Theony Valcana

Nuclear triiodothyronine receptors in the developing rat brain

This study examines whether the high sensitivity of the developing brain to thyroid hormones and the purported decline in sensitivity in adulthood, are correlated with changes in the density and affinity characteristics of specific nuclear T3 receptors. The authors have found that the nuclei of cerebral hemispheres have a high density of T3 receptors at birth (212 +/-28 X 10(-17) mol/microgram DNA) which declines to adult levels by the end of the second postnatal week (115 +/- 7 X 10(-17) mol/microgram DNA), remaining at this level until 6 months of age. Even though no significant changes were detected in the equilibrium dissociation constant (Kd) during the early period of development, comparison neonatal with the adult brain reveals a decrease in Kd (neonatal, 3.9 X 10(-10) M; adult, 2.3 X 10(-10) M). In the developing animal, neonatal thyroidectomy increased the number of binding sites in the nucleus by 36--44%. It is concluded that the high number of nuclear T3 receptors in the first week of postnatal life is correlated with the high dependence of brain tissue on thyroid hormones and that the decline in brain sensitivity may be associated with the decline in nuclear T3 receptors. The high affinity and density of nuclear receptors in adult brain tissue relative to the developing brain and liver, respectively, point to a continued regulatory role of thyroid hormones in brain.

Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA+RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T3-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T4 intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)

Characterization of thyroid hormone transport in synaptosomes from rat brain

In this study we examined the mode of triiodothyronine (T3) and tetraiodothyronine (T4) transport in synaptosomal preparations from cerebral hemispheres of adult rat brain. Our results show that these hormones are transported by different mechanisms: T3 uptake is a saturable process and Hofstee analysis of the data reveals two transport components--a high affinity (Kt approximately 50 pM), low density and a low affinity (Kt approximately 3.1 nM) high density system. The Vmax of both components is influenced by the extracellular/intracellular Na+ gradient. T3 uptake decreases in the presence of ouabain and gramicidin. T3 uptake also shows a temperature dependence and decreases in the presence of KCN. In contrast, T4 uptake is a nonsaturable process and is not influenced by metabolic inhibitors or Na ions. It is proposed that T3 entry into neurons is a carrier-mediated process and depends on Na ions. In contrast, T4 is transported by diffusion which is driven by high extracellular/intracellular differences in T4 concentration, maintained by the high rate of cellular deiodination of T4 to T3, characteristic of this tissue.

Thyroxine deiodination, cytoplasmic distribution and nuclear binding of thyroxine and trnodothyronine in liver and brain of young and aged rats

This study examines (a) the effects of aging on plasma thyroid hormone concentration and (b) in vivo binding and cytoplasmic distribution of thyroid hormones as well as the conversion of thyroxine (T4) to triiodothyronine (T3) in liver and cerebral hemispheric tissue. The results show that (a) in male Long-Evans rats aging decreases plasma T4 concentration but does not affect plasma T3 concentration and (b) the in vivo nuclear T3 binding does not change significantly. However, nuclear T3 binding derived from T4 is decreased as a consequence of reduced T4 to T3 conversion in both tissues. The nuclear T4 binding is also depressed, perhaps due to the decrease in the T4 of the protein free cytoplasmic compartment. Aging was also found to change protein free and protein bound cytoplasmic distribution of T4. That is, an increase was observed in protein bound cytoplasmic T4 and a decrease in the protein free cytoplasmic T4 of both tissues. These results indicate an overall alteration in thyroid hormone production and peripheral tissue binding and processing of thyroid hormones with a consequent suboptimal thyroid state with aging.

Pentylenetetrazole-induced convulsions affect cellular and molecular parameters of the mechanism of action of triiodothyronine in adult rat brain

The aim of the current study was to elucidate whether the response of the adult rat brain to thyroid hormones is affected by the intensity of neuronal activity. For this purpose, the kinetic characteristics of nuclear T3 binding, the relative expression of thyroid hormone receptor (TR) isoforms and the synaptosomal content of thyroid hormones in adult rat brain were examined after administration of a single convulsion dose of pentylenetetrazole (PTZ). Experiments in adult Wistar rats revealed an increase (33%) of the density of specific T3 nuclear receptors in cerebral hemispheres 4h after PTZ-induced seizures while no changes were observed in the dissociation constant. The relative expression of the T3-binding isoforms of TRs was not affected, while there was a gradual decrease of the relative expression of the TR alpha2 variant (non-T3 binding isoform). The above changes were coupled with an increase of the synaptosomal T3 levels during the epileptic seizures. Our study revealed inversely proportional changes between the nuclear T3 binding sites and the TR alpha2 mRNA levels 4 h after PTZ-induced seizures, suggesting that the regulation of the expression of the non-T3 binding variant of TRs determines the nuclear T3 binding sites in adult rat brain, while the synaptosomal T3 levels could play a novel functional role in the signaling from the synapse to the nucleus.

[3H]GABA binding in the cerebellum of the reeler murine mutant

In the cerebellum of the reeler mutant mouse, characterized morphologically by depletion of the granule cell population and abnormal synapse formation, increased GABA concentration and alterations in [(3)H]GABA binding have been observed. This study shows decreased affinity of the Na(+)-independent, high affinity GABA binding component of synaptosomal membranes and an increased affinity of the Na(+)-dependent, high affinity GABA binding component in reeler cerebellar homogenate and synaptic membranes. In contrast to the changes in affinity, the number of both Na(+)-dependent and Na(+)-independent binding sites was not significantly altered. The decreased affinity of the Na(+)-independent GABA binding and the increased affinity of the Na(+)-dependent binding, evidenced only in cerebellar tissue, were interpreted to indicate, respectively, hypo- and hypersensitivity of the postsynaptic and presynaptic elements of cerebellar GABAergic synapses, induced by the depressed excitatory granule cell input and/or the increased mossy fiber contact with the ectopic Purkinje cells.

