The objective of the proposed research is to elucidate the pathways by which the muscarinic cholinergic receptor (mAChR) regulates phospholipid metabolism, the generation of diacylglycerol (DAG), the activation of protein kinase C (PKC) and the induction of c-fos and other proteins in 1321N1 astrocytoma cells. The first specific aim is to determine the mechanisms by which phosphatidylcholine (PC) hydrolysis is activated. Questions to be addressed are whether PC turnover occurs in the absence of phosphoinositide hydrolysis (e.g. in NG108-15 cells which lack mAChR- stimulated PI hydrolysis); whether PI-generated second messengers mediate PC hydrolysis (Ca2+ mobilization, which can be buffered by quin 2; PKC activation, removed by down-regulation); and whether a G-protein can be shown to regulate PC hydrolysis in membranes. The second specific aim is to determine the sources of DAG in hormone-stimulated 1321N1 cells and the mechanisms that regulate its synthesis and metabolism. Specific questions to be addressed are the relative contributions of PI and PC to DAG formation (using differential lipid labelling); the contributions of phospholipase C versus D to DAG formation from PC; and the extent to which DAG kinase and other routes of DAG metabolism regulate DAG levels. The third specific aim it to demonstrate the critical role of agonist-mediated increases in calcium in causing protein kinase C redistribution as well as phosphorylation of PKC substrates ("activation" of PKC). Specific questions are whether PKC redistribution can occur in response to increased Ca2+ under conditions where there is no apparent increase in DAG; whether the redistribution seen with 3H-PDB binding is reflected in changes in membrane-associated PKC protein (by Western analysis and immunocytochemistry); how the time course of PKC activation, as determined by protein phosphorylation (80 kDa protein, EGF receptor) compares with that for association of PKC with membrane; and whether there are specific isozymes of PKC (using specific antibodies) in 1321N1 cells that differ in their pattern of redistribution or susceptibility to down-regulation. The final aim is to describe downstream events that are triggered through the phospholipid/PKC pathway in 1321N1 cells. Specific goals are to determine whether c-fos and other early response genes are induced by mAChR stimulation; what factors regulate the expression of c-fos (PKC, Ca2+); and whether hormonal activation of the phospholipid/PKC pathway leads to induction of glial fibrillary acid protein (GFAP) or of growth factors (e.g. NGF, PDGF, FGF) in 1321N1 cells.