Nuclear T3 Receptors Research Articles

Distribution of the nuclear thyroid-hormone receptor in extraocular and skeletal muscles.

The correlation between the occurrence of Graves' ophthalmopathy and Graves' hyperthyroidism may indicate a role for tri-iodothyronine (T3) hormone in the pathogenesis of Graves' ophthalmopathy. In Graves' ophthalmopathy the recti eye muscles are greatly enlarged whereas skeletal muscles seem unaffected. The distribution of the nuclear T3 receptor was studied in normal human and rat eye and skeletal muscles with immunohistochemistry using mouse (monoclonal) antibodies, and by in-situ hybridization for the detection of mRNA encoding the T3-receptor protein. Nuclear staining with T3-receptor antibodies was found in all types of tissues studied. Cytoplasmic staining occurred predominantly in the muscle fibres of the orbital layer of the eye muscles and was generally absent or very low in skeletal muscle fibres and hepatocytes. Immunostaining could be inhibited by preabsorbing the antibodies with bacterially expressed T3-receptor protein, implying specificity. The presence of nuclear and cytoplasmic hormone-free T3 receptor sites was indicated after preincubation of sections with T3 hormone; T3-receptor immunostaining decreased and T3-hormone staining increased. In-situ hybridization clearly revealed the presence of alpha-1 and beta-1 forms of the T3-receptor mRNA in liver, skeletal muscles, and orbital and intermediate layers of the eye muscles. The data demonstrate the presence of T3 hormone-receptor molecules in the extraocular and skeletal muscles. The different susceptibilities of these muscles to Graves' hyperthyroidism may relate to the quantitative differences in T3 hormone-receptor distribution.

Retinoic acid decreases nuclear triiodothyronine receptor expression and impairs an early step of adipose differentiation in the thyroid hormone-sensitive mouse Ob 17 preadipocyte cell line.

In the murine preadipocyte cell line Ob 17, T3 is known to be necessary at an early step of adipose differentiation for the expression of late phenotypes [lipogenic enzymes such as malic enzyme, glycerol-3-phosphate dehydrogenase (GPDH), etc.] and not necessary for the expression of lipoprotein lipase (LPL), which emerges earlier, at growth arrest. These cells contain nuclear T3 receptors, which mainly belong to products of the c-erbA alpha gene and are down-regulated by T3. In this work, retinoic acid (RA) added to Ob 17 cells at growth arrest impaired morphological differentiation and the development of both late (malic enzyme and GPDH) and early (LPL) phenotypes regardless of whether T3 was added. T3 sensitized the cells to the inhibitory action of RA; the ED50 for GPDH activity was shifted from 0.5 microM to 3 nM in cells cultured with 1.5 nM T3. Later addition of RA (6 days after growth arrest) did not inhibit the differentiation. RA also brought out a marked and fast decrease in nuclear T3 receptors. This was observed whatever the stage of cell development and related to both a rapid decrease in the relative abundance of c-erbA alpha-related mRNA species and an increased disappearance rate, suggesting the involvement of pre- and posttranslational events. RA and T3 acted additively in decreasing the T3 receptor and c-erbA alpha mRNA levels. The effects of RA on T3 receptors were rapidly reversed after RA withdrawal; the reversal was large (75%) when RA was introduced at growth arrest and total when introduced later. The cell sensitivity to RA, considering the T3 receptors, was higher at growth arrest (ED50 for RA, 0.2 and 1.5 microM in assays with RA added at growth arrest and 5 days later, respectively). The results suggest intricated regulatory pathways between RA and T3 at an early step of adipose differentiation and also suggest that among different mechanisms through which RA may impair this differentiation, a decreased level of nuclear T3 receptors at an early period should play a role.

Regulation of glucokinase and proinsulin gene expression and insulin secretion in RIN-m5F cells by dexamethasone, retinoic acid, and thyroid hormone.

