MECHANISMS OF THYROID HORMONE ACTION IN DEVELOPMENT

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The long-term goal of this research program is to elucidate the mechanisms of action of thyroid hormones (TH) at the molecular level through a study of the relationships between hormone metabolism, hormone-receptor interactions and hormone action in developing amphibian species: Rana catesbeiana and Ambystoma mexicanum. For a variety of reasons, including the facts that TH receptors are present in their nuclei and they acquire the ability to convert thyroxine (T4) to 3,5,3'triiodothyronine (T3) in vivo just prior to metamorphosis, these animals offer a unique opportunity to investigate these relationships. Studies 1-5 are all concerned with with enzyme systems responsible for producing T3 from T4 (I5'Ds) in liver and their regulation and significance in TH action. The systems will be characterized in vitro by determining their kinetic properties, and the effects of development, TH, fasting and animal maintenance temperatures will be studied. Activity will be related to T3 and T4 levels in plasma and liver cytosol, and hepatic receptor occupancy. Liver will also be examined for T4 5-monodeiodinase activity. Study 6 will determine whether receptor number in tail tissue is increased, as it is in erythrocytes, during spontaneous and TH-induced metamorphosis. Study 7 is concerned with the reason why tetrac, which has a relatively low affinity for the receptor, is comparable in potency in LT3 in tadpoles. Study 8 will investigate the mechanisms by which prolactin inhibits, and adrenal corticoids augment some actions of TH in tadpoles. Possible mechanisms include effects on TH metabolism and distribution, and receptor number and affinity. Studies 9-12 are concerned with the actions of T3 on transcription and pretranslational events, and the relevance of the putative receptors in TH action. The levels of translatable mRNA for carbamyl phosphate synthetase (CPS) present in liver during spontaneous and induced metamorphosis will be determined and correlated with CPS activity, T3 and T4 levels in plasma and liver cytosol, and receptor occupancy. A cDNA probe for CPSmRNA will be used to determine if T3 increases the amount of CPSmRNA. The relative potencies of T3 analogues in stimulating CPSmRNA activity will be compared with their relative affinities for the receptor. Lastly, the effects of metamorphosis and TH on the entire mRNA activity profile, as determined by 2-dimensional separation on polyacrylamide gels of in vitro translated products of hepatic poly(A+)RNA, will be determined.