Calcium Dynamics in Interstitial Cells of Cajal

Cajal 间质细胞中的钙动态

• 批准号: 8063209
• 负责人: GIANRICO FARRUGIA
• 金额: $ 32.4万
• 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 2014-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8063209
• 关键词: Adult; Antibodies; Apoptosis; Bioinformatics; CDC2 Protein Kinase; Calcium; Cell Culture Techniques; Cells; Complex; Data; Disease; Down-Regulation; Electrophysiology (science); Electroporation; Enteral; Event; Figs - dietary; Future; Gastrointestinal Motility; Gastrointestinal Stromal Tumors; Gastrointestinal tract structure; Grant; Image; Immunohistochemistry; Interstitial Cell of Cajal; Ion Channel; Knock-out; Knockout Mice; Lead; Maintenance; Microarray Analysis; Microelectrodes; Morbidity - disease rate; Mus; Muscle; Muscle function; Nerve; Organ; Paper; Patch-Clamp Techniques; Pathologist; Pathway interactions; Physiology; Process; Proliferating; Proteins; Proteomics; Publishing; Recombinants; Regulation; Reverse Transcriptase Polymerase Chain Reaction; Role; Serotonin; Serotonin Receptor 5-HT2B; Signal Transduction; Smooth Muscle; Subfamily lentivirinae; System; Techniques; Testing; Therapeutic Intervention; Tissues; Transfection; Western Blotting; Wild Type Mouse; Work; base; cell type; channel blockers; clinically significant; inhibitor/antagonist; innovation; knock-down; mortality; motility disorder; novel; public health relevance; reconstruction; research study; selective expression; small hairpin RNA; tumor

DESCRIPTION (provided by applicant): The control of gastrointestinal (GI) motility requires a complex interaction between several cell types such as smooth muscle and enteric nerves. In recent years we have added interstitial cells of Cajal (ICC) to this complex system. ICC regulate smooth muscle function and loss of ICC is associated with several motility disorders. ICC networks are constantly turning over and maintenance of the networks requires tight regulation of ICC proliferation. Ano-1, a Ca2+ activated Cl- channel has recently been found to be selectively expressed on ICC in the muscle layers of the gut as well as in several tumors, including gastrointestinal stromal tumors. The overall objective of this proposal is to determine the role of Ano1 in ICC function. The central hypothesis of this proposal is that Ano-1 has dual, novel roles in ICC physiology. It acts as a key molecule in the regulation of ICC proliferation and as an ion channel required for the repolarization of the slow wave. The central hypothesis will be tested in two specific aims. Specific Aim 1 will test the hypothesis that Ano1 regulates proliferation of ICC and Specific aim 2 will test the hypothesis that Ano1 is required for repolarization of the slow wave. The first specific aim is supported by preliminary data that show that Ano1 in the tunica muscularis is expressed only on ICC, that Ano1 may be used to detect loss of ICC in motility disorders, that a lack of Ano1 is associated with a decrease in proliferation of ICC and that an increase in expression of Ano-1 is associated with an increase in ICC proliferation. The preliminary data also provide evidence, from bioinformatics analysis of microarray data comparing knockout to wild type mice, that key molecules associated with the control of proliferation, including CDK1/cdc2, are down-regulated when Ano1 is absent. The second specific aim is supported by preliminary data that show that Cl- channel blockers alter the duration of the plateau of the slow wave, that the slow wave is prolonged when Ano-1 expression is decreased and that it normalizes after Ano-1 is expressed. The PI will test the central hypothesis by a combination of innovative techniques including 3D reconstructions of immunohistochemical data, Western blots, RT-PCR, single cell PCR, quantitative PCR, lentivirus and si/shRNA knock down techniques, electroporation, knockout mice, organotypic and single cell cultures, bioinformatic analysis of microarray data, proteomics, Ca2+ and Cl- imaging, microelectrode recordings and patch clamp techniques. Successful completion of the proposed studies has both basic significance and clinical impact. Our work on the newly discovered protein Ano1 will provide mechanistic information on both how ICC networks are maintained and on the regulation of the slow wave. Of immediate clinical significance our work will inform clinicians and pathologists on use of antibodies to Ano1 to assess ICC in motility disorders and provide targets for future therapeutic interventions. Our work also has broad implications beyond the GI tract. As Ano-1 is expressed in many organs and tumors including gastrointestinal stromal tumors, our findings will likely apply to several other organs outside of the GI tract. PUBLIC HEALTH RELEVANCE: Gastrointestinal motility disorders are common and associated with considerable morbidity and mortality. A recent finding is that a key cell type, the interstitial cells of Cajal (ICC) is required for normal gastrointestinal motility and is lost in several of these disorders. This proposal will investigate the role of a newly discovered protein, Ano1, in the control of ICC function, including the control of ICC numbers, which may lead to new strategies to replace the lost or damaged ICC and restore normal gastrointestinal motility.

