The thyroid gland is a major source of circulating T3 in the rat.

In rats, the respective contribution of the thyroid and peripheral tissues to the pool of T3 remains unclear. Most, if not all, of the circulating T3 produced by extrathyroidal sources is generated by 5'-deiodination of T4, catalyzed by the selenoenzyme, type I iodothyronine 5'-deiodinase (5'D-I). 5'D-I in the liver and kidney is almost completely lost in selenium deficiency, resulting in a marked decrease in T4 deiodination and an increase in circulating T4 levels. Surprisingly, circulating T3 levels are only marginally decreased by selenium deficiency. In this study, we used selenium deficiency and thyroidectomy to determine the relative contribution of thyroidal and extrathyroidal sources to the total body pool of T3. Despite maintaining normal serum T4 concentrations in thyroidectomized rats by T4 replacement, serum T3 concentrations remained 55% lower than those seen in intact rats. In intact rats, restricting selenium intake had no effect on circulating T3 concentrations. Decreasing 5'D-I activity in the liver and kidney by > 90% by restricting selenium intake resulted in a further 20% decrease in serum T3 concentrations in the thyroidectomized, T4 replaced rats, suggesting that peripheral T4 to T3 conversion in these tissues generates approximately 20% of the circulating T3 concentrations. While dietary selenium restriction markedly decreased intrahepatic selenium content (> 95%), intrathyroidal selenium content decreased by only 27%. Further, thyroid 5'D-I activity actually increased 25% in the selenium deficient rats, suggesting the continued synthesis of this selenoenzyme over selenoproteins in other tissues in selenium deficiency. These data demonstrate that the thyroid is the major source of T3 in the rat and suggest that intrathyroidal T4 to T3 conversion may account for most of the T3 released by the thyroid.

Dissociation of actin polymerization and enzyme inactivation in the hormonal regulation of type II iodothyronine 5'-deiodinase activity in astrocytes.

T4 dynamically regulates the levels of type II iodothyronine 5'-deiodinase in the brain. Using an astrocyte cell culture model, we have shown that thyroxine increases inactivation of this enzyme through a mechanism using the actin cytoskeleton. In the absence of T4, the filamentous actin (F-actin) stress fibers are absent, and deiodinase inactivation is relatively slow. T4 increases inactivation of type II 5'-deiodinase by 1) restoring the F-actin stress fibers, 2) promoting the binding of the enzyme to F-actin, and 3) stimulating enzyme internalization. To determine whether inactivation of the deiodinase is due solely to the restoration of stress fibers by T4 or also involves direct thyroxine-mediated enzyme-F-actin interactions, we examined the effects of retinoids on both actin polymerization and type II 5'-deiodinase activity in cultured astrocytes, as these hormones have been shown to alter cytoskeletal organization in other tissues. In thyroid hormone-deficient astrocytes, retinoic acid increased F-actin levels, with no change in total cell actin. The F-actin content increased approximately 40% within 30 min after the addition of retinoic acid. After a plateau of 6-8 h, the F-actin content increased further to approximately 90% of the total cell actin and was associated with the reappearance of stress fibers. Only this latter retinoid-stimulated increase in F-actin content was blocked by actinomycin-D. Restoration of the F-actin stress fibers by retinoids did not increase the turnover of the type II 5'-deiodinase (t1/2, 1.99 h-1) or promote binding of the enzyme to F-actin in the absence of T4. Similarly, retinoids did not affect the rapid T4-mediated turnover (t1/2, 0.18 h-1) of type II 5'-deiodinase. These data show that an intact F-actin cytoskeleton in the absence of T4 is inadequate to alter the inactivation of type II 5'-deiodinase and that specific T4-enzyme-F-actin interactions are necessary to initiate the rapid inactivation/internalization of this enzyme.

Thyroid hormone-dependent redistribution of the 55-kilodalton monomer of protein disulfide isomerase in cultured glial cells.

