Biomedical subjects

J L Leonard

Publications and source records attributed to J L Leonard.

At least 55 records · Page 3Linked to original sources

PubMed1991

Rapid alteration in circulating free thyroxine modulates pituitary type II 5' deiodinase and basal thyrotropin secretion in the rat.

TSH secretion is decreased by both T4 and T3. This negative feedback control of TSH secretion has been correlated with an increase in pituitary nuclear T3 content, and it is not clear whether T4 exerts its effect directly on the thyrotroph or after its deiodination to T3. However, levels of the pituitary enzyme catalyzing T4 to T3 conversion, 5'D-II, are decreased in the presence of an increased amount of T4. Thus, it is unclear why the thyrotroph would have a mechanism for modulating the production of T3, if T3 is, in fact, the sole bioactive signal providing negative feedback inhibition. To examine this apparent paradox, we administered EMD 21388, a compound which inhibits the binding of T4 to transthyretin resulting in a rapid increase in circulating free T4 levels, to rats pretreated with radiolabeled T4 and T3. We observed increases in pituitary and liver T4 content of greater than 150%, without increases in the respective tissue T3 contents. The EMD 21388-treated rats also exhibited a 25% decrease in pituitary 5'D-II activity (103.8 +/- 15.8 fmol 125I released.mg protein-1.h-1, vs. control, 137.4 +/- 15.9, mean +/- SE), as did rats treated with sodium salicylate, another compound that inhibits T4-TTR binding (100.8 +/- 7.1). TSH levels significantly decreased 2 h after the administration of EMD 21388. These data demonstrate that despite a T4-mediated decrease in pituitary 5'D-II activity, an increase in T4 independently decreases TSH secretion.

Animals↗

PubMed1991

Characterization of a N-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.

Affinity Labels↗

PubMed1990

The Hermaphrodite's Dilemma.

Given sexual conflict, mating encounters between simultaneous hermaphrodites will conform to a new, conditional, non-zero sum game of strategy, the Hermaphrodite's Dilemma; special cases of which include Prisoner's Dilemma and Game of Chicken. The model predicts that hermaphrodite mating systems will be based on reciprocation with cheating in a preferred sexual role. This model suggests that study of hermaphrodite mating systems will provide direct evidence for the existence of sexual conflict and suggests that sexual conflict may act to stabilize hermaphroditism through such mating systems.

Animals↗

PubMed1990

The actin cytoskeleton mediates the hormonally regulated translocation of type II iodothyronine 5'-deiodinase in astrocytes.

Thyroid hormone, specifically thyroxine, alters cytoskeletal organization in astrocytes by modulating actin polymerization and, in turn, regulates the turnover of the short-lived membrane protein, type II iodothyronine 5'-deiodinase. In the absence of thyroxine, approximately 35% of the total cellular actin is depolymerized, and greater than 90% of the deiodinase is found in the plasma membrane and not associated with the cytoskeleton. Addition of thyroxine promotes actin polymerization and decreases the depolymerized actin to approximately 10% of the total actin pool, induces binding of the deiodinase to F-actin, and promotes rapid internalization of the enzyme. These data provide direct evidence that the actin cytoskeleton participates in the inactivation pathway of the deiodinase by translocating this short-lived plasma membrane protein to an internal membrane pool.

Actins↗

PubMed1990

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.

Affinity Labels↗

PubMed1990

Affinity labeling of rat liver and kidney type I 5'-deiodinase. Identification of the 27-kDa substrate binding subunit.

