Modulation of Colonic Response to Stress by Peripheral CRF2 Receptors

摘要 应激和促肾上腺皮质激素释放因子(CRF)对结肠分泌运动功能有深远的影响， 主要通过CRF1受体。CRF2在结肠应激反应中的生理作用尚不清楚。 初步数据显示，首先，外周CRF2的药理学激活可防止CRF1介导的应激反应。 或CRF诱导的结肠肠神经元激活和腹泻，而阻断或缺失CRF2则增强 结肠运动对压力的反应。第二，CRF1的激活增强了内脏的疼痛反应，而CRF2 激活可阻止辣椒素诱导的原代培养腰骶背根神经节神经元钙瞬变。第三，CRF 导致同时表达CRF1/CRF2的细胞比只表达CRF1的细胞产生更少的cAMP。 基于这些关键的观察，我们假设在啮齿类动物中，外周CRF2起着应对压力的作用 抑制应激引起的结肠运动和内脏痛敏的信号，通过直接和间接作用于 外周靶细胞。具体目标1将确立CRF2作为压力应对的生理作用 急、慢性应激诱导结肠动力反应的机制--结肠肠道抑制 神经元。这将通过阻断或删除CRF2以及阻断神经递质来实现 应激和外源性神经刺激在体内诱导CRF配体释放的途径和研究 而且是在体外。特定目标2将测试CRF2阻止CRF1介导的乙酰胆碱释放并促进 纵行肌间神经丛和原结肠组织中抑制性神经递质的释放 肌间神经细胞培养。CRF1-CRF2在天然细胞和转基因细胞中可能的相互作用将被研究以 深入了解CRF1-CRF2信号串扰。具体目标3将决定是否激活 外周CRF2通过抑制盆腔传入和抑制应激诱导的内脏痛敏化 脊髓背根节神经元的在体功能、电生理和分子检测 分离的结肠传入准备和体外培养的背根神经节神经元。阐明了其生理作用和 外周CRF2抑制应激和CRF相关结肠动力改变及机制的研究 内脏高敏感性对肠易激等功能性疾病有重要的临床意义 应激、CRF1信号通路和症状之间的联系日益被认识到。与公共卫生的相关性 这项研究旨在确定结肠中CRF2受体信号作为应激的功能 适应系统，维持结肠运动和疼痛反应的动态平衡。这项研究与Gut有关 由压力引发或加重的疾病，包括肠易激综合征。对这些影响和影响的阐明 外周CRF2激活抑制结肠应激或CRF相关升高的机制 运动活动和内脏疼痛将对IBS等功能性肠道疾病具有重要的临床意义， 因此，人们越来越认识到压力和症状之间的联系。

ABSTRACT Stress and corticotropin-releasing factor (CRF) exert a profound effect on colon secreto-motor function, primarily through CRF1 receptors. The physiological role of CRF2 in the colonic response to stress is unknown. Preliminary data show that first, pharmacological activation of peripheral CRF2 prevents CRF1-mediated stress- or CRF-induced colonic enteric neuron activation and diarrhea while blockade or deletion of CRF2 enhances the colonic motor response to stress. Second, CRF1 activation enhances visceral pain response while CRF2 activation prevents capsaicin induced primary culture lumbosacral DRG neurons Ca2+ transients. Third, CRF causes less cAMP production in cells that express both CRF1/CRF2 than in cells that expresses only CRF1. Based on these key observations, we hypothesize that in rodents, peripheral CRF2 serves as a stress-coping signal that halts stress-induced colonic motility and visceral hyperalgesia through a direct and indirect action on peripheral target cells. Specific aim 1 will establish the physiological role of CRF2 as a stress-coping mechanism in acute and chronic stress-induced colonic motor response, through inhibition of colonic enteric neurons. This will be achieved by blockade or deletion of CRF2 as well as by blocking neurotransmitter pathways and by demonstrating that stress and extrinsic nerve stimulation induce CRF ligand release in vivo and in vitro. Specific aim 2 will test that CRF2 prevents CRF1 mediated acetylcholine release and promotes inhibitory neurotransmitters release in longitudinal muscle myenteric plexus (LMMP) tissue and primary colonic myenteric neuron culture. The putative CRF1-CRF2 interaction in native and transfected cells will be studied to gain insight on the CRF1-CRF2 signaling cross talk. Specific aim 3 will determine whether activation of peripheral CRF2 inhibits stress-induced visceral pain sensitization through the inhibition of pelvic afferents and lumbosacral DRG neurons by performing functional, electrophysiological and molecular assays in vivo in isolated colonic afferent preparation and in vitro DRG neurons. The elucidation of the physiological role and mechanisms through which peripheral CRF2 dampens stress- and CRF-related colonic omotor alterations and visceral hypersensitivity will have important clinical implications in functional disorders such as irritable bowel syndrome, where a link between stress, CRF1 signaling pathway and symptoms are increasingly recognized. RELEVANCE TO PUBLIC HEALTH The proposed study aims at establishing that CRF2 receptor signaling in the colon functions as a stress adaptation system to maintain colonic motor and pain response homeostasis. The study has relevance to gut diseases that are triggered or exacerbated by stress, including IBS. The elucidation of the effects and mechanisms through which peripheral CRF2 activation dampen stress- or CRF-related increases in colonic motor activity and visceral pain will have important clinical implications for functional gut diseases such as IBS, for which a link between stress and symptoms are increasingly recognized.