Localization of Activated cAMP-Dependent Protein Kinase and Gap Junctions

小行星9219998 将使用体外方法研究用已知影响连接形成和促进肾上腺类固醇生成的化合物处理细胞后的间隙连接功能和间隙连接基因调控。 具体而言，将使用间隙连接cDNA克隆研究促肾上腺皮质激素（ACTH）、DbcAMP、毛喉素和cAMP依赖性蛋白激酶（pKA）活性对间隙连接表达的影响。 将使用Y-1小鼠和SW-13人肾上腺皮质细胞系以及成年大鼠肾上腺皮质细胞的原代培养物。 缝隙连接表达将用免疫组织化学、蛋白质印迹和北方印迹分析表征和定位。 细胞通讯的证据将通过荧光黄染料转移实验获得。 将含有针对间隙连接基因产物（Cx43）的cDNA反义和抗体的载体引入细胞中以确定间隙连接表达的抑制作用。 将在用间隙连接cDNA转染的细胞中研究间隙连接的过表达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。 间隙连接是在某些组织中，在某些发育阶段和/或某些功能状态期间将细胞彼此连接的结构。 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相互交流方式的了解。 ***

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. ***