Calcium Dynamics in Interstitial Cells of Cajal

Cajal 间质细胞中的钙动态

• 批准号: 10425251
• 负责人: GIANRICO FARRUGIA
• 金额: $ 35.78万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10425251
• 关键词: Address; Bicarbonates; Binding; Calcium; Cells; Complex; Constipation; Data; Disease; Electrophysiology (science); Enteral; Epigenetic Process; Exons; Frequencies; GLI Family Protein; Gastrointestinal Motility; Gastrointestinal Transit; Gastrointestinal tract structure; Gastroparesis; Genes; Genetic; Genetic Transcription; Grant; Health; Human; Immune system; Interstitial Cell of Cajal; Intestines; Ion Channel; Ions; Irritable Bowel Syndrome; Knockout Mice; Link; Measurement; Mediating; Methods; Microscopy; Mus; Muscle; Nerve; Nervous system structure; Neuroglia; Pacemakers; Pattern; Pharmacology; Physiological; Play; Predisposition; Property; Publishing; Regulation; Resolution; Role; Series; Single Nucleotide Polymorphism; Small Intestines; Smooth Muscle; Speed; Techniques; Testing; Transcription Initiation Site; Transcriptional Regulation; Up-Regulation; Variant; Wild Type Mouse; Work; Zinc Fingers; base; cell motility; cell type; density; differential expression; experimental study; extracellular; gastrointestinal; innovation; motility disorder; mouse model; novel; prevent; promoter; symporter

PROJECT SUMMARY/ABSTRACT The extrinsic nervous system, enteric nerves, glia, the immune system, interstitial cells of Cajal (ICC) and smooth muscle all need to work in concert to enable coordinated motility. The Ca2+ activated Cl- channel, Ano1 is expressed in ICC and is required for ICC electrical activity. In the previous grant cycles it was shown that Ano1 regulates proliferation of ICC, that loss of Ano1 is associated with GI motility disorders, that spontaneous Ca2+ transients in the myenteric ICC network are usually coordinated but are altered and highly uncoordinated in Ano1 knockout mice, and the promoter for Ano1 was identified. How Ano1 regulates Ca2+ transients and how Ano1 is regulated is not known but what is known, though underappreciated, is that Ano1 also conducts HCO3-. The overarching novel hypothesis for this competitive renewal is that Ano1, acting with the transporter Slc4a4, regulates ICC Ca2+ transients that in turn regulate electrical activity and that transcriptional regulation of Ano1 levels in ICC by the zinc finger protein Gli physiologically regulates contractile activity. The hypothesis will be tested in two specific aims (SA). SA1 will determine how pacemaker function in myenteric ICC is regulated by pH through transport of bicarbonate ions by Ano1 and Slc4a4 (NBCe1) and SA2 will determine how transcriptional regulation of Ano1 expression by Gli contributes to normal intestinal pacemaking and GI motility. The SAs are supported by extensive preliminary data. 1) Changing extracellular HCO3- alters intracellular Ca2+ transients but not when Ano1 is blocked; 2) Pacemaker ICC differentially express the electrogenic Na+,HCO3- co-transporter Slc4a4 (NBCe1) variant c; 3) Genetic and pharmacologic block of Ano1 and Slc4a4 alter intracellular Ca2+ transients through entry of HCO3- and modulation of pH; 4) Gli1 and Gli2 bind directly to the promoter of Ano1; 5) Ano1 expression is inversely correlated with Gli levels; 6) Inhibition of Gli markedly increases Ano1 currents; 7) Upregulation of Gli alters downstream smooth muscle electrical activity, contractile patterns and GI transit; and 8) A single nucleotide variant prevents Gli binding to the Ano1 promoter and is linked to susceptibility to irritable bowel syndrome (IBS). To address the overarching hypothesis a variety of methods will be used, spanning from the use of genetically encoded indicator molecules, high speed and high resolution microscopy techniques, intracellular pH measurement, several genetically modified mouse models, electrophysiology, and improvements in the ability to obtain genetic and epigenetic sequencing information from well identified cells. Additional new expertise in genetics, transcriptional regulation, epigenetics and pH regulation has been integrated with the established team to take an innovative approach to understanding GI pacemaking in health and disease. As a result of previous work, the preliminary data, and the proposed experiments, a significant advance in the understanding of the regulation of Ano1 and how Ano1 regulates Ca2+ transients will be achieved with implications in both health and disease within and outside the GI tract.

项目总结/摘要 外源性神经系统、肠神经、神经胶质、免疫系统、Cajal间质细胞（ICC）和 平滑肌都需要协同工作以实现协调运动。Ca 2+激活的Cl-通道Ano 1 以ICC表示，是ICC电活动所必需的。在以往的赠款周期中， Ano 1调节ICC的增殖，Ano 1的缺失与GI动力障碍有关， 肌间ICC网络中的Ca 2+瞬变通常是协调的，但也会改变和高度不协调 在Ano 1敲除小鼠中，并鉴定了Ano 1的启动子。Ano 1如何调节Ca 2+瞬变和 Ano 1是如何被调节的尚不清楚，但已知的是，尽管未被充分认识，Ano 1也传导 HCO3-。对于这种竞争性更新，最重要的新假设是，Ano 1与 转运蛋白Slc 4a 4调节ICC Ca 2+瞬变，进而调节电活动， 通过锌指蛋白Gli生理调节ICC中Ano 1水平的转录调节 收缩活动该假设将在两个特定目标（SA）中进行测试。SA 1将决定如何 肌间ICC中的起搏器功能由pH通过Ano 1转运碳酸氢根离子来调节， Slc 4a 4（NBCe 1）和SA 2将决定Gli如何对Ano 1表达进行转录调控， 正常的肠道起搏和胃肠道动力。SA得到了大量初步数据的支持。第一章 改变细胞外HCO 3-改变细胞内Ca 2+瞬变，但当Ano 1被阻断时不改变; 2）起搏器 ICC差异表达产电Na+，HCO 3-共转运蛋白Slc 4a 4（NBCe 1）变体c; 3）遗传和 Ano 1和Slc 4a 4的药理学阻断通过HCO 3-的进入改变细胞内Ca 2+瞬变， pH的调节; 4）Gli 1和Gli 2直接结合Ano 1的启动子; 5）Ano 1的表达呈反向 与Gli水平相关; 6）Gli的抑制显著增加Ano 1电流; 7）Gli的上调改变了 下游平滑肌电活动、收缩模式和GI转运;和 一种变体阻止Gli与Ano 1启动子结合，并与肠易激综合征易感性有关 （国际商业银行）。为了解决总体假设，将使用各种方法，从使用 遗传编码指示分子，高速和高分辨率显微镜技术，细胞内 pH测量，几种转基因小鼠模型，电生理学，以及 从良好鉴定的细胞获得遗传和表观遗传测序信息的能力。额外的新 在遗传学，转录调控，表观遗传学和pH调节方面的专业知识已与 建立了一个团队，采取创新的方法来理解健康和疾病中的GI起搏。作为 以前的工作，初步数据和拟议的实验的结果，一个显着的进步， 了解Ano 1的调节以及Ano 1如何调节Ca 2+瞬变将通过以下方式实现 对胃肠道内外的健康和疾病都有影响。