplasia. Although kinase activity was present in a crude microsomal membrane preparation, no change was observed during thyroid growth. In contrast, tyrosine kinase activity assayed with the artificial substrate poly(Glu,Na:Tyr) 4:1 was present in normal rat thyroid cytosol and increased 2- to 6-fold during the rapid phase of hyperplasia in the first 5-10 days of PTU treatment. It declined to control values by day 15, when the size and DNA content of the thyroid reached a plateau. Preincubation of the cytosolic preparations with several peptides known to bind to and activate growth factor receptor tyrosine kinases failed to enhance the activity, suggesting, along with the cytosolic localization, that the activity was distinct from these receptors. By gel filtration chromatography and polyacrylamide gel electrophoresis, tyrosine kinase activity was associated with a 55 kDa protein. Partial purification over a poly(Glu,Na:Tyr)4:1-Sepharose column, yielded a protein that appeared capable of autophosphorylation. It is suggested that this tyrosine kinase plays a role in mediating the growth-promoting effects of this model of thyroid cell hyperplasia.

Animals↗

PubMed1989

Metabolism of 35S- and 14C-labeled propylthiouracil in a model in vitro system containing thyroid peroxidase.

In previous communications we described an in vitro model system containing highly purified thyroid peroxidase (TPO) for studying the mechanism of inhibition of thyroid hormone biosynthesis by the antithyroid drugs, 6-propylthiouracil (PTU) and 1-methyl-2-mercaptoimidazole (MMI). We showed that inhibition of iodination of thyroglobulin in this system may be reversible or irreversible depending on the relative concentrations of iodide and drug and the TPO concentration. Metabolism of the drugs occurred under both conditions, but was more limited under irreversible conditions of inhibition. It was of interest to examine the nature of the drug metabolites associated with reversible and irreversible conditions of inhibition. For this purpose we have employed the 35S- and 14C-labeled drugs and a recently developed reverse phase HPLC procedure. Results of a similar study with MMI were reported in an earlier communication. In the present study we report our findings with PTU. Under conditions of reversible inhibition, PTU was readily metabolized and by 15 min was reduced to a few percent of the starting value. The earliest detectable metabolite with both [35S]- and [14C]PTU was the disulfide, which reached a peak in about 15 min and then slowly declined. Coincident with the decline in the disulfide was the appearance of more polar metabolites. In the case of [35S]PTU, these corresponded to sulfate/sulfite, PTU sulfonate, and a product tentatively identified as PTU sulfinate. The latter two were also observed as 14C-labeled metabolites produced from [14C]PTU. Two nonpolar desulfurated 14C-labeled metabolites were also observed. Surprisingly, these did not correspond to either propyluracil or propyldeoxyuracil, the anticipated most likely products of PTU desulfuration. The identity of these desulfurated metabolites of PTU in the TPO model system remains to be determined. Under conditions of irreversible inhibition of iodination, a relatively small fraction of PTU was metabolized. PTU disulfide was, again, the earliest detectable metabolite, and it declined with time. However, only small amounts of other metabolites were observed, in contrast to the results obtained under conditions of reversible inhibition of iodination. As in the case of MMI, the difference in metabolic pattern between reversible and irreversible conditions is primarily related to the rapid inactivation of TPO that occurs under irreversible conditions. In general, the metabolism of PTU by the TPO model system resembled that previously observed with MMI. With both drugs, the disulfide was the earliest detectable metabolite, and under conditions of reversible inhibition of iodination, an appreciable fraction of the sulfur was oxidized as far as sulfate/sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

A reexamination of the proposed inactivation of thyroid peroxidase in the rat thyroid by propylthiouracil.

The antithyroid drug 6-propylthiouracil (PTU) was previously shown in our laboratory to have an unexpectedly prolonged inhibitory effect on iodination in the thyroid glands of rats. Eighteen hours after injection of a relatively small dose, iodination in the thyroid remained inhibited by more than 90%. We previously suggested that the prolonged inhibitory effect might be due to inactivation of thyroid peroxidase (TPO), a reaction previously shown to occur under certain conditions in an in vitro iodinating system containing highly purified TPO. However, the analytical procedure used in our earlier study did not exclude the possibility that sufficient PTU remained in the thyroid even after 18 h to inhibit TPO-catalyzed iodination by a reversible mechanism. Development of an improved analytical procedure, based on HPLC, led us to reexamine the mechanism of the prolonged inhibitory effect of PTU on iodination in rat thyroid glands. Rats were injected with [35S]PTU (1 mumol/100 g BW), and ultrafiltrates prepared from their homogenized thyroid glands were analyzed by HPLC. The major 35S-labeled metabolites were identified as sulfate/sulfite, PTU sulfinate, and PTU sulfonate. However, even after 18 h, a significant amount of unchanged [35S]PTU was also present. The calculated mean concentration of residual PTU was 20 microM, a sufficiently high level to explain the observed inhibition of iodination on the basis of a reversible mechanism. Experiments were also performed to examine the intrathyroidal distribution of 35S at intervals after the injection of [35S]PTU. All of the oxidation products of PTU showed marked increases between 2 and 16 h after injection. Based on our view that TPO is the major mediator of intrathyroidal metabolism of PTU, this observation is inconsistent with our previous proposal that TPO is inactivated after PTU injection. The results of the present study, therefore, lead us to withdraw our previous suggestion that TPO is inactivated after injection of PTU into rats. It is more likely that inhibition of iodination by PTU in the rat thyroid involves competition between PTU and tyrosyl residues of thyroglobulin for oxidized iodine, comparable to the reversible mechanism of inhibition observed in the TPO model system.

Animals↗