The primary objective of this project is to determine the mechanism by which TSH regulates thyroid gland function. TSH rapidly stimulates the adenylate cyclase-cyclic AMP system and most, if not all, of its metabolic effects can be attributed to this effect. Several aspects of TSH action will be examined with major emphasis on the following: 1) TSH-responsive adenylate cyclase. Target size analysis will evaluate the functional units of the system. The role of guanine nucleotides will be examined. Monoclonal antibodies to TSH-responsive thyroid plasma membranes will be developed since they stimulate adenylate cyclase but non-competitively inhibit 125I-TSH binding. Effects of these antibodies on TSH stimulated parameters such as glucose oxidation, iodide organification, colloid droplet formation and 32P incorporation into phospholipids will be investigated in thyroid slices. Phosphorylation (cyclic AMP dependent and independent, Ca++ and phospholipid stimulated) and dephosphorylation will be studied. 2) Cytoskeletal and contractile proteins. The function of this system in TSH stimulation of endocytosis and exocytosis will be investigated. We have purified and characterized myosin, actin, profilin, tropomyosin and calmodulin in thyroid tissue. Antibodies to them will be raised for immunofluorescent studies during TSH stimulation of thyroid cells. Effects of Ca++ and calmodulin on this system will be studied. Actin associated proteins and myosin light chain kinase will be characterized. 3) TSH-induced refractoriness. We have described two metabolic blocks in TSH-induced refractoriness. One involving adenylate cyclase is stimulus specific while that on subsequent metabolic effects is not. The necessity for continued presence of the hormone for refractoriness and new protein synthesis for recovery will be studied. Refractoriness induced by prostaglandins, acetylcholine, dibutyryl cyclic AMP and cholera toxin will be examined. The various components of the adenylate cyclase system and the role of Ca++ in desensitization will be studied. Studies will evaluate TSH stimulation of Hashimoto's thyroid tissue and the cytoskeletal and contractile proteins in functioning and non-functioning thyroid nodules.
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