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.

甘油二酯(DG)作为第二信使起着重要的作用。主修 在理解它的产生方面已经取得了进展。它更远了 人们对新陈代谢知之甚少。使用新方法整合 静息状态下NIH3T3细胞质膜上标记的DGs，新 DGs的代谢模式被发现在其中转化为 磷脂酰胆碱(PC)是一个关键事件，表明DG易位 从PM到细胞内的位置。该项目将扩展这些功能 激活NIH3T3细胞的研究。具体地说，该研究将 检查：1)暴露于不同药理物质的影响 外源放射性标记DG的转化模式，以检测 不同信令系统之间在级别上的串扰交互 DG新陈代谢。接受检测的试剂是：forsklin，它提高了 细胞内cAMP水平，钙离子载体，升高胞浆内钙离子 浓度，以及激活蛋白质的佛波醇酯和梅泽林 细胞将通过脂质体标记外源DG和 暴露于不同时间的毒剂；细胞脂质将 用薄层层析进行分析，通过放射性测量确定转化率。 这些数据将与同类实验进行比较，其中 细胞将暴露于三种激动剂之一：卡巴胆碱、异丙肾上腺素 或者是血小板衍生生长因子。2)内源DG含量变化 并在相同条件下测定脂肪酸组成 NIH3T3细胞和大鼠肝脏胞浆中的DG结合蛋白。 将使用三种方法：i)将根据以下情况开发一种分析方法 其他；脂类配体的现有方法。二)使用常规和 亲和层析纯化程序，它将确定是否 DG结合活性(DGBA)可与脂肪或脂肪分离或合并 酸结合蛋白(FABP)或甾醇载体蛋白2(SCP-2)。DGBA 与肝-FABP、肠-FABP和SCP-2的纯样本相关联 被研究。Iii)肝脏FABP在DG代谢中的作用将是 使用表达这种蛋白质的转基因细胞系进行了研究 数额很大。将比较外源性和内源性DG代谢 在转基因和对照培养中。这些研究将有助于澄清 第二信使新陈代谢的基本方面 关键细胞的功能包括分泌、收缩和增殖。