Previous studies indicate that central or peripheral injection of corticotrophin-releasing factor (CRF) mimics the propulsive effect of restraint stress on colonic motor function and a CRF receptor antagonist injected either centrally or peripherally exerted similar blockade of restraint-induced stimulation of colonic transit. These data suggest that peripheral CRF receptor activation may also be involved in the colonic motor response to stress. The overall objective of the proposal is to establish the mechanisms whereby peripheral injection of CRF stimulates colonic motor function which are so far largely unknown, and their physiological relevance. New advances related to the characterization of distinct CRF receptor subtypes 1 and 2 (CRF- R1 and CRF-R2), the discovery of the novel mammalian CRF-related peptide, urocortin, as the endogenous ligand for CRF-R2, the development of selective CRF receptor subtype antagonists and the generation of CRF-R1 knockout mice provide powerful new tools which will be used to achieve this objective. The first specific aim, derived from our preliminary data, will test the hypothesis that CRF injected intraperitoneally (ip) acts through peripheral CRF-R1 and that stress recruits this pathway. This will be achieved by 1) characterizing the changes in colonic motor function (proximal and distal myoelectrical activity, transit and fecal output) induced by ip injection of CRF-related ligands with a different spectrum of affinity to the CRF-R1 and CRF-R2; 2) establishing the blockade of ip CRF and urocortin and stress actions on the colon by ip astressin and a selective CRF-R1 antagonists and the use of CRF-R1 knockout mice; 3) assessing the role of circulating CRF/urocortin in the activation of CRF receptor during stress using in vivo immuneutralization and RIA measurements of peptides after stress. In the second aim, we will delineate the mechanisms whereby ip CRF stimulates colonic motor function using combined pharmacological and surgical approaches to rule out the possible involvement of endocrine and extrinsic nervous system components and test the hypothesis that CRF may directly activate enteric cholinergic/substance P motor neurons using Fos immunohistochemistry combined with double labeling and localization of CRF-R1 expression by in situ hybridization in the colon. Unraveling the CRF receptor subtype and mechanism through which ip CRF stimulates colonic propulsive activity may have important implications in the understanding of the pathophysiology of stress-related exacerbations of irritable bowel syndrome (IBS) since IBS patients have an enhanced colonic motility response to iv CRF compared with controls.
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