Modulation of Colonic Response to Stress by Peripheral CRF2 Receptors

描述（由申请人提供）：IBS患者具有与排便习惯改变相关的复发性腹痛。IBS的机制知之甚少，缺乏治疗方法。应激反应受损是IBS的关键风险因素。应激的主要介质是肽促肾上腺皮质激素释放因子（CRF）。在最后一个授予期间，我们已经表明，在相反的CRF1，CRF2受体抑制结肠感觉运动反应和一个受损的CRF2功能加剧了结肠应激反应。CRF2如何介导结肠中的应激弹性的机制尚不清楚。在初步数据中，我们显示a）CRF2激活结肠神经元NOS和NO释放并涉及VIP以抑制结肠运动反应，B）人tau过表达小鼠显示对轻度应激的改变的结肠反应，和c）CRF2而不是CRF1激活抑制应激或CRF诱导的肠和初级传入神经元tau磷酸化。基于上一次资助的数据和初步数据，我们假设CRF2在结肠中通过激活肠道NO和调节神经元tau蛋白（一种新的肠道应激途径）来维持应激应对功能，并且改变CRF2信号传导将导致结肠对应激的适应不良感觉运动反应。这将在三个目标下进行测试。目的1将确定CRF2-NO-VIP三联体的功能相互作用。将在未处理和应激大鼠中监测CRF2介导的nNOS分子活化、NO释放、VIP活化和结肠收缩。将评估应激下结肠CRF2受体剪接变体的调节以确定与扰动的结肠运动稳态相关的变体。目的2将表征新的应激-tau-肠途径，并将剖析大鼠中应激诱导的肠神经元tau调节的作用和机制。将研究结肠肠神经元与tau磷酸化的同一性、结肠反射和CRF2对应激诱导的tau磷酸化的抑制。目的3将检验以下假设：应激或CRF诱导的内脏伤害感受的调制涉及肠和脊髓神经元tau蛋白磷酸化。将评估应激和非应激大鼠中的肌间和DRG神经元tau和神经元兴奋性沿着内脏伤害。将确定CRF 2介导的内脏伤害感受抑制是否与tau磷酸化抑制相关。将使用肠和脊髓tau调节和CRF2的位点特异性缺失（siRNA）。总体而言，这些研究将使用药理学，遗传学，组织，细胞和分子工具来剖析CRF2受体激活如何抑制应激相关的结肠感觉运动反应的机制。解开这些CRF2介导的机制，在肠道应激反应将有显着的影响，在理解应激诱导的肠道感觉运动的改变，并在指导未来的新的治疗方法，应激相关疾病，如IBS。

DESCRIPTION (provided by applicant): IBS patients have recurrent abdominal pain associated with altered bowel habits. The mechanisms of IBS are poorly understood and therapies are lacking. A compromised stress response is a key risk factors for IBS. The primary mediator of stress is the peptide cortocotropin releasing factor (CRF). In the last granting period we have shown that in contrast to CRF1, CRF2 receptors suppress the colonic sensorimotor responses and that a compromised CRF2 function exacerbates the colonic response to stress. The mechanism how CRF2 mediates stress resiliency in the colon is not known. In preliminary data, we show that a) CRF2 activates colonic neuronal NOS and NO release and involves VIP to inhibit colonic motor response, b) human tau- overexpressing mice display altered colonic response to mild stress and c) CRF2 but not CRF1 activation suppresses stress or CRF-induced enteric and primary afferent neuronal tau phosphorylation. Based on data from last grant and the preliminary data, we hypothesize that CRF2 in the colon subserves a stress-coping function through activation of enteric NO and modulation of neuronal tau, a novel enteric stress pathway, and that altered CRF2 signaling will lead to maladaptive colonic sensorimotor responses to stress. This will be tested under three aims. Aim 1 will determine the functional interaction of the CRF2-NO-VIP triad. CRF2 mediated molecular activation of nNOS, NO release, VIP activation and colonic contraction in vivo will be monitored in naive and stressed rats. Regulation of colonic CRF2 receptor splices variants under stress will be assessed to determine variants that are associated with perturbed colonic motor homeostasis Aim 2 will characterize a novel stress-tau-gut pathway and will dissect the effects and mechanism of stress-induced enteric neuronal tau modulation in rats. The identity of colonic enteric neurons with tau phophorylation, the colonic reflex and the suppression of stress-induced tau phophorylation by CRF2 will be investigated. Aim 3 will test the hypothesis that stress or CRF-induced modulation of visceral nociception involves enteric and spinal neuronal tau phosphorylation. Myenteric and DRG neuronal tau and neuronal excitability along with visceral nocicption in stressed and non-stressed rats will be assessed. Whether the CRF2 mediated suppression of visceral nociception is associated with the suppression of tau phosphorylation will be determined. Enteric and spinal tau modulation and site specific deletion of CRF2 (siRNA) will be used. Overall, the studies will use pharmacological, genetic, tissue, cellular and molecular tools to dissect the mechanisms how CRF2 receptor activation suppresses stress-related colonic sensorimotor responses. Unraveling these CRF2 mediated mechanisms in the gut response to stress will have significant impact in the understanding of stress-induced gut sensorimotor alteration and in guiding future novel therapeutic approaches to stress related diseases such as IBS.