HORMONAL CONTROL OF HEPATIC METABOLISM AND FUNCTION

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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.