In addition to the effects of thyroid hormone that are mediated through interaction with chromatin-associated receptors, T4 modulates the activity of the cellular content of the membrane-associated protein type II iodothyronine 5'-deiodinase (5'D-II) by regulating its degradation through an actin-dependent extranuclear mechanism. Under the influence of thyroid hormone, the substrate-binding subunit of 5'D-II is translocated from the plasma membrane to an intracellular microfilament-associated pool. In glial cells, a 55-kilodalton (kDa) protein (glial-p55), which was shown to be identical to the 55-kDa monomer of protein disulfide isomerase (PDI) also demonstrates a similar T4-dependent association to the F-actin microfilaments. To explore the role of glial-p55 in the extranuclear effect of thyroid hormone in glial cells, the effects of thyroid hormone on the subcellular localization of glial-p55 were further examined. The current study demonstrates the presence of two pools of glial-p55. While the majority of glial-p55 is associated with endoplasmic reticulum and represents PDI, approximately 25% of glial-p55 is cytosolic in the absence of thyroid hormone. Cytosolic glial-p55 is lost from the cells after mild permeabilization with saponin, and treatment of cells with T4 causes the shift of glial-p55 from the cytosolic pool to the subcellular fractions that contain the actin cytoskeleton. Crude microsomal preparations were prepared which contain membranes, microfilaments, and other particulate cell structures. In the absence of thyroid hormone, glial cells lack an intact actin cytoskeleton, and glial-p55 is easily removed from these preparations by conditions that remove extrinsic membrane proteins like PDI, such as alkaline pH and detergent extraction. In contrast, glial-p55 is not removed from the crude microsomes prepared from thyroid hormone-replete glial cells that contain an intact actin cytoskeleton. Since previous work in our laboratory indicated that glial-p55 becomes actin associated in a thyroid-dependent manner along with the substrate-binding subunit of 5'D-II, this study suggests that the 55-kDa monomer of PDI may play a role in the thyroid hormone-dependent regulation of actin polymerization and the degradation of 5'D-II.

Thyroid hormone regulates the expression of laminin in the developing rat cerebellum.

In the rat cerebellum, migration of neurons from the external granular layer to the internal granular layer occurs postnatally and is dependent upon the presence of thyroid hormone. In hypothyroidism, many neurons fail to complete their migration and die. Key guidance signals to these migrating neurons are provided by laminin, an extracellular matrix protein that is fixed to the surface of astrocytes. Expression of laminin in the brain is developmentally timed to coincide with neuronal growth spurts. In this study, we examined the role of thyroid hormone on the expression and distribution of laminin in the rat cerebellum. We show that laminin content steadily increased 2- to 3-fold from birth to maximal levels on postnatal day 8-10 then steadily decreased to a plateau by postnatal day 12 in the euthyroid cerebellum. Immunoreactive laminin appeared in the molecular layer of the euthyroid cerebellum by postnatal day 4, reached maximal intensity by postnatal day 8-10, and was gone by postnatal day 14. In contrast, laminin content in the hypothyroid cerebellum remained unchanged from birth until postnatal day 10 and then increased to maximal levels over the next two days; maximal levels were approximately 35% less than those levels in the euthyroid cerebellum. Laminin staining did not appear in the molecular layer of the hypothyroid rat cerebellum until postnatal day 10, reached maximal intensity by postnatal day 15 and disappeared by postnatal day 18, despite the continued presence granular neurons in the external granular layer. These data indicate that the disruption of the timing of the appearance and regional distribution of laminin in the absence of thyroid hormone may play a major role in the profound derangement of neuronal migration observed in the cretinous brain.

Thyroid hormone regulates the extracellular organization of laminin on astrocytes.

Astrocytes produce laminin, a key extracellular matrix guidance molecule in the developing brain. Laminin is bound to transmembrane receptors on the surface of astrocytes known as integrins, which are, in turn, bound to the microfilament meshwork inside the astrocyte. Previous studies have shown that T4 regulates the pattern of integrin distribution in astrocytes by modulating the organization of the microfilaments. In this study, the effect of thyroid hormone on the secretion and topology of laminin in astrocytes was examined. Linear arrays of secreted laminin were observed on the surface of the T4-treated astrocytes within 10 h after seeding the cells onto poly-D-lysine-coated coverslips and became an organized meshwork by 24 h. In contrast, little if any laminin was identified on the surface of either hormone-deficient or T3-treated cells until 36 h after seeding and then was restricted to punctate deposits. Secretion of laminin into the medium by hormone-deficient and T3-treated cells was significantly greater than that by T4-treated cells. Conversely, deposition of laminin into the extracellular matrix was significantly greater in T4-treated cells than in hormone-deficient and T3-treated cells. Thyroid hormone had no effect on the production of laminin by astrocytes. These data show that T4 regulates the extracellular deposition and organization of laminin on the surface of astrocytes and provide a mechanism by which this morphogenic hormone can influence neuronal migration and axonal projection in the developing brain.