Extrathyroidal production of 3,3',5-triiodothyronine from the thyroid secretory product, thyroxine, is catalyzed by tissue-specific iodothyronine 5'-deiodinases. Type I 5'-deiodinase (5'D-I) produces greater than 75% of the T3 found in the circulation and in thyroid hormone-responsive tissues and is most abundant in rat liver and kidney. In this study, we used the bromoacetyl derivatives of T4 (N-bromoacetyl-[125I]L-thyroxine, BrAcT4) and T3 (N-bromoacetyl-[125I]3,3',5-triiodothyronine, BrAcT3) as alkylating affinity labels to identify 5'D-I-related protein(s). BrAcT4 and BrAcT3 rapidly and irreversibly inactivated 5'D-I activity in liver and kidney microsomes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity labeled 5'D-I preparations showed that approximately 80% of the affinity label was incorporated into a protein with a Mr of 27,000 (p27). 5'D-I substrates and inhibitors specifically blocked affinity labeling of p27 with a rank order of potency (BrAcT4 greater than BrAcT3 greater than 3,5,3'-triiodothyronine (rT3) approximately flavone EMD 21388 greater than iodoacetate greater than N-acetyl-T4 (NAcT4) greater than N-acetyl-T3 (NAcT3] identical to that determined for inhibition of 5'-deiodination. Hyper- and hypothyroidism-induced increases and decreases in 5'D-I activity, respectively, were matched by comparable changes in the quantity of affinity labeled p27. BrAcT3 was a less effective affinity label for p27 and minor labeling of a new band with 53 kDa was observed. Molecular sieve chromatography of detergent-solubilized 5'D-I showed coincident peaks of p27 and 5'-deiodinating activity with an apparent Mr approximately 51,000. Two-dimensional gel electrophoresis showed that p27 was a single polypeptide with a pI of 6.1. Approximately 2-5 pmol of p27 were present per mg of liver microsomal protein, equal to previous estimates for 5'D-I content. Our results suggest that p27 represents the substrate binding subunit of type I 5'-deiodinase, the enzyme catalyzing the key reaction in the activation of T4 to the thyromimetically active T3.

Affinity Labels↗

PubMed1990

Thyroxine-dependent modulation of actin polymerization in cultured astrocytes. A novel, extranuclear action of thyroid hormone.

Actin depolymerization specifically blocks the rapid thyroid hormone-dependent inactivation of type II iodothyronine 5'-deiodinase. Thyroid hormone appears to regulate enzyme inactivation by modulating actin-mediated internalization of this plasma membrane-bound protein. In this study, we examined the interrelationships between thyroxine-dependent enzyme inactivation and the organization of the actin cytoskeleton in cultured astrocytes. Steady-state enzyme levels were inversely related to actin content in dibutyryl cAMP-stimulated astrocytes, and increases in filamentous actin resulted in progressively shorter enzyme half-lives without affecting enzyme synthesis. In the absence of thyroxine, filamentous actin decreased by approximately 40% and soluble actin correspondingly increased; thyroxine normalized filamentous actin levels without changing total cell actin. Thyroxine treatment for only 10 min resulted in an approximately 50% loss of enzyme and increased filamentous actin 2-fold. Neither cycloheximide nor actinomycin D affected the thyroxine-induced actin polymerization. Astrocytes grown without thyroxine also showed a disorganized actin cytoskeleton, and 10 nM thyroxine or 10 nM reverse triiodothyronine normalized the actin cytoskeleton appearance within 20 min; 10 nM 3,3',5-triiodothyronine had no effect. These data show that thyroxine modulates the organization of the actin cytoskeleton in astrocytes and suggest that regulation of actin polymerization may contribute to thyroid hormone's influence on arborization, axonal transport, and cell-cell contact in the developing brain.

Actins↗

PubMed1990

Regulation of type II iodothyronine 5'-deiodinase by thyroid hormone. Inhibition of actin polymerization blocks enzyme inactivation in cAMP-stimulated glial cells.

