LATS Young Investigator Awards


Paula Bargi de Souza (Área Básica)

Winner 2011


ACUTE T3-TREATMENT ALTERS THE TSH DISTRIBUTION IN THYROTROPHS: A NEW NEGATIVE FEEDBACK LOOP BY NON-GENOMIC ACTION? BARGI-SOUZA, P1; ROMANO, RM1; SALGADO, RM2; SILVA, FG1; BRUNETO, EL1; NUNES, MT1 1Department of Physiology and Biophysics; 2Department of Cell and Developmental Biology, USP, Brazil. Thyroid Hormone Action

INTRODUCTION
The thyroid-stimulating hormone or thyrotropin (TSH) produced by thyrotrophs that represents 5% of the cells in adenohypophysis exerts a central role in the hypothalamus-hypophysis-thyroid (HHT) axis. This glycoprotein is the main regulator of the synthesis and secretion of thyroid hormones (TH) which in turn exert a negative feedback mechanism in the hypothalamus and in pituitary by reducing the synthesis of beta and alpha chains through genomic actions, and as a consequence, the TSH secretion. This classical mechanism depends of the TH interaction with thyroid hormone receptor (TR) and the binding in thyroid response elements (TREs) present in the gene targets, such as beta and alpha TSH subunits and thyrotropin releasing hormone (TRH) and is more evident hours or days after treatment. In the last decades have seen an increasing body of evidence has shown that some TH actions might be elicited in a short period of time (seconds to minutes) and in the presence of gene transcription inhibitors, which indicates that TH can also act non genomically. Recent data published by our laboratory demonstrated that the triiodothyronine (T3) acutely reduces the poli-A tail length of beta TSH subunit mRNA by posttranscriptional mechanisms and impairs the association between this transcripts and ribosome reducing the translational rate (1). These results suggest that T3 could act non genomically on the control of TSH gene expression. Taking this data into consideration and that the TSH secretion is the first step by which TSH serum concentration could be controlled, our hypothesis is that the T3 can alters the TSH secretion and distribution in thyrotrophs of rats T3-treated acutely.

OBJECTIVE
This study aims to evaluate if TH could acutely regulate the TSH distribution in thyrotrophs which might provide evidence that they could act at this step by non genomic mechanisms.

METHODS
For this study, initially, male Wistar rats weighing 200-250g were housed in a room kept at constant temperature (23 * 2 C) and on a 12 h-light/12h-dark (lights on at 0700 h) schedule. Part of the animals were made hypothyroid by surgical thyroidectomy, after being deeply anaesthetized with ketamine and xylazine, and received 0.03% methylmercaptoimidazole (MMI), plus 4.5 mM calcium chloride in drinking water during 20 days. Sham-operated animals were used as control. The animals were then divided into the following groups: 1) euthyroid sham-operated (SO); 2) SO plus supraphysiological doses of T3 (100 μg/100 g BW) (SO+T3100); 3) hypothyroid (Tx); 4) Tx plus T3 in supraphysiological doses, as specified above (Tx+T3100); 5) Tx plus T3 in physiological doses (0.3 μg/100 g BW) (Tx+T30.3) (2). Euthyroid (SO) and hypothyroid (Tx) animals received iv administration of NaCl 0.9% as vehicle. All the animals were killed 30 min after the administration of T3 or vehicle. The pituitaries were rapidly excised and used for the analysis of TSH expression was evaluated by Western blotting; the TSH distribution was analyzed by means of immunofluorescence and electron immunocytochemistry
At least seven animals per group were used, and the experiments were repeated at least three times. The experimental protocol was in agreement with the ethical principles in animal research adopted by the Brazilian College of Animal Experimentation and was approved by the Institute of Biomedical Sciences/University of São Paulo-Ethical Committee for Animal Research.
The results were obtained from at least three experiments and subjected to normality (Kolmogorov-Smirnov) and homocedasticity test (Bartlet), followed by analysis of variance (one way - ANOVA), and Student-Newman-Keuls post test, using the GraphPad Prism 5 Software. The data were expressed as means * SEM and differences were considered significant at P < 0.05.

RESULTS
It was observed a 63% decrease of the TSH protein content in pituitary of hypothyroid rats compared with SO and SO+T3100. The acute T3-treatment, in both doses, promoted a 30% increase in the TSH protein content. These results suggest a possible blocks in the TSH secretion.
To analyze this possibility was performed the immunofluorescence assay. It was observed that pituitaries from Tx rats presented a more evident TSH staining at the cell surface, whereas in the SO group, it was observed whole cytoplasm. The acute treatment of SO rats with T3 (SO+T3 group) apparently did not alter the immunolabeling pattern of the TSH protein, which remained in the cytoplasm, but when the acute treatment was observed in hypothyroid rats, with both dose of T3, there is a weaker staining at the cell surface, compared to Tx. This alteration seemed to be more remarkable when T3 was administered in physiological doses, condition in which one can also observe an increase in the staining for TSH in the whole cytoplasm.
The precise location of the granules containing TSH was detected by electron microscopy Immunogold labeling, which showed a marked increase in TSH content in Tx rats’ thyrotrophs; the vesicles containing TSH were also shown to be closer to the plasma membrane, when compared with SO rats, as observed by immunofluorescence, and it is noteworthy the presence of high amount of TSH labeling in blood vessels. In the SO group, the TSH labeling was more pronounced in vesicles scattered in the cytoplasm. The acute T3 treatment increased the TSH labeling in vesicles dispersed in the whole cytoplasm in the SO+T3100, Tx+T3100 and Tx+T30.3 groups and reduced the staining in blood vessels.

CONCLUSION
The results presented herein demonstrated for the first time that the triiodothyronine acutely blockade TSH secretion, as shown by the alterations of the distribution of TSH granules in thyrotrophs 30 min after their administration, and point to a non-genomic action of TH also at this level.

KEYWORDS
Triiodothyronine - Thyrotropin - Non genomic actions

REFERENCES
Goulart-Silva F, Souza PB, Nunes MT 2011 T3 rapidly modulates TSHβ mRNA stability and translational rate in the pituitary of hypothyroid rats. Molecular and Cellular Endocrinology 332 1-2: 277-282
Dillmann WH, Berry S, Alexander NM 1983. A physiological dose of triiodothyronine normalizes cardiac myosin adenosine triphosphatase activity and changes myosin isoenzyme distribution in semistarved rats. Endocrinology 112: 2081-2087