Control of motility in the lower esophageal sphincter

项目负责人/主要研究者（最后，第一，中间）：Cobine，Caroline，A 项目摘要 下食管括约肌（LES）是远端食管和远端食管之间的增厚的肌肉区域。 胃近端LES产生收缩张力，形成保护屏障并限制酸性内容物 防止胃回流到食道造成损伤LES音调被认为是“肌源性的”， 但是LES肌肉不仅仅由平滑肌细胞（SMC）和神经元组成。相反， Cajal（ICC）与SMC电偶联并调节SMC的兴奋性。例如，我们发现， 对氮能输入的反应，一种吞咽后放松的重要调节剂，在患有以下疾病的动物中减少： ICC损失（W/WV小鼠）。这一概念受到了批评，因为W/WV小鼠的测压测量 显示出氮能松弛的迹象这些研究中的一个重要观察结果，但没有调查或 在机制方面讨论的是，W/WV小鼠的LES是膨胀的，这表明ICC提供了 LES中的重要功能，如音调的发展和维持。该项目旨在更好地 了解LES音调的机制。初步研究结果表明，激活钙依赖的Cl- 在LES中由ICC最高表达的CaCC通道（由Ano 1编码）负责显著的 这部分肌肉的张力。Ano 1通道被ICC中自发的Ca 2+瞬变激活。的 由Ca ~（2+）激活的内向电流引起合胞体的去极化，并增加合胞体的开放概率。 SMC中的电压依赖性Ca 2+通道。这种机制的收缩程度似乎也是 由称为Ca 2+敏化的机制调节，这一过程在以前的论文中提出，但以前的 调查结果可能有其他解释。在这里，我们建议调查以下内容：1。确定 负责机电和药物机械调节的分子和功能装置 在LES中的音调，2。研究来自内在神经元的输入如何调节电机械和 LES张力的药理机制。这项研究将利用各种尖端技术， 技术，如转基因小鼠，Ca 2+瞬变的光遗传学监测和 磷酸化步骤增加或抑制SMC中的Ca 2+敏感性。许多这些临床前研究 将在小鼠上进行，但我们也将在食蟹猴LES上进行平行研究， 与人类有很大的遗传同源性 OMB编号0925-0001/0002（修订版03/2020批准至02/28/2023）页码继续格式页码

Program Director/Principal Investigator (Last, First, Middle): Cobine, Caroline, A Project Summary The lower esophageal sphincter (LES) is a thickened region of muscle between the distal esophagus and proximal stomach. The LES develops contractile tone that forms a protective barrier and restricts acidic contents of the stomach from being refluxed into and damaging the esophagus. LES tone is thought to be ‘myogenic’, but LES muscles are not just composed of smooth muscle cells (SMCs) and neurons. Rather, interstitial cells of Cajal (ICC) are electrically coupled to SMCs and regulate the excitability of SMCs. For example, we found that responses to nitrergic input, an important regulator of relaxation after swallowing, are reduced in animals with loss of ICC (W/WV mice). This concept was criticized because manometric measurements in W/WV mice displayed evidence of nitrergic relaxation. An important observation in these studies, but not investigated or discussed in terms of mechanism, was that the LES in W/WV mice is hypotensive, suggesting that ICC provide important functions in the LES, such as development and maintenance of tone. This project seeks to better understand the mechanism of LES tone. Preliminary findings suggest that activation of Ca2+-dependent Cl- channels (CaCC encoded by Ano1), most highly expressed by ICC in the LES, are responsible for a significant portion of tone in these muscles. Ano1 channels are activated by spontaneous Ca2+ transients in ICC. The inward current activated by Ca2+ causes depolarization of the syncytium and increases the open probability of voltage-dependent Ca2+ channels in SMCs. The degree of contraction by this mechanism appears also to be regulated by a mechanism known as Ca2+ sensitization, a process suggested in previous papers, but previous findings are subject to other explanations. Here we propose to investigate the following: 1. Determine the molecular and functional apparatus responsible for the electromechanical and pharmacomechanical regulation of tone in the LES, 2. Examine how inputs from intrinsic neurons modulate the electromechanical and pharmacomechanical mechanisms underlying LES tone. This study will utilize a variety of cutting-edge techniques, such as transgenic mice, optogenetic monitoring of Ca2+ transients and biochemical analysis of phosphorylation steps that either increase or inhibit Ca2+ sensitivity in SMCs. Many of these pre-clinical studies will be performed on mice, but we will also perform parallel studies on Cynomolgus monkey LES, a primate with significant genetic homology to humans. OMB No. 0925-0001/0002 (Rev. 03/2020 Approved Through 02/28/2023) Page Continuation Format Page