The cellular events mediating the rapid, thyroid hormone-dependent modulation of membrane-bound, type II iodothyronine 5'-deiodinase were studied in dibutyryl cyclic AMP(bt2cAMP)-treated brain astrocytes. Unstimulated cells had undetectable type II 5'-deiodinating activity. Treating the cells with bt2cAMP and hydrocortisone induced enzyme expression by increasing transcription of the enzyme or an essential enzyme related protein(s), with steady-state levels of type II 5'-deiodinase attained after 8 h of bt2cAMP treatment. Glial cells grown in the absence of thyroid hormone had 10-15-fold higher levels of 5'-deiodinating activity than cells grown in the presence of serum. The increased type II 5'-deiodinating activity observed in serum-free cultures was due to a prolonged enzyme half-life with no change in the rate of enzyme synthesis. Addition of thyroxine or 3,3',5'-triiodothyronine to the serum-free culture medium resulted in a concentration-dependent fall in steady-state enzyme levels, with EC50 values of approximately 0.4 nM. 3,5,3'-Triiodothyronine was at least 100-fold less effective. Chloroquine, NH4Cl, tunicamycin, colchicine, and monodansylcadavarine had no effect on the t1/2 of the enzyme, while both carbonyl cynanide m-chlorophenylhydrazone and cytochalasins completely blocked the inactivation of the type II 5'-deiodinase. These data indicate that in glial cells, an intact actin-cytoskeleton is required for thyroid hormone to modulate the energy-dependent regulation of the half-life of the short-lived, membrane-bound enzyme, type II 5'-deiodinase.

Actins↗

PubMed1990

Effect of biological alterations of type I 5'deiodinase activity on affinity labeled membrane proteins in rat liver and kidney.

Type I iodothyronine 5'deiodinase (5'D-I) is a membrane-bound enzyme catalyzing the deiodination of T4 to T3. The affinity label, N-bromoacetyl-thyroxine (BrAcT4), has previously been used to characterize a 27 kilodalton protein (p27) from rat liver and kidney microsomes with characteristics of the catalytic subunit of the 5'D-I. We examined the effect of physiological conditions, known to alter 5'D-I activity, on affinity-labeled proteins in rat liver and kidney microsomes. To confirm that the affinity labeled protein was associated with the deiodinase, we treated rats with the active site directed enzyme inhibitor, propylthiouracil (PTU), in the absence and presence of 100-fold excess methimazole (MMI), an antithyroid drug which blocks PTU inhibition of 5'D-I in vivo. In addition, we used the affinity label as a probe to measure 5'D-I levels in membrane preparations from short and long term fasted rats. Rats were treated ip with PTU (50 micrograms/100 g BW) or MMI (5 mg/100 g BW); in a second experiment, groups of rats were fasted for 4 days (4 D), 1 day (1 D), or fed ad lib (C) and hepatic and kidney microsomes were prepared. 5'D-I activity and 5'D-I content, as judged by specific incorporation of the affinity label into p27, were determined. PTU decreased both 5'D-I activity and BrACT incorporation into p27 by 60-65%. Coadministration of MMI attenuated the effect of PTU on 5'D-I activity and p27 affinity labeling. No other affinity labeled proteins were affected. In fasting experiments, the changes in affinity labeling of p27 paralleled the changes in 5'D-I activity. 5'D-I activity was significantly decreased in hepatic microsomes obtained from 4 D-fasted rats as compared to C rats, but was unchanged in hepatic microsomes from 1 D-fasted rats or in kidney microsomes from 1 D or 4 D-fasted rats as compared to C. Maximal BrAcT4 incorporation into p27 decreased by 45% in hepatic microsomes from 4 D-starved rats as compared to C (6.7 +/- 0.9 vs. 11.9 +/- 1.5 pmol BrAcT4 incorporated/mg microsomal protein, respectively). There was no change in p27 content in hepatic microsomes from 1 D-starved rats (11.2 +/- 1.1). Starvation failed to alter the BrAcT4 labeling of kidney microsomes (16.7 +/- 4.4, 16.2 +/- 6.6, and 14.8 +/- 3.2 pmol BrAcT4 in 4 D, 1 D, and C rats, respectively). In this study, we have demonstrated that alterations in biological activity of 5'D-I correspond to alterations in affinity labeling of p27.

Affinity Labels↗

PubMed1990

Effect of hypophysectomy on growth hormone receptor gene expression in rat tissues.