Thyroxine-dependent regulation of integrin-laminin interactions in astrocytes.

Adhesive interactions among the extracellular matrix protein laminin, cell surface receptors known as integrins, and the microfilament network play a fundamental role in the regulation of neural cell migration during brain development. The disturbed neuronal migration that occurs when thyroid hormone is lacking during early neonatal life contributes to the profound morphological alterations characteristic of the cretinous brain. We have previously shown that thyroid hormone determines the organization of the microfilament network in astrocytes by regulating the polymerization of F-actin fibers. In this paper, we examined whether T4-dependent alterations in microfilament organization affected astrocyte-laminin interactions. We show that T4-treated astrocytes readily attached to laminin, whereas attachment of thyroid hormone-deficient cells to laminin was delayed. T4-dependent cell attachment to laminin was completely abolished by blocking integrin recognition sites with site-specific peptides or by depolymerizing the microfilaments with dihydrocytochalasin B. We also show that T4 was required for integrin clustering and focal contact formation in astrocytes attached to laminin. Thus, T4 dynamically regulates interactions between integrins and laminin via modulation of microfilament organization in astrocytes. The T4-dependent regulation of laminin-integrin interactions provides a mechanism by which this morphogenic hormone can influence neuronal migration and development.

Nuclear thyroid hormone receptor in the rat uterus.

The uterus may be a target organ for T3 action. The present study was done to determine whether uterine nuclei contain receptors for T3 (T3R). Methods were established for the measurement of T3R concentrations in preparations of rat uterine nuclei. The liver was studied concomitantly as a tissue known to contain nuclear T3R. Nuclear fractions were prepared by established procedures and extracted in buffer containing 0.5 M KCl. Specific [125I]T3 binding in the nuclear extract was analyzed by Scatchard plot. In both liver and uterine nuclear extracts, a binding component of high affinity for [125I]T3 (Ka = 1.0-3.0 X 10(9) M-1) was detected. This binding component was lost after heating at 50 C for 10 min. On sucrose density gradients, the principal [125I]T3-binding component in uterine and liver nuclear extracts had a 3.5-4.0 S sedimentation coefficient. Binding specificity was assessed by competition assays for [125I]T3 binding by using stable T3, T4, triiodothyroacetic acid (TRIAC), rT3, and 3,5-diiodothyronine (T2). The order of relative binding affinities (RBAs) in uterine nuclear extracts was T3 greater than TRIAC greater than T4 greater than rT3 greater than T2, and that in liver extracts was TRIAC greater than T3 greater than T4 greater than rT3 greater than T2. In contrast, RBA values derived from dilute serum were distinctly different from both uterine and liver values: T4 greater than T3 greater than rT3 greater than TRIAC greater than T2. The concentrations (femtomoles per mg DNA) of T3R in several tissues were: liver, 595 +/- 162; kidney, 492 +/- 120; uterus, 249 +/- 66; spleen, 48 +/- 10 (mean +/- SE; n = 4). These results provide evidence for the presence of T3R in the nuclear fraction of the rat uterus with properties similar to those of the liver nuclear T3R.

Differential expression of thyroid hormone receptor isoforms in neurons and astroglial cells.

