Control of motility in the lower esophageal sphincter

项目主任/首席调查员(最后、第一、中间)：科宾、卡罗琳、A 项目摘要 下食道括约肌(LES)是位于食道远端和下段之间的肌肉增厚区域。 近端胃。LES产生收缩的色调，形成保护屏障，限制酸性物质的含量。 防止胃被反流到食道并损害食道。莱斯的语气被认为是“肌源性的”， 但LES肌肉不仅仅由平滑肌细胞(SMC)和神经元组成。更确切地说，是间质细胞 Cajal(ICC)与SMC电偶联，调节SMC的兴奋性。例如，我们发现 对氮能输入的反应是吞咽后放松的重要调节因素，在患有 失去ICC(W/WV小鼠)。这一概念受到了批评，因为W/WV小鼠的测压测量 显示出氮能松弛的证据。这些研究中的一个重要观察结果，但不是调查或 从机制上讨论，W/WV小鼠的LES是低血压的，提示ICC为 在LES中的重要功能，如音调的发展和维持。这个项目旨在更好地 了解LES声调的产生机制。初步研究结果表明，依赖于钙离子的氯离子激活 信道(由Ano1编码的CACC)在LES中由ICC最高表达，负责显著的 这些肌肉中的部分肌张力。在ICC中，自发的钙瞬变激活了ANO1通道。这个 钙激活的内向电流导致合胞体去极化，并增加开放概率。 SMC上的电压依赖性钙通道。这一机制的收缩程度似乎也是 受一种称为钙敏化的机制调节，这一过程在以前的论文中提出，但在之前的 这些发现可能会受到其他解释的影响。在这里，我们建议进行以下调查：1.确定 负责机电和药物机械调节的分子和功能装置 2.研究来自内源性神经元的输入是如何调制电、机械和电刺激的 LES音调背后的药理机制。这项研究将利用各种尖端技术 转基因小鼠、钙离子瞬变的光遗传监测和生物化学分析等技术 增强或抑制SMC内钙敏感性的磷酸化步骤。许多这样的临床前研究 将在老鼠身上进行，但我们也将在食蟹猴身上进行平行研究，食蟹猴是一种具有 与人类有显著的基因同源性。 OMB编号0925-0001/0002(批准的第03/2020版至2023年2月28日)续页格式页

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