Characterization of nuclear triiodothyronine (T3) and tetraiodothyronine (T4) binding in developing brain tissue

The main objective of this study was to characterize nuclear T3 and T4 binding in the developing rat brain. More specifically, we sought to determine (a) whether T3 and T4 bind to the same nuclear receptor, (b) whether there are multiple forms of nuclear T3 or T4 receptors, and (c) whether the above parameters are similar in nuclei of cerebral hemispheres and cerebellum of developing rat brain. From in vivo and in vitro binding experiments utilizing gel filtration techniques, we have shown that T3 binds to a main macromolecular fraction of molecular weight (M.W.) approx. 60 000 daltons; however, a minor binding component of M.W. greater than 100 000 daltons was also observed. Utilizing the same techniques it was shown that T4 does not bind with the main T3 binding macromolecule but only with the minor (M.W. greater than 100 000) binding component. Inasmuch as T4 competes with T3 for its binding, we have hypothesized that (a) the stability of the T4-receptor complex requires special stereochemical receptor-chromatin relationships that hold for in vivo or de novo conditions but not in the salt-extracted (0.4 M KCl) nuclear receptor preparation, or (b) T4 interacts with more than one receptor unit and forms unstable T4-receptor complexes corresponding to the high M.W. macromolecular fraction. The T3 and T4 binding characteristics described above were common to both brain regions at both developmental ages examined; however, these tissues were found to differ in quantitative aspects of T3 and T4 binding and with respect to the rate of the in vivo T4 to T3 conversion. We suggest that the nuclear T4 does not contribute to the end biological effects but, rather, it determines the number of free T3 binding sites. The end biological responses may thus be proportional to the binding of T3--derived from plasma and the local cellular conversion of T4 to T3--with its major nuclear binding protein and inversely proportional to the T4 nuclear concentration.

Diazepam affects the nuclear thyroid hormone receptor density and their expression levels in adult rat brain.

Thyroid hormones (THs) are involved in the occurrence of anxiety and affective disorders; however, the effects following an anxiolytic benzodiazepine treatment, such as diazepam administration, on the mechanism of action of thyroid hormones has not yet been investigated. The effect of diazepam on the in vitro nuclear T3 binding, on the relative expression of the TH receptors (TRs) and on the synaptosomal TH availability were examined in adult rat cerebral hemispheres 24 h after a single intraperitoneal dose (5 mg/kg BW) of this tranquillizer. Although, diazepam did not affect the availability of TH either in blood circulation or in the synaptosomal fraction, it decreased (33%) the nuclear T3 maximal binding density (B(max)). No differences were observed in the equilibrium dissociation constant (K(d)). The TRalpha2 variant (non-T3-binding) mRNA levels were increased by 33%, whereas no changes in the relative expression of the T3-binding isoforms of TRs (TRalpha1, TRbeta1) were observed. This study shows that a single intraperitoneal injection of diazepam affects within 24 h, the density of the nuclear TRs and their expression pattern. The latest effect occurs in an isoform-specific manner involving specifically the TRalpha2 mRNA levels in adult rat brain.

Effects of hypothyroidism on RNA synthesis in the adult rat brain

In this study we investigated the effects of hypothyroidism on adult brain RNA synthesis. Our data show that in the cerebral hemispheres of hypothyroid rats there is a decrease in microsomal RNA content and microsomal [3H]uridine incorporation. Sucrose gradient analysis revealed that these changes are mainly associated with free ribosomes and subunits and reflect changes in rRNA. The above changes are accompanied by a decrease in RNA polymerase I activity. All of the above mentioned changes returned to normal after thyroxine (T4) treatment. In contrast to RNA polymerase I, RNA polymerase II activity was not affected. However, electrophoretic analysis of the in vitro poly(A)+RNA translation products revealed that hypothyroidism affects a few mRNAs. These results indicate that thyroid hormones have a role in adult brain tissue metabolism.

Acute LiCl-treatment affects the cytoplasmic T4 availability and the expression pattern of thyroid hormone receptors in adult rat cerebral hemispheres.

We have previously reported that short-term LiCl-treatment affects the kinetic characteristics of thyroid hormone binding in adult rat brain (Bolaris, S., Margarity, M., Valcana, T., 1995. Effects of LiCl on triiodothyronine (T3) binding to nuclei from rat cerebral hemispheres. Biol. Psychiatry 37, 106-111); however, the mechanism underlying the above effects of LiCl administration is yet to be determined. In this study, the effects of lithium within one day after its administration (5 mmol/kg BW) on the relative expression of thyroid hormone receptor isoforms and on the cytoplasmic and synaptosomal thyroid hormone availability in adult rat cerebral hemispheres were examined. Although short-term LiCl-treatment did not affect the levels of triiodothyronine either in the synaptosomal or in the cytoplasmic fraction 24 h after LiCl administration, the cytoplasmic availability of thyroxin was lower. In addition, 24 h after the administration of lithium the mRNA levels of the TRalpha1 isoform (T3 binding) increased while the relative expression of the TRalpha2 variant (non-T3 binding) was decreased. Notably, the decrease of the TRalpha2 mRNA levels was also observed 4h after LiCl administration. The expression levels of the TRbeta1 isoform were unaffected in any interval examined. The present study suggests that short-term lithium treatment regulates the relative expression of TRs in an isoform-specific manner and affects the cytoplasmic availability of thyroxin in adult rat brain.