Localization of Activated cAMP-Dependent Protein Kinase and Gap Junctions

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9219998 Murray In vitro methods will be used to study gap junction function and gap junction gene regulation following treatment of cells with compounds known to influence junction formation and to promote adrenal steroidogenesis. Specifically, the effect of adrenocorticotropin (ACTH), DbcAMP, forskolin, and cAMP-dependent protein kinase (pKA) activity on gap junction expression will be studied with gap junction cDNA clones. Y-1 mouse and SW-13 human adrenocortical cell lines and primary cultures of adult rat adrenal cortical cells will be used. Gap junction expression will be characterized and localized with immunohistochemistry, western blot and northern blot analysis. Evidence of cell communication will be obtained with Lucifer yellow dye transfer experiments. Vectors containing cDNA antisense and antibody directed against gap junction gene products (Cx43) will be introduced into cells to determine the effect of inhibition of gap junction expression. The effect of over-expression of gap junctions on adrenal cell function will be studied in cells transfected with gap junction cDNA. These studies will give insight into the relationship of junctional communication to the hormone response. %%% Gap junctions are structures that connect cells to one another in certain tissues, in certain stages of development, and/or during certain functional states. These structures are pathways by which cells communicate by passage of chemical messengers. This research will use techniques of molecular biology to examine the effect of hormones on gap junction formation and function. The results of these studies will increase our knowledge of how hormones affect the way cells communicate with one another. ***