While it is clear that cells maintain a cytoplasmic free magnesium level well below the magnesium equilibrium potential and require magnesium ion for the normal activity of many enzymes, the role of intracellular magnesium in regulatory events in cellular physiology remains largely unexplored. Attempts to study mechanisms that regulate intracellular magnesium levels and to assess the physiological role of this cation have been limited by the lack of adequate techniques for measurement of intracellular free magnesium. The development of magnesium-sensitive fluorescent indicators that can be loaded into cells has made it possible to monitor intracellular magnesium on a second by second basis for the first time. Having established a technique for utilizing one of these new indicators, Mag-fura2, to measure internal magnesium in BC3H-1 cells, the primary goals of this proposal are: 1) to characterize the kinetic relationship between intracellular magnesium and ATP/creatine phosphate levels in BC3H-1 cells, 2) to develop a technique for determining the magnesium buffer capacity of these cells, and 3) to characterize the effect of acute exposure to epidermal growth factor and other growth factors on ATP levels in BC3H-1 and Swiss 3T3 cells. Since cells maintain their intracellular magnesium levels within certain limits, and require magnesium for the regulation of a variety of critical cellular functions, it seems likely that regulation of intracellular magnesium is a physiologically important process. Recent advances in the development of fluorescent probes for magnesium ion now make it possible to approach this problem. The proposed research will take advantage of these new research tools to provide fundamental new information about the intracellular regulation of magnesium, the most abundant cation found in mammalian cells.