In RIN-5mF cells, treatment with either dexamethasone or retinoic acid resulted in increased glucokinase activity. Treatment with dexamethasone (50 and 500 nM) increased glucokinase activity 140% and 260%, respectively, whereas treatment with retinoic acid (100 and 1000 nM) increased glucokinase activity by 50% and 80%, respectively. An additive effect was observed when 1000 nM retinoic acid was added to either 50 or 500 nM dexamethasone. Treatment with either of these factors resulted in increased glucokinase mRNA levels. In contrast, treatment with thyroid hormone (T3; 0.2 and 10 nM) had no effect on glucokinase activity, but decreased glucokinase mRNA levels. T3 did not affect the response of either activity or mRNA levels to retinoic acid. Binding experiments revealed the presence of a single class of T3 nuclear receptors, with a Kd of 0.46 x 10(-10) and a binding capacity of 3.84 pmol/mg protein in these cells. This suggests that the changes produced by T3 may be due to a physiological effect mediated via nuclear receptors. Proinsulin mRNA levels were increased by retinoic acid (10-100 nM) and dexamethasone (5-500 nM), but decreased by T3 (0.04-10 nM). Insulin secretion in the absence or presence of glucose and potassium was increased about 50% in cells previously treated with 1000 nM retinoic acid. T3 (0.2 nM) did not affect insulin secretion in the absence or presence of glucose or in the presence of secretagogues (potassium and glyceraldehyde). Dexamethasone (50 nM) did not significantly increase insulin secretion in the absence or presence of glucose or potassium. In contrast, dexamethasone decreased glyceraldehyde-induced insulin secretion by 60% (P less than 0.05). We conclude that the glucokinase and proinsulin genes respond in parallel to dexamethasone and retinoic acid (both increased) and to T3 (both decreased).

Benzodiazepines inhibit temperature-dependent L-[125I]triiodothyronine accumulation into human liver, human neuroblast, and rat pituitary cell lines.

L-T3 (T3) accumulates into cells in a temperature-dependent saturable manner through a purported iodothyronine membrane carrier protein. We report energy-dependent uptake of picomolar [125I]T3 into differentiated cell lines derived from human liver, human neuroblast, and rat pituitary malignancies. Furthermore, this cellular uptake is inhibited by classical and nonclassical benzodiazepine-type drugs (BZs); the apparent half-maximal inhibitory concentrations range from 50 nM to 50 microM, varying with drug and cell type. The site of this T3-BZ interaction was explored with cross-competitive radioligand binding to rat liver cell fractions. No interaction was seen in experiments cross-competing unlabeled T3 (10(-9)-10(-5) M) against [3H]Ro5 4864, a peripheral BZ receptor ligand, for binding sites in a crude rat liver mitochondrial fraction. As well, lormetazepam and triazolam, BZs that potently inhibit cellular uptake of [125I]T3, have no effect on [125I]T3 binding to rat liver nuclear sites. Studies of [3H]diazepam and [3H]Ro5 4864 show very little temperature-dependent uptake into HepG2 cells (less than 0.5% over 90 min) and no effect from coincubation of unlabeled T3 (1 microM). Thus, the possibility that BZs are substrates for the T3 carrier protein and are causing the reduced cellular hormonal accumulation via competitive uptake and dilution of the radiolabeled cellular T3 is unlikely. In summary, 1) drugs from the BZ class inhibit high affinity temperature-dependent cellular accumulation of thyroid hormone into cell lines from rat and human species; 2) the site of action of BZ inhibition does not involve direct antagonism of the T3 nuclear receptor, nor is it likely that the peripheral BZ receptor is the iodothyronine carrier. BZs could be interacting with the purported iodothyronine carrier protein itself to block uptake.

Ontogeny of the liver nuclear T3-receptor during the last days of incubation and posthatch in the chick embryo.

The characteristics of the nuclear T3 receptor in the liver of the chick embryo were studied from incubation day 18 until day 1 posthatching. Treatment of the nuclei with 3 mol.l-1 MgCl2, which removed the endogenously bound hormone, was used in order to determine the total amount of receptors. The affinity constant Ka decreased between incubation day 18 (0.996 +/- 0.276.10(9) M-1) and day 19 (0.247 +/- 0.072.10(9) M-1), remained the same thereafter until hatching and increased again on day 1 posthatching (1.846 +/- 0.928.10(9) M-1). The total amount of receptors tended to increase from incubation day 18 to day 20 non-pipping (np) (from 4.40 to 11.55 fmol/micrograms DNA) and decreased thereafter to 2.38 fmol/micrograms DNA on day 1 posthatching. The amount of free binding sites reached a maximum on day 19 (6.91 fmol/micrograms DNA) and then decreased drastically until posthatching (0.19 fmol/micrograms DNA). The maximal specific binding was found on day 20 (np), just prior to penetration of the air chamber. During the time at which the level of T3 remains high in the plasma, a reduction in the amount of receptor was observed, which may be the consequence of a down-regulation by T3 itself.

