Our present understanding of the regulation of cellular metabolic events is limited by our ability to reproduce subcellular organizational components in vivo. In the present proposal, we outline a series of experiments designed to investigate the organization of the hormone- responsive phosphoinositide cycle in the rat cell line, WRK-1. We have previously reported the existence of both vasopressin-sensitive and insensitive phosphoinositides in these cells, and more recent data suggests a physical compartmentation of the cycle, as well. Our present goals are three fold: 1) Using two different agonists, vasopressin and bradykinin, we will determine whether a common or distinct phosphoinoitide lipid pool is involved in the cyclic metabolic response to different stimuli. The results of these experiments will indicates how closely the receptor is associated with hormone-responsive lipid pools. 2) To determine the degree to which the phosphoinositide cycle is a closed metabolic phenomenon within the cell, we will carry out experiments designed to test whether or not exogenous substrate intermediates can participate in hormone-stimulated cycling. A negative result in this instance would suggest that all the enzymes of the cycle are arranged in such a manner that the product from one reaction is channeled directly to the next enzyme in the pathway. 3) We will examine the mechanism by which phosphatidylinositol resynthesis is stimulated during cycling using a combination of whole cell and broken cell protocols. We will determine whether the hormone induced increase in synthesis of phosphatidylinositol is the result of increased substrate concentrations or enzyme activation or both. The combined results of these studies should provide insight into the organizational aspects of phosphoinositide cycling, giving us a more accurate picture of the biochemical events involved in cellular regulation. It has for a number of years been appreciated that one of the major pathways of transmembrane signaling by hormones involves the breakdown of particular phospholipid components of the cell membrane. Dr. Monaco's prior work has demonstrated that only a fraction of the total phospholipid present in the membrane is susceptible to hormone- stimulated breakdown. Thus, only that portion is capable of serving as a source of second messenger in the cellular response to hormone. The continuation of this research is directed toward further identification of the mechanisms that, in effect, compartmentalize the cell membrane phospholipids into hormone-sensitive and insensitive fractions. The results of this research will contribute to our fundamental understanding of the complex mechanisms by which cells respond to extracellular signals.