描述（由申请人提供）：胃肠道（GI）运动的控制需要多种细胞类型（例如平滑肌和肠神经）之间复杂的相互作用。近年来，我们将卡哈尔间质细胞 (ICC) 添加到这个复杂的系统中。 ICC 调节平滑肌功能，ICC 丧失与多种运动障碍有关。 ICC 网络不断更新，网络的维护需要对 ICC 扩散进行严格监管。 Ano-1 是一种 Ca2+ 激活的 Cl- 通道，最近被发现在肠道肌肉层以及多种肿瘤（包括胃肠道间质瘤）的 ICC 上选择性表达。该提案的总体目标是确定 Ano1 在 ICC 功能中的作用。该提案的中心假设是 Ano-1 在 ICC 生理学中具有双重、新颖的作用。它是调节 ICC 增殖的关键分子，也是慢波复极化所需的离子通道。中心假设将在两个具体目标中进行检验。具体目标 1 将检验 Ano1 调节 ICC 增殖的假设，具体目标 2 将检验 Ano1 是慢波复极化所必需的假设。第一个具体目标得到初步数据的支持，初步数据表明肌层中的 Ano1 仅在 ICC 上表达，Ano1 可用于检测运动障碍中 ICC 的缺失，Ano1 的缺乏与细胞增殖的减少有关。 ICC 且 Ano-1 表达的增加与 ICC 增殖的增加相关。通过对基因敲除小鼠和野生型小鼠进行比较的微阵列数据的生物信息学分析，初步数据还提供了证据表明，当 Ano1 缺失时，与增殖控制相关的关键分子（包括 CDK1/cdc2）会下调。第二个具体目标得到了初步数据的支持，初步数据表明，Cl-通道阻滞剂改变了慢波平台期的持续时间，当 Ano-1 表达减少时，慢波延长，并且在 Ano-1 表达后，慢波恢复正常。 。 PI 将通过创新技术的组合来测试中心假设，包括免疫组织化学数据的 3D 重建、蛋白质印迹、RT-PCR、单细胞 PCR、定量 PCR、慢病毒和 si/shRNA 敲除技术、电穿孔、基因敲除小鼠、器官型和单细胞培养、微阵列数据的生物信息分析、蛋白质组学、Ca2+ 和 Cl- 成像、微电极记录和膜片钳技术。拟议研究的成功完成既具有基础意义，又具有临床影响。我们对新发现的蛋白质 Ano1 的研究将提供有关 ICC 网络如何维持和慢波调节的机制信息。我们的工作具有直接的临床意义，将告知临床医生和病理学家如何使用 Ano1 抗体来评估运动障碍中的 ICC，并为未来的治疗干预提供目标。我们的工作还具有胃肠道以外的广泛影响。由于 Ano-1 在许多器官和肿瘤中表达，包括胃肠道间质瘤，因此我们的发现可能适用于胃肠道以外的其他几个器官。 公共卫生相关性： 胃肠动力障碍很常见，并且与相当大的发病率和死亡率相关。最近的一项发现是，一种关键的细胞类型——卡哈尔间质细胞 (ICC) 是正常胃肠运动所必需的，但在其中几种疾病中会丢失。该提案将研究一种新发现的蛋白质Ano1在控制ICC功能中的作用，包括控制ICC数量，这可能会带来新的策略来替代丢失或损坏的ICC并恢复正常的胃肠道运动。