Protein radical formation on thyroid peroxidase during turnover as detected by immuno-spin trapping.

Thyroid peroxidase (TPO) is a 933 amino acid residue, heme-containing, integral membrane glycoprotein that catalyzes two steps in the maturation of the thyroid hormone precursor. As with other peroxidases, these reactions require hydrogen peroxide and initial enzyme oxidation. Previous researchers studied the oxidative state of the TPO heme moiety using spectrophotometric and catalytic analyses. We use a novel antiserum to 5,5-dimethyl-1-pyrroline N-oxide (DMPO) to detect radical-derived DMPO spin-trapped TPO. Our work reveals that TPO generates radical adducts in the presence of H2O2, but that the generation of these adducts can be suppressed by the addition of substrates and inhibitors. Chemical alteration of the tyrosine residues of TPO greatly reduces the generation of TPO-DMPO adducts. Iodide strongly suppresses the H2O2-generated production of TPO radical adducts and protects the enzyme from loss of enzyme activity. Because the normal catalytic mechanism of TPO involves the production of radical species, TPO is potentially more susceptible to oxidative damage than most enzymes which do not require H2O2 as a substrate. We hypothesize that oxidatively damaged TPO may trigger the production of anti-TPO autoantibodies, resulting in the development of autoimmune thyroid disorders. Evidence that correlates iodine deficiencies with development of thyroid autoimmune disorders supports this conjecture.

Inhibition of follicle-stimulating hormone induction of aromatase activity in porcine granulosa cells by thyroxine and triiodothyronine.

We examined the effects of T3 and T4 on basal and FSH-induced aromatase activity in granulosa cells isolated from medium-sized follicles (4-6 mm) of prepubertal pigs. Treatment of cells with T3 or T4 alone during an initial 48-h induction period did not result in any significant change in aromatase activity, as measured by 17 beta-estradiol accumulation during the subsequent 6-h test period, when testosterone (0.5 microM) was added as substrate. However, when cells were cultured with FSH and T3 or T4 during the induction period, a definite dose-dependent inhibition of FSH-induced aromatase activity was demonstrated. This inhibition was not altered by the presence of a binding protein (BSA). The inhibition of FSH-induced aromatase activity by T4 and T3 is a true biological effect, as inactive iodocompounds (MIT and DIT) and iodide had no significant effect on the gonadotropin-induced aromatase activity. Furthermore, the viability of cells was unaffected by the thyroid hormones, and total cellular protein did not change significantly. These results indicate that thyroid hormones might play an important role in modulating FSH-induced aromatase activity, and that the elevated plasma estrogen levels in some cases of hyperthyroidism are not due to increased ovarian secretion.

Cycloleucine transport in isolated rat thymocytes: in vitro effects of triiodothyronine and thyroxine.

Thymocytes obtained from suckling or young adult rats were used as a model system to study the action of thyroid hormones in vitro. In this tissue, L-triiodothyronine (T3) increased the uptake of the non-metabolizable amino acids, alpha-aminoisobutyrate and cycloleucine. A detectable effect of T3 on the uptake of cycloleucine was seen at a concentration of 0.1 muM and maximum effects were seen at 20 muM. Thyroxine (T4) also increased cycloleucine uptake with about one-third the potency of T3, and this effect could not be ascribed to conversion of T4 to T3. In contrast, L-monoidotyrosine and L-diiodotyrosine were without effects on transport. Kinetic studies indicated that T3 enhanced uptake by inhibiting amino acid efflux; no effect was seen on influx. The effect of T3 on amino acid uptake was evident within 1 min, and was not inhibited by either prior treatment of the cells with cycloheximide or by lowering the incubation temperature from 37 to 24 C. In other studies, when T3 was injected into rats in vivo at a dose of 20 mug/100 g, the uptake of cycloleucine was enhanced in thymocytes obtained 1 h later. These data suggest that thyroid hormones can directly influence amino acid transport in rat thymocytes. This effect is prompt, is independent of new protein synthesis, and may reflect a direct interaction with specific components of the cell membrane.

