Localization of Activated cAMP-Dependent Protein Kinase and Gap Junctions

[9219998] Murray：在用已知影响连接形成和促进肾上腺甾体生成的化合物处理细胞后，将使用体外方法研究间隙连接功能和间隙连接基因调控。具体来说，促肾上腺皮质激素（ACTH）、DbcAMP、forskolin和camp依赖性蛋白激酶（pKA）活性对间隙连接表达的影响将通过间隙连接cDNA克隆进行研究。Y-1小鼠和SW-13人肾上腺皮质细胞系及成年大鼠肾上腺皮质细胞原代培养物将被使用。通过免疫组织化学、western blot和northern blot分析来表征和定位间隙连接的表达。细胞通讯的证据将通过路西法黄色染料转移实验获得。将含有cDNA反义和针对间隙连接基因产物（Cx43）的抗体的载体导入细胞，以确定抑制间隙连接表达的效果。我们将在转染间隙连接cDNA的细胞中研究过表达间隙连接对肾上腺细胞功能的影响。这些研究将深入了解连接通讯与激素反应的关系。间隙连接是在特定组织、特定发育阶段和/或特定功能状态下连接细胞的结构。这些结构是细胞通过化学信使进行交流的途径。本研究将利用分子生物学技术来研究激素对缝隙连接形成和功能的影响。这些研究的结果将增加我们对激素如何影响细胞之间交流方式的认识。＊＊＊

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. ***