Two cDNA probes derived from the nucleic acid sequence for the rabbit GH receptor were used to study RNA samples from normal and hypophysectomized (hypox) rat tissues by Northern analysis. Results obtained with a probe that contained a nucleotide sequence corresponding to part of the extracellular domain of the GH receptor indicated that rat liver, gastrocnemius muscle, and epididymal fat each contain a 4.4-kilobase (kb) message and one or more shorter messages that appear to be homologous to the rabbit GH receptor message. The other probe, which contained a nucleotide sequence that corresponds to the intracellular domain of the GH receptor, detected only one 4.4-kb message in these rat tissues. These results suggest that rat tissues may synthesize several forms of the GH receptor, but only one form that contains a region homologous to the intracellular domain of the rabbit liver GH receptor. Hypophysectomy increased the abundance of the 4.4-kb message 5-fold in muscle and reduced it by a factor of 2 in adipose tissue. No significant difference was seen between GH receptor message levels of normal and hypox rat liver when the results were expressed as a fraction of the total RNA. The level of the beta-actin message was also measured in liver, muscle, and fat from normal and hypox rats. No significant differences were found when the message levels in normal rats were compared to those for the corresponding tissue in hypox rats. When normalized to the beta-actin message levels, a significant increase was seen in the relative amount of the GH receptor mRNA in muscle and liver of hypox rats. The increased levels of the GH receptor message in muscle and liver and the simultaneous decreased level in fat suggest that GH receptor synthesis may be regulated selectively in these tissues by hormonal factors that are altered by hypophysectomy.

Adipose Tissue↗

PubMed1989

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.

Affinity Labels↗

PubMed1989

Reexamination of the gill withdrawal reflex of Aplysia californica Cooper (Gastropoda; Opisthobranchia).

The gill withdrawal reflex (GWR), an important model system for neural mechanisms of learning, varies in form and amplitude within as well as between preparations and is therefore a heterogeneous collection of action patterns, not a reflex. At least 4 action patterns occur in response to mechanical stimulation of the siphon. It is often impossible to categorize a particular movement unambiguously. All may occur spontaneously. Gill movements may be described as combinations of 10 actions; 4 involving vein movements are described here. All actions and action patterns can occur in preparations lacking the central nervous system. Some vein movements may generate considerable force without markedly altering gill area. It is suggested that this explains why some early studies failed to identify the important role of the peripheral nervous system in the GWR. Studies based on the assumption that the GWR involves a single type of movement controlled by cells of the parietovisceral ganglion require reevaluation.

Animals↗

PubMed1988

Dibutyryl cAMP induction of type II 5'deiodinase activity in rat brain astrocytes in culture.

Dibutyryl cAMP treatment of cultured rat astrocytes results in the rapid appearance of T4 to T3 conversion catalyzed by type II iodothyronine 5'deiodinase, without altering other deiodinating pathways. Induction of enzyme activity was time-dependent with a lag period of 60 min, reaching plateau levels after 6-8 hours, and required continued synthesis of mRNA and new protein. Isoproterenol also induced T4 to T3 converting activity through beta-adrenergic receptor mediated interactions. These data suggest that dibutyryl cAMP stimulated astrocytes provide an excellent model for the study of the molecular and cellular events modulating T4 to T3 conversion in the brain.

Adrenergic beta-Agonists↗

PubMed1986

[Epidemiologic study of human listeriosis in France, 1984].

To estimate the incidence rate of listeriosis in France during one year, a census of all the isolates of Listeria monocytogenes was made in 1984 by the microbiologists of 78% of the French hospitals. 630 cases of listeriosis were registered giving a yearly incidence rate of 11.3 cases for 1 million inhabitants. 353 cases (56%) were perinatal listeriosis (positive cultures for L. monocytogenes from the mother, the child or the placenta). Compared with earlier data, listeriosis seems to be more frequent in France now than ten years ago. A higher incidence rate in and around the Ile de France region and a seasonal increase in October were observed in 1984.

Female↗

PubMed1985

Thyroid hormone metabolism in primary cultures of fetal rat brain cells.

