TRANSPORT AND ACTIONS OF THYROID HORMONES

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The long range objective of this research is a detailed understanding of the mechanism of action of thyroid hormones. The specific goals of the proposal are to define the mechanism of thyroid hormone uptake into pituitary cells and to evaluate the possibility that thyroid hormones exert some of their effects in pituitary cells by an extra-nuclear action. Three types of preliminary data suggest that thyroid hormones are transported into pituitary tumor cells, GH-cells, by a carrier mediated transport system. First, uptake of L-triiodothyronine (T3) into GH-cells can be inhibited by dansylcadaverine cytochalasin b and chloroquine but not by closely related drugs like cytochalasins d and e and didansylcadaverine. Second, thyroid hormone uptake is markedly temperature dependent in a manner that is inconsistent with a simple diffusional mechanism. Third, kinetic analysis of T3 transport reveals a saturable uptake mechanism. It is proposed to study thyroid hormone transport in detail, analyzing the kinetics, substrate specificity and energy dependence of transport into intact cells and isolated membrane vesicles. The hypothesis that thyroid hormones enter cells by receptor mediated endocytosis will be tested by comparing the behavior of rhodamine-labeled and native T3. The thyroid hormone transporter(s) will be isolated by reconstitution into liposomes and photoaffinity labeling. In GH-cells thyroid hormones induce growth hormone and regulate the concentration of receptors for thyrotropin-releasing hormone and the dose-response curves for these actions differ by factors of 10 to 100 for various thyroid hormone analogs. To test the possibility that regulation of TRH receptors results from a membrane effect of thyroid hormones, T3 responses will be measured in membrane vesicles and enucleated GH-cells. Finally, in an attempt to assess the physiological significance of thyroid hormone transporter(s) and possible membrane sites of action of the hormones, mutants of GH-cells unable to transport thyroid hormones will be selected using T3-ricin A chain and characterized in detail. These studies should add to our understanding of the mechanism of action of an important regulatory hormone.