The objective of this study is to characterize the second messenger system which mediates the actions of calcium- dependent hormones in liver cells. These hormones, which include epinephrine and vasopressin, stimulate the breakdown of inositol lipids in the plasma membrane with a resultant release of two second messengers; diacylglycerol which activates protein kinase C and inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) which mobilizes intracellular calcium. The mechanism by which Ins(1,4,5)P3 releases calcium will be investigated and special emphasis will be placed on the regulation of this system by other cellular constituents and hormones. Of particular interest in this respect is the apparent potentiation by GTP of Ins(1,4,5)P3-induced calcium release, which we and others have described. The calcium release system will be studied in preparations of permeabilized hepatocytes and isolated sub-cellular fractions. The hypothesis will be investigated that the calcium mobilization system consists of an Ins(1,4,5)P3-activated calcium channel in the endoplasmic reticulum, with associated regulatory proteins whose activity can be modulated by guanine nucleotides. This proposal will be tested by measurements of the kinetics of calcium release and reuptake using fluorescent calcium indicators and by examining the communication between different intracellular pools of calcium. In addition to macroscopic calcium transport measurements, microscopic and single channel calcium currents will be measured using microsomes reconstituted into planar lipid bilayers. These studies will give insight into the physiological responses to the wide range of calcium-dependent hormones which have important effects in almost all mammalian cell types. Attempts will be made to identify the protein components of the calcium release system by using photoaffinity labels, with a view to longer term plans to reconstitute the Ins(1,4,5)P3-sensitive calcium channel. A further focus of this investigation will be the mechanisms by which the calcium- mobilizing hormones interact with other classes of hormones such as insulin.