Studies on the action mechanism of the antihemostatic effect of lodopeptides.

A synthetic iodopeptide having a glutamic acid-diiodotyrosine molar ratio of 1:1 has been shown to be an effective anticoagulant both in vivo and in vitro. Contrasted with heparin the following general conclusions may be made regarding its action. The iodopeptide does not act through the inactivation of thrombin in plasma. Iodopeptide does interact with fibrinogen to form a complex which, in vitro, is not soluble in buffered saline at physiological pH. At pH 8, iodopeptide interacts with fibrinogen to form a soluble complex in the presence of 0.9% NaCl that is not coaguable either by thrombin or Crotalus venom enzymes. All the available evidence indicates that the fibrinogen to fibrin conversion is not inhibited under these conditions, but that fibrin, once formed, is not able to polymerize due to interference by iodopeptide. Similar results were obtained with heparin in vitro with thrombin-fibrinogen mixtures in the absence of NaCl. Studies with Russell's viper venom in native PRP strongly suggest that the iodopeptide also interferes with processes in the early coagulation pathway associated with prothrombin activation.

Inotropic effects of thyroxine and related compounds on guinea-pig left atria in vitro.

Triiodothyronine, diiodothyronine and diiodotyrosine have positive inotropic activity on normal guinea-pig left atria in vitro. The increases produced by triiodothyronine and diiodothyronine are small but their detection shows that the inotropic responses to these agents can be studied in vitro. Thyroxine, thyronine and monoiodotyrosine are inactive. Reserpine pre-treatment reduces the inotropic effect of diiodotyrosine but does not reduce those of triiodothyronine and diiodothyronine.

Exogenous free iodotyrosine inhibits iodide transport through the sequential intracellular events.

We describe a new function of exogenous iodotyrosine as a regulator of iodide transport. Porcine thyroid follicles in culture were preincubated with 0-20 mumol/l monoiodotyrosine or diiodotyrosine (DIT) in the presence of bovine thyrotropin (TSH) for 24 h; these iodotyrosines inhibited iodide uptake in a dose-response manner. Extracellular [125I]DIT was actively transported to the thyroid follicle in the presence of TSH or (Bu)2cAMP. Inhibition of iodide uptake by iodotyrosine required preincubation with iodotyrosine in the presence of TSH; without TSH, iodotyrosine was ineffective. Follicles preincubated with DIT for 24 h inhibited TSH-mediated cAMP production, which is an important signal for iodide transport. Inhibition of iodide uptake and cAMP generation by iodotyrosine was negated characteristically by 3-nitro-L-tyrosine, an inhibitor of iodotyrosine deiodinase, or by methimazole, an inhibitor of thyroid peroxidase. Our findings suggest that iodotyrosine regulates iodide transport through the following sequential intracellular events: TSH-dependent iodotyrosine transport into the thyroid cell; deiodination of iodotyrosine and release in iodide; iodine organification by the peroxidase system; inhibition of cAMP generation by organified iodine; and inhibition of iodide transport. Thus, exogenous iodotyrosine can serve as an inhibitor of thyroid hormone formation only when TSH is present.

Thyroid hormones reversibly suppress somatostatin secretion and immunoreactivity in cultured neocortical cells.