The metabolism of thyroxine 3,5,3',5'-tetraiodothyronine, (T4) and 3,5,3'-triiodothyronine (T3) was studied in primary cultures of dispersed fetal rat brain cells. Cultured brain cells actively metabolized both T4 and T3 by enzyme catalyzed deiodination reactions which increase (type II 5'-deiodinase) or decrease (type I 5'-deiodinase and type III 5-deiodinase) the bioactivity of thyroid hormone. Homogenates of cultured brain cells showed both type I and type II 5'-deiodinating activities and these two enzymes tended to differ in their time course of appearance. Cultures exposed to 10 microM cytosine arabinoside for 16 h showed up to a 70% reduction in type I activity without decreasing the type II enzyme suggesting that the type II enzyme is associated with non-dividing neuronal cells. The predominant pathway for T4 and T3 metabolism in situ was tyrosyl-ring or type III 5'-deiodination which followed first order kinetics with a t1/2 of 70 min. T4 to T3 conversion by the type II enzyme was consistently observed after correcting for the degradation of newly formed T3 by the type III enzyme. In situ, both type II and type II enzymes were thiol-dependent and both activities were inhibited by iopanoic acid. Type III 5-deiodination of T4 produced 34 fmol 3,3,5'-triiodothyronine (rT3)/h per 10(6) cells at 10 mM dithiothreitol (DTT) and 97 fmol of rT3/h per 10(6) cells at 50 mM DTT. T3 production by the type II enzyme was 1.2 and 4.4 fmol of T3/h per 10(6) cells at 10 and 50 mM DTT, respectively. Thyroid hormone deficient culture conditions increased type II enzyme activity by 4-5-fold within 48 h and this was prevented in a dose-dependent fashion by supplementing the media with increasing amounts of T3. These data indicate that primary cultures of dispersed brain cells mimic the intact cerebral cortex with respect to the metabolism of thyroid hormone and the regulatory mechanisms which defend cerebrocortical T3 levels. The vigorous metabolism of both T4 and T3 by these cultures may explain some of the difficulties in demonstrating thyroid hormone-dependent biochemical changes at physiologically relevant levels of thyroid hormone.

Animals↗

PubMed1985

Regulation of rat cerebrocortical and adenohypophyseal type II 5'-deiodinase by thyroxine, triiodothyronine, and reverse triiodothyronine.

To further understand the regulation of type II iodothyronine 5'-deiodinase (5'D-II) in the central nervous system and pituitary, we examined the response of this enzyme to the acute administration of T4, T3, and rT3 in hypothyroid rats. Enzyme levels were correlated with serum concentrations of T4 and T3 in thyroidectomized rats after acute administration of either iodothyronine and in animals with hypothyroidism of increasing severity induced by methimazole administration. Estimates of the tissue concentrations of the three iodothyronines, nuclear T3, and serum TSH levels were used to assess mechanisms and intrinsic potencies of the three iodothyronines. In four experiments, doses of T4 that reduced 5'D-II activity by 50% (ID50) ranged from 0.18-0.39 micrograms/100 g BW in the cortex and from 0.34-1.05 in the pituitary, whereas the corresponding ID50 values of rT3 were 1.0 and 3.5, and those of T3 were 4.0 and 5.0 micrograms/100 g BW. T3 doses that saturated nuclear receptors and fully suppressed TSH showed only modest suppression of 5'D-II levels in the cortex and pituitary. Based on estimates of the tissue hormone levels resulting in 5'D-II suppression, T4 and rT3 were much more potent than T3 in decreasing 5'D-II. These findings support the concept that the effect of these iodothyronines on 5'D-II is not mediated by the nuclear T3 receptor. The correlation of serum T4 and T3 with enzyme levels after acute injections of T4 or after chronic treatment with methimazole suggested that plasma T4 is probably the main physiological signal regulating 5'D-II. It is conceivable that rT3 produced locally from T4 also plays a role in the regulation of the enzyme.

Animals↗

PubMed1985

Alterations in the pituitary-thyroid axis and 5'-deiodinase activity in mice with muscular dystrophy.