Pituitary resistance to thyroid hormones.

Pituitary thyroid hormone resistance (PRTH) refers to a particular form of thyroid hormone refractoriness that is accompanied by peripheral hyperthyroidism, as only the TSH-secreting pituitary cells appear to be resistant to the effects of thyroid hormones. The presence of PRTH is suspected and diagnosed on the basis of the finding of high free thyroid hormone levels along with unsuppressed TSH, clinical signs and symptoms of hyperthyroidism and values of at least one of the parameters evaluating peripheral thyroid hormone action in the hyperthyroid range. However, most patients with PRTH present with clinical signs and symptoms of thyroid dysfunction, particularly goiter and tachycardia, overlapping those recorded in patients with generalized thyroid hormone resistance (GRTH), i.e. refractoriness to thyroid hormones at both pituitary and peripheral tissue level. Moreover, most of them display normal values of other parameters evaluating the peripheral effects of thyroid hormones and bear mutations in the gene encoding for T3 nuclear receptors similar to those found in patients with GRTH. These findings are questioning the existence of PRTH as a separate clinical entity and support the view that the various forms of thyroid hormone resistance may be part of a spectrum of disease with variable expression in different issues.

Prolonged Fasting Reduces Rat Hepaticβ1 Thyroid Hormone Receptor Protein without Changing the Level of Its Messenger Ribonucleic Acid*

The level of hepatic nuclear T3-binding capacity falls in rats subjected to fasting. To define the mechanism underlying these changes, we have assayed in liver the concentration of the mRNA coding for the beta 1-receptor (beta 1-TR) isoform, the total nuclear T3-binding capacity, and the fraction of the total binding capacity that can be specifically immunoprecipitated with an anti-beta 1-TR immunoglobulin G preparation. Although no changes in beta 1-TR mRNA concentration were noted, we observed a 60% fall in total binding capacity. beta 1-TR mRNA levels were preserved despite a 50% fall in total poly(A)+ RNA. The fall in beta 1-TR protein, however, was consistent with a generalized decrease in total hepatic protein content. This study provides yet another instance in which measurement of receptor mRNA is not consonant with the behavior of the nuclear T3 receptor protein.

Characterization of seven novel mutations of the c-erbA beta gene in unrelated kindreds with generalized thyroid hormone resistance. Evidence for two "hot spot" regions of the ligand binding domain.

Genetic analysis in our laboratory of families with generalized thyroid hormone resistance (GTHR) has demonstrated tight linkage with a locus, c-erbA beta, encoding a nuclear T3 receptor. Three point mutations and two deletions in this locus have previously been reported in affected individuals in unrelated families as potential molecular bases for this disorder. In the present study, we have used direct sequencing of polymerase chain reaction-amplified exons of the c-erbA beta gene to rapidly identify novel point mutations from seven previously uncharacterized kindreds with GTHR. Six single base substitutions and one single base insertion were identified and found to be clustered in two regions of exons 9 and 10 in the ligand binding domain of the receptor: in the distal ligand-binding subdomain L2 and across the juncture of the taui and dimerization subdomains. Reduction of T3-binding affinity in each of four mutations tested as well as segregation of all mutations to clinically affected individuals strongly supports the hypothesis that these changes are the cause of GTHR in these kindreds. In view of the diversity of clinical phenotypes manifested, the distinct topographic clustering of the mutations provides an invaluable genetic tool for the molecular dissection of thyroid receptor function.

