The objective of this project is to determine the mechanism by which TSH regulates thyroid gland function. TSH rapidly stimulates the adenylate cyclase-cAMP system and most, but not all, of its metabolic effects can be attributed to this action. Hydrolysis of phosphatidylinositol 4,5 bisphosphate (PIP2) with formation of inositol trisphosphate (IP3) and diacylglycerol is another intracellular signalling system in many tissues. Several aspects of TSH action will be examined with major emphasis on the following: 1) Effects of TSH and other agonists on polyphosphatidylinositol turnover and the formation of inositol phosphates (IPs) as an alternative signalling system in thyroid tissue. Dog thyroid slices, dog thyroid cells grown in tissue culture and FRTL-5 cells prelabelled with 3H-inositol will be stimulated with TSH, norepinephrine, carbamylcholine and phorbol esters to measure formation of IPs. Incubation conditions will be used to facilitate the labelling of the TSH responsive pool of PIP2. The phosphatidylinositol phosphodiesterase (phospholipase C) will be characterized using a membrane preparation prepared from tissues pre-labelled with 3H-inositol. 2) Cytoskeletal system and contractile proteins. In thyroid cells pre-labelled with 32P, TSH rapidly caused a reduction of 32p in two polypeptides having many characteristics of myosin light chains (MLC). Antibodies to myosin and MLC and 2-D SDS-PAGE will be used to further identify the polypeptides as myosin light chains. The amino acids which are dephosphorylated will be characterized to determine whether they are the same for TSH and other agonists. Studies will examine the role of Ca++ and calmodulin and whether the decreased phosphorylation reflect decreased activity of kinases or increased activity of phosphatases. 3) TSH-induced refractoriness. We have preliminary data that incubation of thyroid slices with TSH causes a decrease in pertussis toxin ADP-ribosylation of its substrate in a membrane preparation. The possibility that this reflects increased endogenous ADP-ribosylation mediated by TSH prior to addition of pertussis toxin or an alteration in the substrate will be studied. The effects of other agonists which induce refractoriness and the relationship of this phenomenon to TSH- induced desensitization will be investigated. 4) Comparison of metabolic responses to TSH and other agonists in thyroid slices, isolated follicles and cells grown in tissue culture, including FRTL-5 cells.