This proposal investigates the regulatory mechanisms of feedback modulation of nitric oxide (NO) formation. Specifically, the hypothesis which will be tested is: physiologically produced NO regulates its own synthesis by modulating components of the intracellular pathways involved in stimulation of neuronal NO synthase. The hypothesis will be tested using m1 muscarinic receptors since these receptors couple to NO signaling and this pathway has a putative role in learning and memory. It is therefore important to study regulation of NO synthesis. Three specific aims will be pursued: 1) Determine if NO generated by m1 receptor activation results in dampening of receptor coupling to phosphoinositide hydrolysis and calcium signaling. There is evidence that NO from NO donors attenuates muscarinic receptor-mediated phosphoinositide hydrolysis. 2) Determine the cellular mechanisms of NO- induced regulation of coupling of m1 receptors to these second messenger pathways, and 3) Determine if receptor-mediated formation of NO is involved in agonist-induced long-term changes in receptor sensitivity. Experiments will be done using cells that either co-express neuronal NO synthase add ml receptors or express m1 receptors, but not NO synthase. Comparing responses produced by the two populations of cells will provide information on cellular mechanisms involved in regulating NO synthesis via modulation of related signaling pathways.