Thyroid hormone effects on brain somatostatin-like immunoreactivity (SRIF-LI) were studied in an in vitro model system. Serum was removed from the nutrient culture medium of fetal day-18 rat cerebral cortex cells maintained in primary, long-term, dispersed monolayer culture. Chronic administration of either T3 or T4 in serum-free medium was associated with suppressed release of SRIF-LI into the culture medium (36-43 h accumulation), cell content of peptide and acute release in response to potassium-induced depolarization. Suppression was dose-dependent with an IC50 of less than 1 nM for T3. The most dramatic effects were observed for K+-induced release. Thirty-five to 50% suppression was typically observed with T3 at a near maximum dose (3 nM). Reverse T3 and diiodotyrosine were less potent and effective than T3. TRIAC and diiodothyronine also possessed significant suppressive activity. T3 suppression of release depended on duration of pretreatment. Administered for less than 16 h, T3 failed to significantly suppress K+-induced release, but significant suppression was observed for pretreatment periods of 16 h or longer. Indirect fluorescent immunohistochemical examination revealed a reduction in the number of cells positively stained for SRIF-LI in T3-treated dishes relative to controls. Upon removal of T3 and subsequent recovery in serum supplemented medium for 24 h, T3-treated and control cells exhibited similar levels of SRIF-LI release and cell content. T3-treated and control cells incorporated [3H] leucine into trichloracetic acid precipitable counts to similar extents. Dexamethasone and several sex steroids failed to modify the effects of T3 and did not independently influence SRIF-LI levels. Acute cycloheximide administration did not reverse T3 effects. The data indicate that primary brain cell cultures may be useful models to examine direct peripheral hormone actions on nervous tissue. Thyroid hormones suppress SRIF-LI levels in a dose, time and structure-dependent manner, which appears to be reversible. The findings are consistent with a possible integration of peripheral hormone and brain peptide physiology.

Excess iodine induces apoptosis in the thyroid of goitrogen-pretreated rats in vivo.

Previously, we observed that excess iodide rapidly suppressed the elevated ornithine decarboxylase activity in the thyroid of propylthiouracil (PTU)-pretreated rats. Excess iodide also induces involution of goitrous thyroids. These findings led us to study effects of excess iodide on apoptosis of rat thyroids. When given to PTU-pretreated rats, excess potassium iodide (KI) (13 mg/kg body weight, 10 mg as iodine) induced DNA fragmentation in the thyroid at the first 3 hours after its treatment. The percentage of DNA fragmentation was also maximal at 3 hours after KI treatment. In methimazole-pretreated rats, the kinetic of DNA fragmentation was nearly the same; apoptosis increased for the first 6 hours and then decreased at 12 hours after KI administration. Other iodinated compounds such as amiodarone and diiodotyrosine have also shown apoptosis-inducing activity, but their effect was observed later than KI. Iopanoic acid had no such effect. Apoptotic changes were also observed with the use of flow cytometry. PTU or methimazole alone had some stimulatory effect on thyroid apoptosis. Iodine effect was not observed in rats treated with either perchlorate or thiocyanate. These results suggest that excess iodine induces thyroid involution in goitrogen-treated rats at least partially by apoptosis.

Involvement of selection and amplification mechanisms in hormone receptor development in a unicellular model system.

It was demonstrated earlier, that long lasting exposure of Tetrahymena to a hormone (histamine) resulted in an increased responsiveness to a later re-exposure. However, it was difficult to establish whether selection or amplification plays a role in receptor differentiation. As diiodotyrosine (T2) enhances the growth of Tetrahymena, in the present experiment the effect of T2-treatment on a long-term culture of Tetrahymena pyriformis was analysed by mathematical-statistical methods to differentiate the effects of selection and amplification mechanisms on hormone receptor development. Although continuous and periodic treatment with T2 enhanced cell division equally, the resulting populations differed in structure. On continuous treatment the population tended to become inhomogenous. The variance tended to increase for 9 days and decreased afterwards without, however, returning to the control level. On periodic treatment the variance was the same as in the control group, but the second and third exposure were significantly more effective than the first treatment, suggesting that the primary encounter with the hormone had given rise to lasting alterations (hormonal imprinting). It follows that continuous exposure involves a selection process which does not, however, account for a steady increase of the growth rate; for initial amplification, taking place also in this condition, and selection which takes effect later, compensate one another's effects. Regarding the unicellular experimental system as a phylo- and ontogenetic model, the conclusion lies close at hand that the selection and amplication mechanisms promote hormone receptor development by joint rather than alternate action.

