The long-term objective of these studies is to define the physiological differences between the cellular Ca[unreadable]2+[unreadable] homeostasis of normal and neoplastic tissues. The rationale for this project is based on observations that tumor cells exhibit abnormal growth characteristics with respect to extracellular Ca[unreadable]2+[unreadable] and that their maintenance and regulation of intracellular Ca[unreadable]2+[unreadable] may also be altered. The specific aims of this research are: (1)\to determine the cytosolic-free Ca[unreadable]2+[unreadable] concentration and the intracellular distribution of Ca[unreadable]2+[unreadable] in hepatocytes (isolated from normal and regenerating liver) as well as malignant hepatoma cells; (2)\to determine the Ca[unreadable]2+[unreadable] buffering characteristics of normal liver and hepatoma mitochondria and endoplasmic reticulum under physiologically realistic conditions; and (3)\to compare the effects of Ca[unreadable]2+[unreadable] on the cytoskeleton of normal and malignant tissues. Recent progress along these lines include: (1)\development of a substantially improved technique for isolating mitochondria from ascites tumor cells and comparing their respiratory characteristics with those of digitonin-permeabilized cells; (2)\demonstration that AS30-D hepatoma mitochondria do not release Ca[unreadable]2+[unreadable] as normal liver mitochondria do in response to the presence of t-butyl hydroperoxide and that this is due to a lack of net NADPH oxidation because of the presence of a NADP[unreadable]+[unreadable]-reducing malic enzyme in the tumor mitochondria; (3)\demonstration that AS30-D hepatoma mitochondria buffer the free Ca[unreadable]2+[unreadable] concentration of a cytosol-like medium at a significantly higher level than normal liver mitochondria do and that this is due to an abnormally high rate of Ca[unreadable]2+[unreadable] efflux; and (4)\evidence that microsomal vesicles from AS30-D hepatoma cells accumulate more Ca[unreadable]2+[unreadable] and release Ca[unreadable]2+[unreadable] in response to inositol trisphosphate at a faster rate than that observed with normal liver microsomes. (E)