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.

项目概要/摘要 外在神经系统、肠神经、神经胶质、免疫系统、卡哈尔间质细胞 (ICC) 和 所有平滑肌都需要协同工作才能协调运动。 Ca2+ 激活 Cl- 通道，Ano1 以 ICC 表示，并且是 ICC 电活动所必需的。在之前的拨款周期中表明 Ano1 调节 ICC 的增殖，Ano1 的缺失与胃肠道运动障碍有关，自发性 肌间 ICC 网络中的 Ca2+ 瞬变通常是协调的，但会发生改变且高度不协调 在 Ano1 基因敲除小鼠中，Ano1 的启动子被鉴定出来。 Ano1 如何调节 Ca2+ 瞬变和 Ano1 是如何受到监管的尚不清楚，但已知的是 Ano1 还进行 HCO3-。这种竞争性更新的总体新颖假设是 Ano1，与 转运蛋白 Slc4a4，调节 ICC Ca2+ 瞬变，进而调节电活动， 锌指蛋白 Gli 对 ICC 中 Ano1 水平的转录调节 生理调节 收缩活动。该假设将在两个特定目标（SA）中进行检验。 SA1将决定如何 肌间 ICC 中的起搏器功能通过 Ano1 和碳酸氢根离子的转运受 pH 值调节 Slc4a4 (NBCe1) 和 SA2 将决定 Gli 对 Ano1 表达的转录调控如何促进 正常的肠道起搏和胃肠蠕动。 SA 得到了广泛的初步数据的支持。 1） 改变细胞外 HCO3- 会改变细胞内 Ca2+ 瞬变，但当 Ano1 被阻断时不会改变； 2) 起搏器 ICC 差异表达产电 Na+,HCO3- 协同转运蛋白 Slc4a4 (NBCe1) 变体 c； 3）遗传和 Ano1 和 Slc4a4 的药理学阻断通过 HCO3- 和 HCO3- 的进入改变细胞内 Ca2+ 瞬变 pH值调节； 4）Gli1和Gli2直接与Ano1的启动子结合； 5) Ano1表达式为反 与 Gli 水平相关； 6) 抑制Gli显着增加Ano1电流； 7) Gli改变的上调 下游平滑肌电活动、收缩模式和胃肠道转运； 8) 单个核苷酸 变体阻止 Gli 与 Ano1 启动子结合，并与肠易激综合征的易感性相关 （肠易激综合症）。为了解决总体假设，将使用多种方法，包括使用 基因编码指示分子、高速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 从明确识别的细胞中获取遗传和表观遗传测序信息的能力。额外新增 遗传学、转录调控、表观遗传学和 pH 调控方面的专业知识已与 建立的团队采用创新方法来了解健康和疾病中的胃肠道起搏。作为一个 先前工作的结果、初步数据和拟议的实验，在 了解 Ano1 的调节以及 Ano1 如何调节 Ca2+ 瞬变将通过以下方式实现 对胃肠道内外的健康和疾病的影响。