Modulation of Colonic Response to Stress by Peripheral CRF2 Receptors

描述（由申请人提供）：应激和促肾上腺皮质激素释放因子（CRF）主要通过CRF1受体对结肠分泌运动功能产生深远影响。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的药理激活可阻止crf1介导的应激源crf诱导的结肠肠道神经元激活和腹泻，而阻断或删除CRF2可增强结肠对应激的运动反应。其次，CRF1激活增强内脏疼痛反应，而CRF2激活阻止辣椒素诱导的原代培养腰骶DRG神经元Ca2+瞬态。第三，在同时表达CRF1/CRF2的细胞中，与仅表达CRF1的细胞相比，CRF导致cAMP的产生更少。基于这些关键观察结果，我们假设在啮齿动物中，外周CRF2作为应激应对信号，通过直接和间接作用于外周靶细胞，阻止应激诱导的结肠运动和内脏痛觉过敏。具体目的1将通过抑制结肠肠道神经元，确定CRF2在急性和慢性应激诱导的结肠运动反应中作为应激应对机制的生理作用。这将通过阻断或删除CRF2、阻断神经递质通路以及证明应激和外源性神经刺激诱导体内和体外CRF配体释放来实现。特异性目的2将测试CRF2在纵肌肌丛（LMMP）组织和原代结肠肌丛神经元培养中阻止CRF1介导的乙酰胆碱释放并促进抑制性神经递质释放。将研究原生细胞和转染细胞中CRF1-CRF2相互作用，以深入了解CRF1-CRF2信号串扰。具体目标3将通过在离体结肠传入和体外DRG神经元中进行功能、电生理和分子分析，确定外周CRF2的激活是否通过抑制盆腔传入和腰骶DRG神经元来抑制应激诱导的内脏疼痛敏化。阐明外周CRF2抑制应激和crf相关的结肠运动改变和内脏超敏反应的生理作用和机制，将对肠易激综合征等功能性疾病具有重要的临床意义，在这些疾病中，应激、CRF1信号通路和症状之间的联系越来越被认识到。

DESCRIPTION (provided by applicant): 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 stressor 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. Public Health Relevance: 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.