Characterization of aN-Bromoacetyl-L-Thyroxine Affinity-Labeled 55-Kilodalton Protein as Protein Disulfide Isomerase in Cultured Glial Cells

In glial cells, thyroid hormone regulates the polymerization state of the actin cytoskeleton by a mechanism that does not require protein synthesis or the nuclear T3 receptor. Using the affinity label N-bromoacetyl-L-T4, we identified a thyroid hormone-binding protein of 55 kDa (glial-p55) in cultured glial cells that is unique from the type II iodothyronine 5'-deiodinase and which exhibits a T4-dependent shift from a membrane-associated pool to the F-actin cytoskeleton. In a number of other cell types, a 55-kDa thyroid hormone-binding protein has been identified and shown to be a subunit of the enzyme protein disulfide isomerase (PDI). In this study we have characterized glial-p55 and compared it with purified rat hepatic PDI. Glial-p55 appears to be identical to PDI by peptide fragmentation analysis, using multiple methods. The hydrodynamic properties of glial-p55, determined by molecular sieve chromatography and sucrose density centrifugation, showed that this protein has a native molecular mass of 115 kDa, which is in close agreement with that reported for PDI. Like PDI, glial-p55 is an acidic protein with a pI of 4.8 or less. Thus, the 55-kDa thyroid hormone-binding protein in glial cells appears to be PDI.

C-erb-A mRNA content and triiodothyronine nuclear receptor binding capacity in rat liver according to vitamin A status

Vitamin A is required for normal growth and development and synergistically acts with thyroid hormones in these processes. Effects of retinol dietary intake (0, 5, 50 U retinol/g food) on the triiodothyronine (T3) nuclear receptor were examined in rat liver. Properties of this receptor have been investigated by an in vitro binding method. The amount of c-erb-A mRNA has been deduced by reverse transcription and amplification methods (PCR). Results show that both retinol deficiency and excess leads to a reduced amount of c-erb-A mRNA and to a decreased T3 binding capacity, suggesting that retinol, or retinoic acid which is its natural metabolite, contributes to the regulation of the T3 nuclear receptor properties.

Thyroid hormones transcriptionally regulate the beta 1-adrenergic receptor gene in cultured ventricular myocytes

Exposure of ventricular myocytes in primary culture to triiodothyronine (T3) increased the number of beta 1-adrenergic receptors per cell by 2.1 +/- 0.3-fold (n = 7) within 48 h. Immunoblots of membranes prepared from myocytes revealed a marked increase by T3 in the 64-kDa species of the beta 1-adrenergic receptor. Steady-state levels of beta 1- and beta 2-adrenergic receptor mRNAs quantified by DNA-excess solution hybridization were 0.26 +/- 0.06 and 0.81 +/- 0.05 amol of beta-adrenergic receptor mRNA/micrograms of total cellular RNA, respectively (n = 4). beta 1-Adrenergic receptor mRNA increased to 0.90 +/- 0.07 amol/micrograms RNA by 2-4 h after exposure to T3 and then declined by 6 h to twice that of control cells. beta 2-Adrenergic receptor mRNA levels were unaffected by T3. Northern blot hybridization also showed a rapid and sustained increase of 2.2 +/- 0.4-fold (n = 4) in beta 1-adrenergic receptor mRNA. The rate of beta 1-adrenergic receptor gene transcription assessed by nuclear run-on transcription assays increased by 3.4 +/- 0.4-fold in cells treated for 30 min with T3. Ventricular cells contained nuclear T3 receptors and mRNAs of c-erbA genes that encode T3 binding proteins. These studies indicate that thyroid hormones regulate the cardiac beta-adrenergic receptor-adenylate cyclase system by controlling the rate of transcription of the beta 1-adrenergic receptor gene. This regulation involves nuclear T3 receptors and appears to be exerted in a tissue-specific manner.

Demonstration of nuclear 3,5,3'-triiodothyronine receptor proteins in gonadotrophs and corticotrophs in rat anterior pituitary by double immunohistochemical staining.

