Stress-Induced Activition of Colonic Motor Function

描述(申请人提供)：促肾上腺皮质激素释放因子(CRF)和尿皮质激素(UCN)1与两个Gs偶联的CRF1和CRF2受体相互作用。近年来，新的CRF相关多肽UCN-2和UCN-3被发现作为CRF-2的选择性激动剂。大脑中CRF信号通路的激活再现了对压力的整体内分泌、自主神经、内脏和行为反应。在最后的授予期间，我们确定，除了大脑，肠道是外周注射CRF配体的反应靶点，这提供了CRF2介导的胃抑制和CRF介导的结肠刺激运动功能的第一个证据。此外，我们还发现，外周注射CRF拮抗剂可以减轻束缚和低剂量内毒素引起的肠运动改变。该方案的总体目标是建立肠道CRF受体信号系统，作为应激对大鼠肠道功能的双重胃抑制和结肠刺激影响的局部效应肢体。目的1通过激光捕获胃、结肠组织显微解剖切片的RT-PCR、免疫化学定位、自主神经和糖皮质激素阻断实验，验证CRF配体(CRF、UCN 1、UCN 2和UCN 3)及其受体(CRF1和CRF2)基因在胃和结肠组织中的表达受这些应激源调控的假设。目的2探讨外周CRF刺激结肠功能的机制包括利用Fos、双重标记、监测递质释放和新型外周作用的CRF1激动剂和拮抗剂，直接激活结肠胆碱能细胞和肠嗜铬细胞(EC)上的CRF1受体。目的3阐明外周UCN2抑制胃排空的机制包括超声显微技术显示的胃运动和括约肌运动的改变，以及通过RNAi特异性沉默CRF2受体确定的胃壁内兴奋性胆碱能和抑制性神经传递的调节。总体而言，这些研究将提供胃和结肠CRF配体和同源受体的准确表达，它们在应激状态下对这些部位的调节，以及它们对胃和结肠双重局部作用的机制。此外，它还将锚定肠道CRF信号系统，作为应激相关肠道运动改变中生理效应肢体的一部分。这可能为功能性肠病的管理提供新的策略。

DESCRIPTION (provided by applicant): 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.