The brain has abundant nuclear T3-binding sites and contains messenger RNAs (mRNAs) encoding multiple thyroid hormone receptor (TR) isoforms; the cellular distribution of these different TR isoforms is unknown. To determine whether the TR isoforms are differentially expressed in neuronal and astroglial cells, we examined the relative abundance of the mRNAs encoding TR alpha 1, c-erbA alpha 2, and TR beta 1 in primary cultures of fetal rat brain and in several cell lines of neural and glial origin. Additionally, the TR isoform polypeptides were identified by immunocytochemistry using isoform-specific antibodies. Northern blot analysis showed that fetal brain cell cultures contain mRNAs encoding the T3-binding isoforms TR alpha 1 and TR beta 1 as well as the mRNA encoding the non-T3-binding c-erbA alpha 2. c-erbA alpha 2 mRNA was most abundant, comprising more than 85% of the TR mRNAs in the primary cultures. Neuronal enrichment by antimitotic selection increased TR beta 1 mRNA approximately 3-fold, decreased c-erbA alpha 2 mRNA 70%, and had little or no effect on TR alpha 1 mRNA. Neuronal depletion resulted in the complete loss of TR beta 1 mRNA without changing c-erb alpha 2 or TR alpha 1 mRNA levels. Primary cultures of rat astrocytes, the astrocytoma cell line C6, and the pheochromocytoma cell line PC12 contained only the c-erbA alpha 2 mRNA. Immunocytochemistry using isoform-specific anti-sera revealed that TR beta 1 was exclusively localized to neuronal nuclei, and c-erbA alpha 2 was only found in the nuclei of astrocytes. These results show that TR beta 1 is localized to the nuclei of neuronal cells, and that c-erbA alpha 2 is restricted to the nuclei of astrocytes.

The mammalian homolog of the frog type II selenodeiodinase does not encode a functional enzyme in the rat.

Type II iodothyronine deiodinase is a short-lived, membrane-bound enzyme found in rat brain, brown adipose tissue, and cAMP-stimulated astrocytes. Recently, a full-length complementary DNA (cDNA) encoding a 30-kDa, type II-like selenodeiodinase was cloned from frog, and a homologous partial cDNA (rBAT 1.1), containing two in-frame selenocysteine codons (UGA), was isolated from rat brown adipose tissue. Importantly, the rBAT 1.1 cDNA was derived from a 7.5-kb messenger RNA (mRNA) and did not encode a functional selenoenzyne unless an enabling selenocysteine insertion sequence was appended to the presumed coding region and this cDNA. In this study we determined whether the native 7.5-kb SeD2 mRNA in rat tissues programmed the synthesis of the native type II deiodinase using specific antibodies that were raised against the C-terminus of full-length, 30-kDa SeD2 protein and against the catalytic core of SeD2. Direct analysis of the translation products programmed by the native SeD2 mRNA in cAMP-stimulated astrocytes was performed using antisense deoxynucleotides and hybrid selection strategies. (Bu)2cAMP-stimulated rat astrocytes expressed both type II deiodinase activity (approximately 2500 U/mg protein) and contained abundant levels of the 7.5-kb SeD2 mRNA. However, no immunoreactive 30-kDa SeD2 protein was identified by Western analysis, immunoprecipitation, or immunocytochemistry, and the specific C-terminus antiserum failed to immunoprecipitate deiodinase activity from (Bu)2cAMP-stimulated astrocytes, brown adipose tissue or brain. Instead, the native 7.5-kb SeD2 mRNA encoded a 15-kDa protein that terminated at the first UGA codon and contained the catalytically inactive, N-terminal 129 amino acids of SeD2. These data show that the native 7.5-kb SeD2 mRNA in stimulated astrocytes does not encode D2.

Effects of selenium deficiency on thyroid hormone economy in rats.

In selenium-deficient rats, peripheral T4 to T3 conversion is markedly decreased due to the loss of the selenoprotein, type I iodothyronine 5'-deiodinase (5'D-I). Despite the marked increase in circulating T4 that results from this loss of 5'D-I, serum T3 concentrations in selenium-deficient rats remain in the normal range. To determine the physiological mechanism(s) that maintains circulating T3 when peripheral T4 to T3 conversion is impaired, we examined the interrelationships between selenium intake and the metabolism of T3 and T4 in the rat. In euthyroid rats, selenium deficiency caused the expected loss of 5'D-I, with a 52% increase in serum T4, which paralleled an increase in the T4 biological half-life. Consistent with the prolonged t1/2 of T4, short term thyroidectomy (48 h) in selenium-deficient rats failed to decrease serum T4 concentrations to the levels observed in short term thyroidectomized, selenium-supplemented rats. Short term thyroidectomy also caused an expected 33% decrease in liver 5'D-I and a 44% increase in brain type II iodothyronine 5'-deiodinase (5'D-II) activities in selenium-supplemented rats. However, in selenium-deficient rats, short term thyroidectomy did not affect 5'D-I or 5'D-II activities. In contrast to the selenium-dependent changes in circulating T4 levels, little or no change in circulating T3 concentrations occurred. There was a 20% increase in the T3 half-life in selenium-deficient rats. The serum T3 sulfate concentration was increased, and T3 deiodination was reciprocally decreased in the selenium-deficient rats. These data suggest that increased T3 sulfate generation in selenium-deficient rats may lead to greater T3 availability through enterohepatic recycling of the iodothyronine and may explain why there are only minor changes in serum T3 concentrations in selenium-deficient rats.

