The long term objective of this proposal is to study the mechanisms which regulate the peripheral tissue uptake and metabolism of thyroxine (T4), the main secretory product of the thyroid gland, and 3,5,3'- triiodothyronine, (T3), the most potent thyroid hormone as well as other iodothyronine metabolites. These studies will examine the role of renal production and tissue compartmentation of T3 and T4 with the circulation. Three general phenomena relating to thyroid hormone uptake will be examined in the proposed studies 1) vascular, 2) cell membrane 3) cellular or cytosolic. The perfused kidney system, established in this laboratory, provides the opportunity to study thyroid effect of other tissues. Using single-pass and recirculating kidney perfusion, the mechanisms regulating cellular thyroid hormone influx, and efflux will be explored. Free thyroid hormone fractions in cytosol and perfusate medium will be determined by equilibrium dialysis. The mechanisms underlying the extensive single-pass renal uptake of thyroid hormone will be sought and related to steady-state tissue thyroid hormone concentrations and metabolic rates. With this information, the dynamic uptake process will be mathematically modeled to aid the distinction between uptake by simple diffusion of free hormone and facilitated or active transport. Modeling of the translocation process is considerably simplified in this system as parameters such as hormone carrier protein type and concentration, ligand concentration, tissue transit time, and the significance of excretory and metabolic pathways can be controlled. These techniques will be employed to distinguish alterations in renal flux and steady-state distribution of iodothyronines in the presence of drugs competing with thyroid hormone binding to circulating or cellular proteins. Light microscopic autoradiography will be performed to identify the tissue and cellular loci of renal thyroid hormone exchange. A study will probe for differences in tissue/perfusate compartmentation of T3 generated by local 5'-deiodination of T4 compared to T3 derived from the circulation. THe effects of in vitro 5 '-deiodinase inhibitors, drugs competing with cellular hormone binding, and altered thyroid status will be examined. Nondeiodinative metabolic pathways such as conjugation and deconjugation will be examined by analysis of metabolites by high performance liquid chromatography. Using purified proximal tubular cells, the metabolic requirements of renal thyroid hormone uptake will be studies and this system will be used to address the hypothesis that thyroid hormone uptake and steady-state distribution may be acutely regulated by factors such as intracellular calcium, pH and redox potential.
