Identification and Activation Mechanisms of Vagal and Spinal Nociceptors in Esophageal Mucosa

项目摘要 胃食管反流病（GERD）是美国的一个主要健康问题， 20%的人口。在至少四分之一的这些患者中，由GERD引起的症状，如 胃灼热和胸痛不能通过质子泵抑制剂抑制胃酸来控制 （PPI）。该PPI难治性GERD患者人群未接受有效的药物治疗 选项.不了解这种情况下持续症状的机制是一个主要的问题。 知识差距，限制了临床治疗选择。持续症状的一个潜在原因是 PPI难治性GERD患者为弱酸性（pH>5.0）反流。会引起疼痛- 介导神经（C-纤维伤害感受器）在食管粘膜，但分子机制还没有 已明确阐明。此外，尚不清楚胆汁酸是否也直接激活食管癌。 C-纤维和介导GERD的伤害性症状。我们的总体假设是， PPI难治性GERD（弱酸和胆汁酸）激活食管伤害性C纤维亚型， 导致难治性GERD的持续症状。确定传入神经的身份 亚型和导致PPI难治性症状的关键受体对于发展至关重要 针对这一人群的具体目标。在过去的十年中，我们已经确定了3种类型的C-纤维在 食管表达不同受体和神经递质，具有不同的中枢神经系统， 预测。有趣的是，我们已经开发了新的数据，表明两个因素在弱 酸性反流强烈刺激食管C-纤维：1）胆汁酸（其经常在食管中发现）， PPI治疗的反流），和2）低质子浓度的酸（pH=6.0，在此称为弱酸）。我们 已发表的论文和初步数据表明，关键受体TGR 5和TASK 1/ASIC 3可能是 涉案在此基础上，我们将首先在目标1中确定伤害性传入神经的亚型 食管粘膜中的神经末梢更容易受到反流的影响。然后我们将阐明 受体介导的C-纤维激活的弱酸在目的2。我们将讨论弱酸 通过抑制钾双孔结构域（K2 P）家族的组合激活C-纤维亚型 通道（TASK 1）和ASIC家族的阳离子通道（ASIC 3）的激活。在目标3中，我们 阐明受体介导的C-纤维激活胆汁酸。我们假设胆汁酸激活C- 纤维亚型，其由胆汁酸受体TGR 5介导。我们将应用基因的方法 表达分析、膜片钳记录和起源于C纤维的神经活动记录 神经末梢研究将在动物模型中进行，其中神经元选择性基因 通过我们验证的体内shRNA沉默策略敲减。阐明其机制， 胆汁酸和弱酸激活C纤维将有助于开发PPI难治性GERD的新靶点。

Project Summary Gastroesophageal reflux disease (GERD) is a major health problem in the United States, impacting over 20% of the population. In at least a quarter of these patients, symptoms caused by GERD such as heartburn and chest pain cannot be controlled by gastric acid suppression with proton pumps inhibitors (PPIs). This population of patients with PPI-refractory GERD have no effective medical treatment options. Failure to understand mechanisms of persistent symptoms in this condition is a major knowledge gap, and limits clinical therapeutic options. One potential cause of persistent symptoms in PPI-refractory GERD patients is weakly acidic (pH>5.0) reflux. This can cause activation of pain- mediating nerves (C-fiber nociceptors) in esophageal mucosa, but the molecular mechanisms have not been clearly elucidated. Furthermore it is not clear whether bile acid also directly activates esophageal C-fibers and mediates nociceptive symptoms in GERD. Our overall hypothesis is that the refluxates in PPI-refractory GERD (mild acid and bile acids) activate esophageal nociceptive C-fiber subtypes and contribute to persistent symptoms in refractory GERD. Determining the identity of the afferent nerve subtype(s) and the key receptor(s) responsible for PPI refractory symptoms are crucial for development of specific targets for this population. Over the past decade, we have identified 3 types of C-fibers in the esophagus which express differential receptors and neurotransmitters and have different central projections. Intriguingly, we have developed new data which demonstrated that two factors in weakly acidic refluxate robustly stimulate esophageal C-fibers: 1) bile acids (that are quite often found in the refluxates on PPI therapy), and 2) acid in low proton concentrations (pH=6.0, termed here mild acid). Our published papers and preliminary data suggest that the key receptors TGR5 and TASK1/ASIC3 are likely involved. Based on these progresses, we will first determine in aim 1 the subtypes of nociceptive afferent nerve terminals in esophagus mucosa which are more vulnerable to refluxates. We will then elucidate receptors mediating C-fiber activation by mild acid in aim 2. We will address the hypothesis that mild acid activates C-fiber subtypes via combination of inhibition of potassium two-pore domain (K2P) family channel (TASK1) and activation of cationic channels from the ASIC family (ASIC3). In aim 3, we will elucidate receptors mediating C-fiber activation by bile acids. We hypothesize that bile acids activate C- fiber subtype(s), which is mediated by the bile acid receptor TGR5. We will apply the approaches of gene expression analysis, patch clamp recordings, and recordings of nerve activity originating in the C-fiber nerve terminals. Studies will be carried out in in animal models in which neuron-selective gene knockdown by our validated in vivo shRNA silencing strategy. Elucidation of the mechanisms by which bile acids and mild acid activate C-fibers will help to develop novel targets for PPI--refractory GERD.