Selenium deficiency and type II 5'-deiodinase regulation in the euthyroid and hypothyroid rat: evidence of a direct effect of thyroxine.

Selenium deficiency in rats is characterized by elevated serum T4 and decreased serum T3 concentrations, and low liver type I (5'D-I) and brain type II (5'D-II) iodothyronine 5'-deiodinase activities. These findings are partially explained by the demonstration that type I 5'D is a selenoprotein; however, 5'D-II does not contain selenium. Since 5'D-II varies inversely with serum T4 concentrations, and serum T4 is elevated in selenium deficiency, the decreased cerebrocortical 5'D-II activity may be secondary to the increased serum T4 levels. To determine the mechanism(s) by which selenium influences 5'D-II activity, we examined the effects of altered selenium intake on brain 5'D-II levels and enzyme turnover in euthyroid and thyroidectomized rats. Rats were fed a selenium-supplemented or selenium-deficient diet for 5 weeks from weaning; half of the animals were also thyroidectomized 3 weeks before death. Selenium deficiency was confirmed by decreased liver and brain glutathione peroxidase activities. In euthyroid rats, selenium deficiency caused a 38% increase in serum T4, and 91% and 39% decreases in 5'D-I and 5'D-II, respectively, compared to those in selenium-supplemented rats. In the thyroidectomized hypothyroid rats, selenium deficiency caused a 60% decrease in 5'D-I, but had no effect on 5'D-II activity, fractional turnover of the enzyme, or the calculated enzyme synthesis rate. The lack of effect of selenium deficiency on 5'D-II levels in hypothyroid rats is consistent with the finding that 5'D-II is not a seleno-enzyme. Thus, the decrease in brain and pituitary 5'D-II activity in selenium-deficient euthyroid rats is due to the T4-dependent increase in the turnover of the enzyme polypeptide.

Total suppression of cortisol excretion by ketoconazole in the therapy of the ectopic adrenocorticotropic hormone syndrome.

Ketoconazole, an antifungal imidazole derivative, has been shown to inhibit adrenal steroidogenesis in vitro and in vivo. This has led to its use clinically as an effective treatment for various forms of Cushing's syndrome. The clinically effective doses have been reported to be between 800 to 1,200 mg per day, usually without glucocorticoid replacement. Herein is reported the first case of Cushing's syndrome due to ectopic adrenocorticotropic production from a metastatic carcinoid tumor of the thymus that was treated with ketoconazole. Urinary cortisol excretion was totally suppressed at the initial dose and optimal control was achieved with relatively low doses of ketoconazole (200 to 400 mg per day), along with dexamethasone replacement. Use of glucocorticoid replacement is advisable in this setting to avoid symptomatic hypoadrenalism.

Modulation of myocardial L-triiodothyronine receptors in normal, hypothyroid, and hyperthyroid rats.

To characterize the nuclear receptor believed to mediate the thyroid hormones' actions on the heart, binding of L-[125I]T3 to extracts of myocardial cell nuclei from normal, propylthiouracil, and T4-treated rats has been investigated. Analysis of in vitro iodothyronine binding to this solubilized nuclear preparation revealed multiple saturable, specific binding sites for T3. High affinity binding for T3 (Kd = 4.2 +/- 1.0 X 10(-10) mol/L), and lower affinity (Kd approximately 10(-8) mol/L) binding activity appeared to be independent (Hill plot slope = 1). The mean maximal binding capacity of the high affinity binding site for T3 in euthyroid rats (84 +/- 8 fmol/mg DNA) was increased by approximately 50% in hypothyroidism (120 +/- 6 fmol/mg DNA) and unchanged in hyperthyroidism (88 +/- 25 fmol/mg DNA). The molecular weight of this T3 binding site is estimated to be 50,000-55,000 daltons. The properties of this solubilized binding activity from rat myocardial nuclei are consistent with its putative role as the biologic thyroid hormone receptor. The increase in binding capacity with hypothyroidism suggest regulation by thyroid hormone of its nuclear receptor in myocardium.