Previously we raised an antiserum (4BII) against nuclear T3 receptor (NT3R), which recognizes c-erb A/NT3R alpha 1 and beta, but not alpha 2. An immunohistochemical study using 4BII revealed that among the tissues examined, the cerebral cortex, anterior pituitary, and thyroid gland exhibited strong immunostaining in the nuclei. In the present study we examined the localization of NT3R proteins in individual cell types of rat anterior pituitary, using the technique of double immunostaining with 4BII and antibodies against pituitary hormones. The cryostat sections and paraffin sections of rat pituitary were incubated with 4BII at 4 C overnight. The NT3R proteins were visualized as brown in the nuclei by avidin-biotin peroxidase staining. The control sections incubated with an antiserum which had previously absorbed with c-erb A peptide or an inactive antiserum showed very poor nuclear staining under the same condition. After being washed with 0.1 M glycine-HCl buffer (pH 2.2) for 2 h to remove 4BII, the sections were incubated with antiserum against individual anterior pituitary hormones at 4 C overnight. Using the method of alkali phosphatase/naphthol/Fast blue staining, the peptide hormones in the cytosol were stained blue. Double staining with 4BII and anti-FSH beta or anti-LH beta anti-serum clearly demonstrated that gonadotrophs contained the NT3R proteins. Similarly, the NT3R proteins in corticotrophs were demonstrated by the immunostaining with 4BII and anti-ACTH antiserum. As expected, the NT3R proteins were present in somatotrophs, thyrotrophs, and lactotrophs. On the other hand, folliculo-stellate cells, which are nonhormone secreting cells and are identified by their S-100 protein immunoreactivity, were stained by 4BII only faintly. The present study demonstrated the existence of NT3R proteins not only in somatotrophs, thyrotrophs and lactotrophs but also gonadotrophs and corticotrophs, suggesting some action of thyroid hormone through the receptor in these cells.

Central Role of Brown Adipose Tissue Thyroxine 5′-Deiodinase on Thyroid Hormone-Dependent Thermogenic Response to Cold*

As judged by the response of uncoupling protein and key enzymes, brown adipose tissue (BAT) is highly dependent upon the local generation of T3 catalyzed by the tissue type II T4 5'-deiodinase (5'-D-II). In hypothyroid rats treated with T3 or T4, the capacity to withstand cold seems better correlated with the normalization of BAT responses than with the liver thyroid status. 5'D-II is activated by cold via sympathetic nervous system (SNS) stimulation, and the activation generates enough T3 to nearly saturate BAT nuclear T3 receptor (NTR) in euthyroid rats. In hypothyroidism, 5'D-II is highly stimulated by the SNS and hypothyroxinemia. In the present studies we have taken advantage of this situation to test 1) the capacity of 5'D-II to maintain nuclear T3 in rats with various degrees of hypothyroxinemia, and 2) the hypothesis that thyroid hormone-dependent BAT-facultative thermogenesis, rather than the effect of thyroid hormone on obligatory thermogenesis (basal metabolic rate), is the basic mechanism by which thyroid hormone confers protection against acute cold exposure. We treated methimazole-blocked rats (undetectable plasma T4 and T3) for a week with either subreplacement doses of T4 (0.5, 1, 2, and 4 micrograms/kg.day) or replacement doses of T4 or T3 (8 or 3 micrograms/kg.day, respectively). Sources and content of BAT nuclear T3 were studied at 25 C and after 48 h at 4 C by labeling the plasmaborne T3 (T3[T3]) with [131I]T3 and the locally generated T3 (T3[T4]) with [125I]T4. Neither the kinetics of nuclear-plasma exchange of T3[T3], the time of appearance of T3[T4] in BAT nuclei, nor NTR maximal binding capacity (0.71 ng T3/mg DNA) was affected by hypothyroidism. Kinetic analyses indicated a maximal BAT NTR occupancy of 40% at euthyroid serum T3 concentrations if T4 is not present. Replacement with T4 normalized both serum T4 and T3, while replacement with T3 normalized serum T3; for all other doses of T4, serum T4 and T3 concentrations were predictably related to the dose. 5'D-II activity decreased with increasing doses of T4, but for each dose of T4, this activity was 2-4 times greater at 4 C than at 25 C. BAT NTR occupancy normalized with 2 micrograms T4/kg in rats maintained at 25 C and with 4 micrograms T4/kg in cold-exposed rats, although in neither condition were serum T4 and T3 normalized nor more than 30% of NTR occupied by plasma T3.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of nuclear T3 receptors in human neuroblastoma cells SH-SY5Y: Effect of differentiation with sodium butyrate and nerve growth factor

