HORMONAL AND IONIC CONTROL OF METABOLISM

新陈代谢的激素和离子控制

• 批准号: 3432696
• 负责人: HOWARD RASMUSSEN
• 金额: $ 2.37万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-09-30 至 1995-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3432696
• 关键词: 3T3 cells; affinity chromatography; calcium flux; carbachol; cations; cell cycle; clone cells; cyclic AMP; diacylglycerols; enzyme activity; fatty acids; forskolin; gas chromatography; hormone regulation /control mechanism; ionophores; isoproterenol; liposomes; liver; metabolism; phorbols; platelet derived growth factor; protein kinase C; thin layer chromatography; transfection

Diacylglycerol (DG) plays an important role as a second messenger. Major advances have taken place in understanding its generation. Its further metabolism is less well understood. Using new methods for incorporating labeled DGs in the plasma membrane (PM) of resting NIH 3T3 cells, new metabolic patterns for DGs were found in which conversion to phosphatidylcholine (PC) is a key event, indicating that DGs translocate from the PM to intracellular sites. This project will extend these studies to activated NIH 3t3 cells. Specifically, the research will examine: 1) Effects of exposure to different pharmacological agents on the patterns of conversions of exogenous radiolabeled DGs, to detect cross-talk interactions between different signalling systems at the level of DG metabolism. The agents to be tested are: forskolin, which raises cellular cAMP levels, Ca++ ionophores, which elevate cytoplasmic Ca++ concentrations, and phorbol esters and mezerein, which activate protein kinase C. Cells will be labeled with exogenous DGs via liposomes and exposed for different times to the agents; cellular lipids will be analyzed by TLC and conversions determined by radioactivity measurements. The data will be compared with experiments of the same kind in which the cells will be exposed to one of three agonists: carbachol, isoproterenol or platelet derived growth factor. 2) Changes in endogenous DG content and fatty acid composition will be determined under the same conditions by a DG binding proteins in the cytosol of NIH 3T3 cells and rat liver. Three approaches will be used: i) An assay will be developed bases on existing methods for other ;lipid ligands. ii) Using conventional and affinity chromatography purification procedures, it will determined if DG binding activity (DGBA) can be separated or coelutes with either fatty acid binding proteins (FABPs) or sterol carrier protein 2 (SCP-2). DGBA associated with pure samples of liver-FABP, intestine-FABP and SCP-2 will be studied. iii) the role of liver FABP in DG metabolism will be investigated, using a transfected cell line that expresses this protein in high amounts. Exogenous and endogenous DG metabolism will be compared in transfected and control cultures. These studies will help to clarify basic aspects of the metabolism of a second messenger involved in critical cell functions as secretion, contraction and proliferation.

二酰基甘油（DG）作为第二使者起着重要的作用。 主要的 在理解它的一代方面已取得进步。 进一步 代谢不太了解。 使用新方法合并 静止的NIH 3T3细胞的质膜（PM）中的DGS，新的 发现了DGS的代谢模式，其中转换为 磷脂酰胆碱（PC）是一个关键事件，表明DGS转运器 从PM到细胞内部位。 这个项目将扩展这些 研究活化的NIH 3T3细胞。 具体而言，研究将 检查：1）暴露于不同药理剂对 外源性放射性标记的DGS转换的模式，以检测 级别的不同信号系统之间的串扰相互作用 DG代谢。 要测试的特工是：福斯科林 蜂窝营地水平，Ca ++离子载体，升高细胞质Ca ++ 浓度，以及激活蛋白质 激酶C.细胞将通过脂质体和外源性DG标记 在不同时间暴露于代理商；细胞脂质将是 通过TLC分析和通过放射性测量确定的转化。 数据将与相同类型的实验进行比较 细胞将暴露于三种激动剂之一：卡尔巴乔，异丙肾上腺素 或血小板得出的生长因子。 2）内源性DG含量的变化 在相同条件下将确定脂肪酸成分 通过NIH 3T3细胞和大鼠肝脏的胞质醇中的DG结合蛋白。 将使用三种方法：i）将开发基础的测定 其他;脂质配体的现有方法。 ii）使用常规和 亲和色谱纯化程序，它将确定是否 DG结合活性（DGBA）可以分离或用任何一种脂肪分离 酸性结合蛋白（FABPS）或固醇载体蛋白2（SCP-2）。 DGBA 与肝癌，肠FABP和SCP-2的纯样品有关 被研究。 iii）肝FABP在DG代谢中的作用将是 研究了，使用表达该蛋白的转染细胞系 大量。 将比较外源性和内源性DG代谢 在转染和控制文化中。 这些研究将有助于澄清 第二使者的代谢的基本方面 关键细胞起分泌，收缩和增殖的作用。