Thyroid hormone-regulated actin polymerization in brain.

Thyroid hormones play an important role in the growth and development of the brain. Central to the proper integration of neuronal circuitry is the ability of the growing neurite to interpret guidance cues during its migration. The action cytoskeleton is especially rich in the growth cone, and is a likely target for thyroid hormone regulation. This brief review summarizes work showing that thyroxine, but not T3, dynamically regulates the polymerization of the actin cytoskeleton in astrocytes. The ability of T4 to enhance actin polymerization, without directly affecting gene expression, has a profound effect on the ability of the cell to interact with laminin, the major extracellular matrix protein in the developing brain. T4 also regulates the formation of key cell contacts with extracellular matrix guidance cues. These processes are likely to participate in thyroid hormone's regulation of brain development.

Inflammatory thyroid disorders.

Inflammatory disorders of the thyroid, including autoimmune thyroiditis, are among the most common endocrine abnormalities encountered in clinical practice. The association of pain with these disorders, however, is relatively uncommon. Despite this observation, painful thyroid disorders comprise a significant component of the spectrum of thyroid disease. A rational approach to such patients, including history, physical examination, laboratory evaluation, radionuclide or ultrasonographic imaging, and fine needle aspiration biopsy, will allow the appropriate diagnosis to be made in the vast majority of cases.

Identification of a 27-kDa protein with the properties of type II iodothyronine 5'-deiodinase in dibutyryl cyclic AMP-stimulated glial cells.

Type II iodothyronine 5'-deiodinase (5'D-II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3), producing greater than 90% of the bioactive thyroid hormone in the cerebral cortex. In cultured glial cells, expression of this enzyme is cAMP dependent. Exploiting the cAMP-dependent nature of this enzyme in these cells and utilizing N-bromoacetyl-L-3'- or 5'-[125I]thyroxine (BrAc[125I]T4) to affinity label cellular proteins, a 27-kDa protein with the properties of this enzyme was identified. Intact cells labeled with BrAc[125I]T4 showed three prominent radiolabeled bands of proteins of Mr 55,000, 27,000, and 18,000 (p55, p27, p18, respectively) which incorporated approximately 80% of the affinity label. All three affinity-labeled proteins were membrane associated. One protein (p27) increased 5-6-fold after treating the cells for 16 h with dibutyryl cAMP; maximal specific incorporation of affinity label into the stimulated p27 was approximately 2 pmol/mg of cell protein in intact cells. Alterations in the steady-state levels of 5'D-II resulted in parallel changes in the quantity of p27. In cell sonicates, the rate of enzyme inactivation by BrAcT4 equaled the rate of affinity label incorporation into stimulated p27, whereas p55 and p18 showed little or no specific dibutyryl cAMP-stimulated labeling. Enzyme substrates T4 and 3,3'5'-triiodothyronine (rT3) specifically blocked p27 labeling, whereas T3 and the competitive 5'D-II inhibitor EMD 21388 (a synthetic flavonoid) were much less effective. Iopanoate, an inhibitor of all deiodinase isozymes, was ineffective in blocking p27 labeling. Inhibition kinetics revealed that iopanoate was a noncompetitive inhibitor of dibutyryl cAMP-stimulated glial cell 5'D-II, suggesting that it interacts at a site distant from the substrate-binding site. These data identify a cAMP-inducible membrane-associated protein (p27) that has many of the properties of 5'D-II.

Thyroxine targets different pathways of internalization of type II iodothyronine 5'-deiodinase in astrocytes.

