Modulation of Colonic Response to Stress by Peripheral CRF2 Receptors

描述(申请人提供)：IBS患者反复腹痛与排便习惯改变有关。肠易激综合征的发病机制目前知之甚少，治疗方法也很缺乏。折衷的应激反应是IBS的关键风险因素。应激的主要介质是促肾上腺皮质激素释放因子(CRF)。在最后的授予期间，我们已经证明，与CRF1相反，CRF2受体抑制了结肠的感觉运动反应，并且CRF2功能受损加剧了结肠对应激的反应。CRF2如何在结肠中调节应激恢复的机制尚不清楚。在初步数据中，我们发现：a)CRF2激活结肠神经元型一氧化氮合酶和一氧化氮的释放，并参与VIP抑制结肠运动反应；b)人tau过表达的小鼠表现出对轻度应激的结肠反应的改变；c)CRF2而不是CRF1的激活抑制了应激或CRF诱导的肠源性和初级传入神经元tau的磷酸化。根据上一次授予的数据和初步数据，我们假设结肠中的CRF2通过激活肠内NO和调节神经元tau(一种新的肠道应激途径)来发挥应激应对功能，并且CRF2信号的改变将导致对应激的不良适应结肠感觉运动反应。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。CRF2介导的nNOS分子激活、NO释放、VIP激活和结肠收缩在幼鼠和应激大鼠体内将得到监测。将评估应激状态下结肠CRF2受体剪接变异体的调节，以确定与扰乱的结肠运动稳态相关的变异体。目标2将描述一种新的应激-tau-肠道通路，并将剖析应激诱导的大鼠肠神经tau调节的作用和机制。我们将研究结肠肠神经元与tau蛋白磷酸化的一致性、结肠反射以及CRF2对应激诱导的tau蛋白磷酸化的抑制。目的3将验证应激或CRF诱导的内脏伤害性感受的调制涉及肠神经和脊髓神经元tau磷酸化的假说。应激和非应激大鼠的肠肌和背根神经节神经元tau和神经元兴奋性以及内脏伤害性将被评估。CRF2介导的内脏伤害性抑制是否与tau磷酸化抑制有关还有待确定。将使用肠道和脊髓tau调节以及CRF2(SiRNA)的位点特异性缺失。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介导的肠道应激反应机制，将对理解应激诱导的肠道感觉运动改变和指导未来应激相关疾病(如IBS)的新治疗方法具有重要影响。

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.