Electron microscopic localization and hormonal (diiodotyrosine) induction of adenylate cyclase in the Tetrahymena.

Normal Tetrahymena cells exhibited adenylate cyclase activity exclusively in association with pinocytotic vesicles, whereas those treated with diiodotyrosine (T2) for growth stimulation showed it in association with the cell membrane, and intracellularly inside many dense bodies. It appears that hormonally activable adenylate cyclase is an inherent component of the Tetrahymena.

Influence of hormone treatment applied or begun in different phases of the cell cycle on hormonal imprinting in Tetrahymena.

Primary exposure to a hormone (hormonal imprinting) alters--in the case of the Tetrahymena increases--cellular response to re-exposure(s) to the same hormone. The intensity of hormonal imprinting depends on the phase of the cell cycle in which the primary exposure has taken place. The effect of imprinting was greater on the cells exposed to the hormone in phase G1 than on those exposed in phase S or G2. The response pattern of the progeny generations corresponded to that of the primarily exposed (imprinted) ancestor cell, irrespective of their own pre-exposure in phase G1, G2 or S of their cycle.

Influence of low and high temperature on diiodotyrosine imprinting in Tetrahymena.

Hormonal imprinting takes place at the primary interaction between target cell and hormone, and alters cellular response to the hormone for lifetime (at the unicellular level in many subsequent generations). Imprinting induced in Tetrahymena cells by diiodotyrosine at the optimum temperature of 25 degrees C took effect on re-exposure to the hormone at 25 degrees C and 15 degrees C, but failed to take effect if the cells were first exposed to the hormone at 15 degrees C or 32 degrees C.

[Effect of thyroid hormones on the calcium pump in sarcoplasmic reticulum]. / O vliianii tireoidnykh gormonov na kal'tsievyi nasos sarkoplazmaticheskogo retikuluma.

In vitro thyroxine inhibited accumulation of Ca2+ by fragments of sarcoplasmic reticulum, isolated from rabbit sceletal muscles. The inhibitory effect of thyroxine was responsible for its direct action on Ca2+ dependent ATPase. Half-maximal inhibition of the enzymatic activity occurred at the same concentrations of thyroxine using both membrane-bound and highly purified solubilized forms of Ca2+-ATPase (15--20 micrometer and 10 micrometer of the hormone, respectively). Triiodothyronine was similar in the effect but not diiodothyrosine, which did not possess the hormonal activity. The data obtained suggest that thyroid hormones affect the mechanism of dephosphorylation in ATP-hydrolase reaction.

[Ultrastructural changes in thyrocytes exposed to the antithyroid preparations betazin and diiodotyrosine]. / Ul'trastrukturnye izmeneniia tirotsitov pri vozdeistvii antitireoidnykh preparatov--betazina i diiodtirozina.

Ultrastructure changes in the thyroid gland cells of rats were studied when feeding of bethazine and diiodthyrozine to rats at doses 0.01, 0.1 and 1.0 mg/kg of body's weight for 12 months. The ultrastructure changes in thyrocytes typical both for hyper- and for hypofunction of the gland were revealed.

[Biological value of the meat from animals implanted with betazine and diiodotyrosine and its effect on the body]. / Biologicheskaia tsennost' miasa zhivotnykh, implantirovannykh betazinom i diiodtirozinom, i ego vliianie na organizm.

The biological value of beef and pork of the animals implanted with antithyroid preparations did not differ from that of controls in varying tests. A long-term feeding of rats with diets containing meat of young bulls and pigs, implanted with betazine and diiodotyrosine, did not influence the content of free and bound cholesterol and total phospholipids in the liver, and produced no effect on the intensity of oxidative phosphorylation in the mitochondria which was proved by the results of the morphological, histochemical and ultrastructural investigations.