STRESS-INDUCED ACTIVATION OF COLONIC MOTOR FUNCTION

以往的研究表明，中央或外周注射促肾上腺皮质激素释放因子（CRF）模仿的束缚应激对结肠运动功能的推进作用和CRF受体拮抗剂注射无论是中央或外周产生类似的封锁限制诱导的刺激结肠运输。 这些数据表明，外周CRF受体激活也可能参与结肠运动应激反应。 该提案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。 与不同CRF受体亚型1和2（CRF-R1和CRF-R2）的表征相关的新进展、作为CRF-R2的内源性配体的新型哺乳动物CRF相关肽尿皮质素的发现、选择性CRF受体亚型拮抗剂的开发和CRF-R1敲除小鼠的产生提供了将用于实现该目标的强大的新工具。 第一个具体的目标，来自我们的初步数据，将测试的假设，腹膜内注射（IP）的CRF通过外周CRF-R1的行为和应激招聘这一途径。 这将通过1）描述结肠运动功能的变化来实现（近端和远端肌电活动，运输和粪便输出）诱导的CRF相关配体与CRF-R1和CRF-R2的亲和力不同的频谱ip注射; 2）通过ip astressin和选择性CRF-R1拮抗剂建立ip CRF和尿皮质素的阻断以及对结肠的应激作用，以及使用CRF-R1基因敲除小鼠; 3）使用体内免疫中和和应激后肽的RIA测量来评估循环CRF/尿皮质素在应激期间CRF受体活化中的作用。 第二个目标，我们将使用联合的药理学和手术方法来描述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，并使用Fos免疫组织化学结合CRF的双重标记和定位来检验CRF可能直接激活肠胆碱能/P物质运动神经元的假设。R1在结肠中的原位杂交表达。 由于肠易激综合征（IBS）患者与对照组相比，对静脉注射CRF的结肠动力反应增强，因此，揭示CRF受体亚型和ip CRF刺激结肠推进活动的机制可能对理解应激相关性肠易激综合征（IBS）加重的病理生理学具有重要意义。

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.