Nuclear receptors for the thyroid hormone triiodothyronine (T3) have been identified in vivo in brain tissues and in vitro in mouse and rat neuroblastoma and glioma cells. The present study characterizes nuclear T3 receptors in human neuroblastoma SH-SY5Y cells and compares their level before and after differentiation. Undifferentiated cells, grown in DME/HAM F-12 medium supplemented with 10% fetal calf serum, show an abundant single type of nuclear receptor, indicated by a straight Scatchard plot, with a Kd of 0.11 nmol/l. After treatment with sodium butyrate (0.5 mM for 4 days) or NGF (2 nM for 6 days), the cells showed neuronal-like patterns (extension of neurites, slowing of growth, increased tyrosine hydroxylase activity), with a decrease in the number of nuclear T3 receptors. As sodium butyrate and NGF treatments differentiate neuroblastoma SH-SY5Y cells, these data suggest a down-regulation of T3 receptors with cell maturation.

Effect of retinoids on protein kinase C activity and on the binding characteristics of the tri-iodothyronine nuclear receptor

Retinoids and thyroid hormones exert profound effects on the development, growth and homeostasis of vertebrates. The receptor proteins which bind retinoic acid, tri-iodothyronine (T3) or steroid hormones and, as a result of this binding, interact with DNA to stimulate expression of specific genes, belong to the same recently discovered superfamily. The functionality of thyroid and steroid hormone receptors is thought to be related to a phosphorylation-dephosphorylation cycle. In the present work, the action of two retinoids (retinol and retinoic acid) was studied on the properties of T3-nuclear receptors and on protein kinase C (PKC) activity in the rat liver (PKC is known to be a phosphorylating enzyme for various proteins). The influence of 12-O-tetradecanoyl phorbol-13-acetate (TPA; known to enhance PKC activity) on the properties of T3-nuclear receptors was also investigated. Measurements of binding characteristics and enzyme activity were performed 4 or 12 h after a single i.p. injection of retinol or retinoic acid (6 mg/kg body weight) or 1 h after a single i.p. injection of TPA (0.7 mg/kg). The activity of PKC was increased 4 h after administration of the retinoids, and the affinity of the T3-nuclear receptor protein was increased markedly after 12 h. The activity of PKC and the affinity of nuclear T3 receptor were both increased 1 h after administration of TPA. These observations provide indirect evidence that retinoids, particularly retinoic acid, induce an increase in PKC activity and a subsequent increase in the affinity of the T3-nuclear receptor protein.

Increased nuclear tri-iodothyronine binding and thyroid hormone-stimulated glucose consumption in mononuclear blood cells from patients with liver cirrhosis

Nuclear tri-iodothyronine (T3) maximal binding capacity (MBC) and thyroxine- and T3-stimulated cellular oxygen consumption and glucose consumption were examined in mononuclear blood cells from six patients with liver cirrhosis (LC), in six patients with alcoholic hepatitis (AH), and in six healthy control subjects. Serum T3 was decreased in patients with LC. The MBC of T3 was increased significantly (P less than 0.05) in cells from patients with LC compared with patients with AH and controls, whereas the equilibrium association constants did not differ. Unstimulated glucose consumption was slightly increased (P less than 0.05) in cells from patients with AH and LC compared with controls. Thyroid hormone-stimulated glucose consumption was significantly (P less than 0.05) increased in cells from patients with LC compared with controls and patients with AH. Unstimulated oxygen consumption did not differ between the groups, but thyroid hormone-stimulated oxygen consumption was depressed in cells from patients with AC (P less than 0.05) compared with patients with LC and with controls. We conclude that both thyroid hormone-stimulated glucose consumption and T3 nuclear receptor binding in cells from patients with LC are increased, and suggest that the observed changes are responsible for maintenance of euthyroidism in the face of reduced circulating T3.

Effects of triiodothyronine (T3) on differentiation of rat cortical neurons in primary cultures

Some of the events which characterize neuronal terminal differentiation have been studied in rat cortical neurons cultured in a selective synthetic medium for a period which corresponds to terminal brain maturation in vivo. In particular, we have studied the effect of T3 on the synthesis of nuclear proteins and the expression of the mRNAs which encode different variants of T3 nuclear receptors (c erb A proteins). We have shown that: a) T3 stimulates the turnover of nuclear proteins, with a more evident effect on the non-histone component; b) for the whole lifespan of cultures the predominant form of c erb Aα mRNA is the α2 variant (which encodes a protein unable to bind T3); whatever the function of α2 protein this finding suggests that its predominance on α1 is settled very early during mammalian brain maturation.

