The purpose of this proposal is to study the biochemical events mediating intracellular thyroid metabolism, specifically thyroxine (T4) to 3,5,3'triiodothyronine (T3) conversion in both the kidneys and the brain. It is widely recognized that extrathyroidal T4 to T3 conversion is the major source of circulating bioactive hormone, and as such is an essential first step in the mechanism of thyroid hormone action in mammals. Recent studies have shown that target tissues differ in their handling of the T3 produced intracellularly, and this is due, in part, to the presence of two isozymes of the enzyme catalyzing 5'deiodination, Iodothyronine 5'deiodinase (5'D). The kidneys and liver """"""""export"""""""" most of the newly formed T3 and contain the most abundant form of the enzyme (type I), while the brain retains almost all of the T3 produced within its cells and contains the other isozyme (type II). Isolation of the two isozymes of 5'D will make use of cultures of both renal and glial cells to explore the inter-relationships of the isozymes and to characterize the molecular events that regulate tissue-specific T4 to T3 conversion. Identification of the 5'D-I will employ photoaffinity labeling using derivatives of inhibitors and substrates. Anti-enzyme antibody will be identified in an existing panel of monoclonals directed against a model of the substrate binding site. Cultured renal epithelial cells will serve as the source of 35S-labeled 5'D-I for the characterization of anti-enzyme antisera and the electrophoretic properties of the enzyme. Kidney cells will also be used to determine the distribution of the 5'D along the rat nephron. Characterization of the molecular events that regulate the rapid modulation of brain 5'D-II will be done in cultures of rat glial cells. Identification of the 5'D-II will utilize the ability of cyclic nucleotides and glucocorticoid to modulate the expression of the enzyme molecule and pulse-chase amino acid labeling techniques. Regulatory pathways, specifically the cellular events mediating the rapid inactivation of 5'D-II, will be explored. Molecular approaches aimed at isolating the mRNA encoding 5'D-II will use a glial cell cDNA library and the ability to modulate enzyme expression. Comparisons of the 5'D isozymes at the protein and mRNA levels will clarify the inter-relationships between these tissue-specific enzymes and provide a basis for understanding the mechanisms by which mammalian tissues modulate their responsiveness to thyroid hormone.
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