In the brain, thyroid hormone dynamically regulates levels of the short-lived plasma membrane protein, type II iodothyronine 5'-deiodinase. In cultured astrocytes, thyroxine modulates deiodinase levels by activating cytoskeletal-plasma membrane interactions that increase the rate of inactivation of the enzyme. Here we characterized the effects of these thyroxine-dependent cytoskeletal interactions upon the route of internalization of the deiodinase by following the intracellular transit of the affinity-labeled substrate-binding subunit of the deiodinase (p29). Thyroxine rapidly induced the inactivation of the deiodinase and initiated the binding of p29 to F-actin. By 40 min, > 75% of the p29 had been transported to an endosomal pool, which was followed by dissociation of the F-actin-p29 complex. There was no significant accumulation of p29 in the dense lysosomes seen in the presence of thyroxine. In the absence of thyroxine, p29 was internalized and transported to the dense lysosomes at a rate parallel to the inactivation rate of the deiodinase (t1/2 0.75 and 0.64 h, respectively) without involvement with the microfilaments. These data demonstrate that thyroxine targets type II iodothyronine 5'-deiodinase to an endosomal pool by activating specific protein-F-actin interactions involved in microfilament-mediated intracellular protein trafficking.

Evidence that type II 5'-deiodinase is not a selenoprotein.

Brain type II 5'-iodothyronine deiodinase and liver type I 5'-iodothyronine deiodinase activities are decreased in rats fed a Se(2+)-deficient diet suggesting that both enzymes are Se(2+)-dependent proteins. Since serum thyroxine (T4) concentrations are twice normal in the Se(2+)-deficient animals, it is unclear whether the Se2+ deficiency or the increased circulating T4 account for the decrease in the brain enzyme. In order to separate these two possibilities, the effects of Se2+ on 5'-deiodinase in glial cells (type II) and LLC-PK1 cells (type I) were examined. LLC-PK1 and glial cells were grown in serum-free defined medium containing 0, 1 pM, 10 nM, and 40 nM Se2+ for 3-5 days or in medium containing 75Se2+ for 24 h. Deiodinase isozymes were determined by measuring catalytic activity and by quantification of the BrAc[125I]T4 affinity-labeled substrate binding subunits. Se2+ deficiency was confirmed by measuring the activity of the selenoprotein, glutathione peroxidase. Se2+ caused a concentration-dependent increase in glutathione peroxidase activity in both cell types, as well as in the type I enzyme, but had no effect on the type II enzyme. LLC-PK1 cells contained multiple 75Se(2+)-labeled proteins including the 27-kDa substrate binding subunit of the type I 5'-deiodinase. Glial cells contained seven 75Se(2+)-labeled proteins ranging in size from 12 to 62 kDa, none of which corresponded to the type II substrate binding subunit. these data show that, unlike the type I enzyme, the type II enzyme does not contain a selenocysteine or selenomethionine, further emphasizing the differences between these two isozymes.

Catalytic activity of type II iodothyronine 5'-deiodinase polypeptide is dependent upon a cyclic AMP activation factor.

Type II iodothyronine 5'-deiodinase is an approximately 200-kDa multimeric enzyme in the brain that catalyzes the deiodination of thyroxine (T4) to its active metabolite, 3,5,3'-triiodothyronine. In astrocytes, cAMP stimulation is required to express catalytically active type II iodothyronine 5'-deiodinase. The affinity ligand N-bromoacetyl-L-T4 specifically labels the 29-kDa substrate-binding subunit (p29) of this enzyme in cAMP-stimulated astrocytes. To determine the requirements for cAMP-induced activation of this enzyme, we optimized N-bromoacetyl-L-T4 labeling of p29 in astrocytes lacking type II iodothyronine 5'-deiodinase activity and examined the effects of cAMP on the hydrodynamic properties and subcellular location of the enzyme. We show that the p29 subunit is expressed in unstimulated astrocytes and requires 10-fold higher concentrations of N-bromoacetyl-L-T4 to achieve incorporation levels equal to those of p29 in cAMP-stimulated cells. Gel filtration showed that p29 was part of a multimeric membrane-associated complex in both cAMP-stimulated and unstimulated astrocytes and that cAMP stimulation led to an increase of approximately 60 kDa in the mass of the holoenzyme. In unstimulated astrocytes, p29 resides in the perinuclear space. Cyclic AMP stimulation leads to the translocation of p29 to the plasma membrane coincident with the appearance of deiodinating activity. These data show that cAMP-dependent activation of type II iodothyronine 5'-deiodinase activity results from the synthesis of additional activating factor(s) that associates with inactive enzyme and leads to the translocation of enzyme polypeptide(s) from the perinuclear space to the plasma membrane.