Creatine (Cr) and phosphocreatine (PCr), essentials constituents of heart, brain,-skeletal muscle, and macrophages, are involved in a variety of cellular processes including energy transduction, mitosis, and glycolysis. Since these cells cannot synthesize Cr, they must rely on Cr in plasma for their supply of intracellular Cr. Muscle cells and macrophages have specific sodium dependent Cr transporters in their plasma membranes. I have found that muscle cells and macrophages regulate the number of these transporters in response to changes in extracellular Cr concentrations and to their state of differentiation. In this way, these cells directly regulate their intracellular Cr stores and indirectly regulate their PCr stores. The goal of this proposal is to characterize the mechanisms by which Cr transport and possibly Cr efflux, regulate intracellular Cr and PCr stores in L6 muscle cells, human macrophages and J774 macrophages. Another goal of this proposal is to examine the structural requirements for a molecule to enter cells via the Cr transport system. This will provide the foundation for developing experimental designs to identify and isolate the Cr transporter. I also have preliminary data suggesting the existence of other control pathways which regulate intracellular Cr stores. I have found that there is a dramatic induction of Cr transport activity during myoblast and monocyte differentiation. Moreover, L6 myoblasts appear to contain an inactive form of creatine kinase which becomes activated during differentiation. The characterization of these putative regulatory molecules which activate creatine kinase will be another major objective of the research plans. Understanding how cells regulate intracellular Cr stores may provide insights into the relationship between abnormalities in Cr metabolism and muscle degeneration observed in response to certain diseases, drugs and hormones. Thus, regulation of Cr transport and phosphorylation integrates the biosynthesis of Cr with its functions in the cells.
