HORMONAL CONTROL OF HEPATIC METABOLISM AND FUNCTION

本项目的总体目标是阐明 激素调节肝功能。 项目的具体目标 期间是研究激素的作用机制，如 血管紧张素II，提供刺激和抑制输入， cell. 刺激性输入是通过增加 磷脂酰肌醇4，5二磷酸，产生两个细胞内 信使、Ca ~（2+）离子和甘油二酯（DAG）。 这些消息激活 不同的蛋白激酶如磷酸化酶激酶和蛋白激酶C 然后调节细胞中的不同功能。 抑制性输入 通过与胰高血糖素刺激的腺苷酸直接相互作用产生 环化酶通过镍鸟嘌呤核苷酸的调节成分腺苷酸 环化酶 提出了四种实验方法。 第一个项目 将探索已知对Ca 2+离子有反应的蛋白激酶的能力 和DAG磷酸化和磷酸化糖原合酶，一个重要的 由多位点磷酸化调节的酶。 磷酸化位点 通过完整细胞中的每种激酶以及对酶的最终影响， 活动将相互关联。 在第二个项目中， 和Ca 2+信号对质膜的磷酸化将是 探讨了 这些研究将通过解析膜蛋白来进行 来自对照和激素处理的32 P标记肝细胞， 三维凝胶并用放射自显影法使其可视化。 密度 放射自显影照片上的信息将在一个 电脑 在第三个项目中，血管紧张素II和 蛋白激酶C调节抗血管紧张素原的肝合成 将被探索。 这项研究将通过测量 蛋白质的合成，并通过使用cDNA探针来测量 血管紧张素原mRNA水平。 在第四个项目中， N1鸟嘌呤核苷酸结合蛋白与 血管紧张素II受体将通过重组纯化的Ni 蛋白质进入已经用GTP-γ-S处理的膜中， 受体的亲和力。 回收高亲和力状态的 受体预期在Ni的重建之后。

The overall goal of this project is to elucidate the mechanisms by which hormones regulate hepatic function. The specific goals of the project period are to examine the mechanism of action of hormones such as angiotensin II that provide both stimulatory and inhibitory inputs to the cell. The stimulatory inputs are generated by increased breakdown of phosphatidylinositol 4,5 bisphosphate which produces two intracellular messengers, Ca2+ ion and diacylglycerol (DAG). These messages activate distinct protein kinases such as phosphorylase kinase and Protein Kinase C which then regulate different functions in the cell. The inhibitory inputs are generated by a direct interaction with glucagon stimulated adenylate cyclase through the Ni guanine nucleotide regulatory component of adenylate cyclase. Four types of experimentation are proposed. The first project will explore the ability of protein kinases known to respond to Ca2+ ion and DAG to phosphorylate and inactivate glycogen synthase, an important enzyme regulated by multisite phosphorylation. The site(s) phosphorylated by each kinase in the intact cell and the resultant effect on enzyme activity will be correlated. In the second project, the effects of the DAG and Ca2+ signals on the phosphorylation of plasma membranes will be explored. These studies will be carried out by resolving membrane proteins from control and hormone treated 32P-labelled hepatocytes on two dimensional gels and visualizing them with autoradiography. The density information on the autoradiographs will be integrated with the aid of a computer. In a third project, the possible role of angiotensin II and Protein Kinase C in regulating the hepatic synthesis of antiotensinogen will be explored. This study will be carried out both by measuring the synthesis of the protein and by using a cDNA probe to measure angiotensinogen mRNA levels. In the fourth project, the molecular interactions between the Ni guanine nucleotide binding protein and the angiotensin II receptor will be explored by reconstituting purified Ni protein into membranes that have been treated with GTP-Gamma-S to reduce the affinity of the receptor. Recovery of a high affinity state of the receptor is expected following reconstitution of Ni.