Increases in cytoplasmic Ca2+ are known to trigger secretory exocytosis in a wide variety of endocrine, exocrine, neural and other cells. This proposal is directed at elucidating the biochemical mechanisms through which elevations in cytoplasmic Ca2+ stimulate secretion. We will employ GH3 pituitary cells in which regulated secretion of prolactin (PRL) has been well- studied. Previous work has implicated a Ca2+-dependent (as well as a protein kinase C-mediated) pathway in the stimulation of PRL secretion by a variety of secretagogues. The proposed work will utilize a newly-developed method (cell cracking) for rendering cells permeable to macromolecules. This method allows preparation of washed """"""""cracked"""""""" GH3 cells which are structurally preserved cellular """"""""ghosts"""""""" devoid of cytoplasm. Addition of micromolar Ca2+, millimolar MgATP and a cytosolic protein to GH3 cell ghosts results in the release of PRL and other secretory proteins at 30 degrees. This system will provide a means for elucidating the biochemical events involved in Ca2+-activated secretion. The proposed work will: 1. Extend the biochemical and structural characterization of cracked cells; 2. Purify and characterize the cytosolic protein required for Ca2- activated PRL release; 3. Directly examine several hypothesized mechanisms for Ca2+ regulation of secretion (protein kinases, Ca2+-binding proteins, phospholipases) in order to facilitate identification of the cytosolic factor; 4. Determine whether the characterized cytosolic protein is required for Ca2+-regulated secretion in intact cells (Gh3, Paramecium and sea urchin eggs); and 5. Elucidate secretory granule- and membrane-associated mechanisms involving the cytosolic factor (using Gh3 cells lactotrophs and chromaffin cells). The outcome of this work will provide direct biochemical characterization of Ca2+-regulated secretion and evaluate the possibility that secretory regulation in several model systems share common features.
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