STRESS-INDUCED ACTIVATION OF COLONIC MOTOR FUNCTION

先前的研究表明，中枢或外周注射促肾上腺皮质激素释放因子（CRF）模拟了抑制应激对结肠运动功能的促进作用，并且中枢或外周注射CRF受体拮抗剂对抑制诱导的结肠运输刺激具有类似的阻断作用。这些数据表明外周CRF受体的激活也可能参与结肠对应激的运动反应。该建议的总体目标是建立外周注射CRF刺激结肠运动功能的机制，这些机制迄今为止在很大程度上是未知的，以及它们的生理相关性。不同的CRF受体亚型1和2 （CRF- R1和CRF- r2）的特征、新型哺乳动物CRF相关肽尿皮质素作为CRF- r2的内源性配体的发现、选择性CRF受体亚型拮抗剂的开发以及CRF-R1基因敲除小鼠的产生等方面的新进展，将为实现这一目标提供强大的新工具。根据我们的初步数据，第一个具体目标将验证腹腔注射CRF （ip）通过外周CRF- r1起作用的假设，并且应激激活了这一途径。这将通过以下方式实现：1)描述ip注射与CRF-R1和CRF-R2具有不同亲和力谱的crf相关配体诱导的结肠运动功能（近端和远端肌电活动、转运和粪便输出）的变化；2)建立ip应激素和选择性CRF- r1拮抗剂对ip CRF和尿皮质素的阻断和结肠应激作用，并使用CRF- r1敲除小鼠；3)利用体内免疫中和和应激后肽的RIA测量，评估应激时循环CRF/尿皮质素在CRF受体激活中的作用。在第二个目标中，我们将通过药物和手术相结合的方法来描述ip CRF刺激结肠运动功能的机制，以排除内分泌和外源性神经系统成分的可能参与，并通过Fos免疫组织化学结合结肠原位杂交对CRF- r1表达的双重标记和定位来验证CRF可能直接激活肠胆碱能/ P物质运动神经元的假设。揭示CRF受体亚型及其刺激结肠推进活性的机制可能对理解肠易激综合征（IBS）应激相关恶化的病理生理学具有重要意义，因为与对照组相比，IBS患者对iv CRF的结肠运动反应增强。

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.