The goal of this proposal is to elucidate the cellular mechanisms involved in regulation of the synthesis and release of a factor similar to endothelium-derived relaxing factor (EDRF) in neurons. Such mechanisms are currently unclear. More specifically, the relationship between muscarinic receptor-mediated increase in phosphoinositide hydrolysis and EDRF formation will be ascertained. This represents a logical pursuit of the findings obtained during the current project period. EDRF was first shown to be synthesized in endothelial cells, followed by its diffusion to neighboring smooth muscle to effect their relaxation. Extensive evidence has accumulated indicating that EDRF or EDRF-like mediators are released in a variety of tissues, including brain, in a Ca2+-dependent fashion, resulting in increased cyclic GMP formation. There is currently active research aiming at elucidating the possible roles such a second messenger might play in altering neuronal function and in mediating communication between different neurons, perhaps through the activation of guanylate cyclase. We will continue using mouse neuroblastoma N1E-115 cells for the proposed studies since they have served as an excellent model to unfold the mechanisms of coupling of neuronal muscarinic receptors to signal transduction pathways, particularly the activation of guanylate cyclase. We have also shown recently that activation of muscarinic receptors in these cells stimulates the formation of an EDRF-like intercellular messenger. The specific aims of the proposed research are as follows: 1) determination of the cellular source of Ca2+ involved in muscarinic receptor-mediated increase in the formation and release of EDRF; 2) assessment of the relationship between the ability of a muscarinic agonist to increase phosphoinositide hydrolysis and intracellular Ca2+ and to enhance the formation and release of EDRF; 3) studying the cellular mechanisms involved in desensitization of muscarinic receptor-mediated EDRF synthesis and release, and the potential regulatory role of protein kinase C, arachidonic acid, cyclic GMP, or EDRF itself.