The goals of this proposal are to determine: (1) the role of anterior pituitary (AP) follicular cells in PRL regulation and (2) the role of the cytoskeleton in mediating distal intracellular events involved in the actions of TRH and dopamine (DA). We have recently developed a culture technique for bovine follicular cells of the AP. These cells in culture form a contact inhibited polarized monolayer which exhibits all the characteristics of a transport epithelium. Cultures will be used to determine its follicular cells of the AP plan a function in the regulation of PRL secretion. The two hypotheses to be tested are that: (1) follicular cells regulate the ionic and biochemical mileu of secretory cells, and (2) they play a role as paracrine mediators of PRL secretion. The first hypothesis will be tested by measuring ion and glucose flux across cell monolayers grown on filters and mounted in a Ussing chamber. The receptor mediating beta adrenergic induced changes in ion flux will be characterized by ligand binding as well as by its coupling to adenylate cyclase. Regulation of ion transport by secretogogues, e.g. DA and TRH, affecting PRL secretion will also be tested. The polarization of follicular cells relative to secretory and vascular elements within the AP will be studied by immunocytochemical localization of Na+/K+ ATPase. The release and regulation of paracrine mediators of PRL from cultured bovine follicular cells will be studied. Two candidates for these mediators are cAMP and S-100 protein. Both substances will be radioimmunoassayed in cells and conditioned media. In perifused AP cells kept under constant DA inhibition the effects of TRH stimulation, removal of DA inhibition or both on cytoskeletal organization will be determined biochemically and morphologically. Organization of microtubules, microfilaments and associated proteins will be visualized by double immunofluorescence staining techniques. Changes in organization will be correlated with changes in PRL secretion. The role of increasing cytosolic ca++, protein kinase c and cAMP in mediating the actions of TRH and DA will be assessed pharmacologically. The possibility will be tested that the actions of TRH and DA on the cytoskeleton are mediated by phosphorylation. Regulated phosorylated proteins will be separated by two dimensional gel electrophoresis and stained by the immunoblot technique. Cytoskeletal proteins to be studied include alpha particle and beta tubulin, actin, non-muscle myosin, fodrin, vimentin, viniculin, alpha actinin, tau and MAPs.
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