Diacylglycerol (DG) plays an important role as a second messenger. Major advances have taken place in understanding its generation. Its further metabolism is less well understood. Using new methods for incorporating labeled DGs in the plasma membrane (PM) of resting NIH 3T3 cells, new metabolic patterns for DGs were found in which conversion to phosphatidylcholine (PC) is a key event, indicating that DGs translocate from the PM to intracellular sites. This project will extend these studies to activated NIH 3t3 cells. Specifically, the research will examine: 1) Effects of exposure to different pharmacological agents on the patterns of conversions of exogenous radiolabeled DGs, to detect cross-talk interactions between different signalling systems at the level of DG metabolism. The agents to be tested are: forskolin, which raises cellular cAMP levels, Ca++ ionophores, which elevate cytoplasmic Ca++ concentrations, and phorbol esters and mezerein, which activate protein kinase C. Cells will be labeled with exogenous DGs via liposomes and exposed for different times to the agents; cellular lipids will be analyzed by TLC and conversions determined by radioactivity measurements. The data will be compared with experiments of the same kind in which the cells will be exposed to one of three agonists: carbachol, isoproterenol or platelet derived growth factor. 2) Changes in endogenous DG content and fatty acid composition will be determined under the same conditions by a DG binding proteins in the cytosol of NIH 3T3 cells and rat liver. Three approaches will be used: i) An assay will be developed bases on existing methods for other ;lipid ligands. ii) Using conventional and affinity chromatography purification procedures, it will determined if DG binding activity (DGBA) can be separated or coelutes with either fatty acid binding proteins (FABPs) or sterol carrier protein 2 (SCP-2). DGBA associated with pure samples of liver-FABP, intestine-FABP and SCP-2 will be studied. iii) the role of liver FABP in DG metabolism will be investigated, using a transfected cell line that expresses this protein in high amounts. Exogenous and endogenous DG metabolism will be compared in transfected and control cultures. These studies will help to clarify basic aspects of the metabolism of a second messenger involved in critical cell functions as secretion, contraction and proliferation.
