Corticotropin-releasing factor (CRF) and urocortin (Ucn) 1 interact with two Gs coupled CRF1 and CRF2 receptors. Recently, new CRF-related peptides, Ucn 2 and Ucn 3, were discovered as selective CRF2 agonists. Activation of CRF signaling pathways in the brain reproduces the overall endocrine, autonomic, visceral and behavioral responses to stress. During the last granting period, we established that, in addition to the brain, the gut is a target responsive to peripheral injection of CRF ligands providing the first evidence of CRF2-mediated gastric inhibition and CRF-mediated colonic stimulation of motor function. In addition, we showed that peripheral injections of CRF antagonists alleviate gut motor alterations induced by restraint and a low dose of endotoxin. The overall objective of the proposal is to establish that the gut CRF receptor signaling system serves as a local effector limb for the dual gastric inhibitory and colonic stimulatory influence of stress on gut function in rats. Aim 1 will test the hypothesis that there is a differential profile of CRF ligand (CRF, Ucn 1, Ucn 2 and Ucn 3) and receptor (CRF1 and CRF2) gene expression in the stomach and colon that is regulated by these stressors using RT-PCR in laser capture microdissected layers of gastric and colonic tissues, immunochemical localization, and autonomic and glucocorticoid blockade. Aim 2 will establish that the mechanisms through which peripheral CRF stimulates colonic function involve direct activation of CRF1 receptors on colonic enteric cholinergic and enterochromaffin (EC) cells, using Fos, double labeling, monitoring of transmitter release, and novel peripherally acting CRF1 agonist and antagonists in vivo and in vitro. Aim 3 will show that mechanisms of peripheral Ucn 2-mediated inhibition of gastric emptying involve alterations of gastric and sphincter motility delineated by ultrasonomicrometry and modulation of excitatory cholinergic and inhibitory neurotransmission at sites within the gastric wall identified using specific silencing of CRF2 receptor with RNAi. As a whole, these studies will provide accurate expression of gastric and colonic CRF ligands and cognate receptors, their regulation at these sites under stress, and mechanisms underlying their dual local actions on the stomach and colon. In addition, it will anchor the gut CRF signaling system as part of the physiological effector limb in stress-related gut motor alterations. This could underpin novel strategies for management of functional bowel disorders.