Mice with hereditary muscular dystrophy have reduced levels of serum T3. To determine possible causes of T3 deficits, we evaluated pituitary thyrotroph ultrastructure by electron microscopy, thyroid gland morphology by light microscopy, and T4 to T3 conversion by measuring iodothyronine 5'-deiodinase activity. Differences were not evident between dystrophic and normal littermates in either the structure of pituitary thyrotrophs or thyroid tissues. Dystrophic mice, however, had only 50% the normal hepatic 5'-deiodinase activity. Cerebral 5'-deiodinase, which does not appear to contribute significantly to serum T3, was similar in normal and dystrophic mice. Submandibular gland concentrations of nerve growth factor and epidermal growth factor are reduced in dystrophic mice but can be increased by T4 treatment. To distinguish whether growth factor deficits are due to reductions in serum T3 or to deficiencies in T4 5'-deiodinase activity and subsequent T3 utilization within the salivary gland, we measured submandibular deiodinase activity. Gland homogenates were active in the deiodinase assay, but no differences were detected between normal and dystrophic mice. In order to evaluate tissue responses to reductions in circulating T4, we treated mice with methimazole. Structural analyses revealed that thyrotrophs in dystrophic mice were less stimulated than thyrotrophs in similarly treated normal littermates. Likewise, thyroid follicular cells appeared less active, and thyroid weights increased only 40-50% as much as in normals. Liver 5'-deiodinase activity decreased in both normal and dystrophic mice. Cerebral 5'-deiodinase activity increased more than 4-fold in normal females but only 2-fold in dystrophic females; 2- to 3-fold increases occurred in both normal and dystrophic males. In summary, the structure of pituitary and thyroid glands in dystrophic mice is similar to that of tissues from normal littermates, but hepatic conversion of T4 to T3 is reduced. When challenged by methimazole-induced reductions in serum T4, pituitary and cerebral tissues in dystrophic mice respond abnormally.

Animals↗

PubMed1985

Studies of nuclear 3,5,3'-triiodothyronine binding in primary cultures of rat brain.

Primary cultures of enzymatically dispersed cells from 17-day-old fetal rat cerebral hemispheres were used to detect the presence of nuclear T3 receptors. Cells grown in Minimum Essential Medium supplemented with 10% fetal bovine serum were grown in parallel with cytosine-arabinoside (ARA-C)-treated counterparts which had been exposed to the antimetabolite for 18 h on culture days 3 and 5 or 4 and 6. Five days after the second ARA-C treatment, phase contrast photomicrographs showed substantial loss of the proliferating basal cells, corresponding to an 85% decrease in cell number. Immunocytochemical studies using antiglial fibrillary acidic protein (anti-GFAP) and antineurofilament (anti-NF) antisera demonstrated loss of GFAP-positive cells (astrocytes) and preservation of NF-positive cells (neurons), the latter considered to be a nondividing population under the culture conditions. Nuclei obtained from the brain cell cultures at this time by Triton washing bound T3 with properties similar to those observed in vivo. Scatchard analysis showed a single, high affinity, limited capacity nuclear T3 receptor with a maximal binding capacity (MBC) of 0.53 +/- 0.10 ng T3/mg DNA and a Kd of 0.19 +/- 0.02 nM (+/- SE; n = 4). ARA-C treatment resulted in a mean decrease in DNA per culture dish of 54%, with an accompanying 2-fold enrichment of the MBC, and no change in the Kd. In untreated cultures grown for 20 days, DNA per dish increased until day 14 and subsequently remained stable at approximately 100 micrograms/dish. The MBC also increased from days 0 to 7, and remained stable until day 14. On day 20, the MBC had declined by approximately 60% to 0.21 ng T3/mg DNA, at which time the neuron population was decreased. The extracted nuclear receptor from brain cell cultures had a sedimentation coefficient of 3.6S. The relative binding affinities of the nuclear receptor for T3 and several analogs were identical to those found in vivo, making significant contamination of the nuclei with cytosolic or serum binding proteins very unlikely. Nuclei isolated from long term, neuron-free glial cell cultures failed to show any consistent high affinity saturable T3 binding.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