Regulation of Rat Pancreatic Nuclear Triiodothyronine Receptor by Glucocorticoid

The nuclear T3 receptors in rat pancreas exhibit a characteristic maturation pattern during development and are subjected to autologous regulation by thyroid hormones. To see if glucocorticoids also regulate T3 receptors in the pancreas, rats at various age groups were subjected to experimental conditions that altered their glucocorticoid status and the corresponding changes in nuclear T3 receptors, and exocrine enzymes in their pancreata were evaluated. Hydrocortisone administration to normal suckling and weaning rats did not change total T3 binding capacity or the dissociation constant as measured at 30 degrees C (Kd30). A significant increase in the degree of occupancy of the T3 receptor was found at 5-10 days after treatment with hydrocortisone (42.2 + 4.1% vs. 19.2 + 2.0%). T3 binding capacity and exocrine enzyme concentrations were significantly reduced in both adrenalectomized (Adx) pups and adults, indicating a continuous dependency on glucocorticoid from preweaning to adulthood. In adrenalectomized rat pups, either T4 or glucocorticoid replacement alone restored T3 binding capacity and exocrine enzyme concentrations. T4 and glucocorticoid were given together to Adx rats, the level of stimulation of both pancreatic T3 binding capacity and exocrine enzyme concentrations was found to be equal to the sum of the stimulation by each of these hormones when given alone. Furthermore, a good correlation was found between Bmax30 (Bmax measured at 30 degrees C, representing total sites) for T3 binding and exocrine enzyme activities in different groups following various experimental treatments. These findings provide further evidence that thyroxine can act directly on the rat pancreas presumably through the T3 receptor in regulating the postnatal development of the exocrine enzymes.

Thyroidal involvement in the expression of avian muscular dystrophy

We showed previously that propylthiouracil (PTU), a thyroid inhibitor, could alleviate several major signs of hereditary muscular dystrophy in chickens. The goals of the present investigation were to: (1) determine whether a nearly athyroid condition (achieved within two days after hatching by surgical thyroidectomy plus PTU) during an 11-day period beneficially affects the dystrophic condition when followed by triiodothyronine (T3) replacement to 33 days of age; (2) determine the beneficial effects on the expression of avian dystrophy when the thyroidectomized-PTU-treated chickens received a wide range of moderate to low T3 replacement doses beginning by two days after thyroidectomy; and (3) examine the thyroid hormone receptor system in dystrophic muscle for a possible abnormality. Thyroid deprivation increased muscle function (righting ability) and reduced plasma creatine kinase activity in dystrophic chickens. The major thyroid-related abnormality in dystrophic pectoralis muscles was an increased maximum binding capacity of solubilized nuclear T3 receptors.

Evidence for the Presence of Protein Kinases Which Stimulate Phosphorylation of c-erb A Protein in Rat Kidney Nuclei.

Protein kinases were separated from rat kidney nuclear extract by hydroxylapatite column chromatography. Five (I-V) different protein kinases were isolated when histone was used as a substrate. Two (I and III) of them stimulated phosphorylation of c-erb A-beta protein (50 kDa) expressed in Escherichia coli. The c-erb A product has an activity of high affinity T3 binding. One (I) of the kinases was dependent on cyclic adenosine 3',5'-monophosphate (cyclic AMP). The other kinase (III) was not dependent on cyclic nucleotides. The latter kinase was eluted from hydroxylapatite column with 0.05 M PO4 at pH 7.4. The sedimentation coefficient(s) estimated by continuous sucrose density gradient centrifugation was approximately 6.0 Km values for ATP were estimated by double reciprocal analyses, which gave 110.0 microM in the protein kinase I (in the presence of 10(-6) M cyclic AMP) and 25 microM in the protein kinase III, respectively. The data showed that 1.0 mol phosphate was incorporated into 80 mol of c-erb A protein (50 kDa) either in the presence of protein kinase I (with 10(-6) M cyclic AMP) or in the presence of protein kinase III. These results suggested that there are protein kinases for c-erb A protein, whose functional properties are similar to those of nuclear T3 receptor, in rat kidney nuclei.