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）运动的控制需要几种细胞类型（例如平滑肌和肠神经）之间的复杂相互作用。近年来，我们将Cajal（ICC）的间质细胞添加到了这个复杂的系统中。 ICC调节平滑肌功能和ICC的丧失与多种运动障碍有关。 ICC网络不断翻转，网络的维护需要严格对ICC扩散的调节。 ANO-1，Ca2+活化的Cl-通道最近被发现在肠道肌肉层以及包括胃肠道间质肿瘤在内的几种肿瘤中有选择地表达在ICC上。该提案的总体目的是确定ANO1在ICC函数中的作用。该提议的中心假设是ANO-1在ICC生理学中具有双重新作用。它是ICC增殖调节的关键分子，也是慢波重极化所需的离子通道。中心假设将以两个具体的目的进行检验。具体目标1将检验以下假设：ANO1调节ICC的扩散，并且特定目标2将检验以下假设：慢波重极化需要ANO1。第一个具体目的是由初步数据支持的，该数据表明，肌肉曲霉中的ANO1仅在ICC上表达，ANO1可用于检测运动障碍中ICC的损失，缺乏ANO1与ICC的增殖降低有关，并且ICC的增殖降低，并且ANO-1的表达与ICC刺激量的增加有关。初步数据还提供了证据，从对敲除与野生型小鼠进行比较的微阵列数据的生物信息学分析，当不存在ANO1时，与包括CDK1/CDC2在内的增殖相关的关键分子（包括CDK1/CDC2）相关。第二个特定目的是由初步数据支持的，该数据表明Cl-通道阻滞剂会改变慢波平稳的持续时间，当ANO-1表达降低时，慢波会延长，并且在表达ANO-1后它归一化。 PI将通过创新技术（包括3D重建免疫组织化学数据，Western blots，RT-PCR，单细胞PCR，单细胞PCR，定量PCR，慢病毒和SI/SHRNA敲击技术，电型技术，电型小鼠和单个细胞培养数据，Mic Mickotic Clor-note Clotial Clor-note clo），Mic Mickotic Clotig，Mic Mickotic Clotic，Mic Mickotic Clogiation，Mickotic clotic，Mic Mickotic Clotic，Mic Mickotic Clotor-nike+成像，微电极记录和斑块夹技术。成功完成拟议的研究具有基本意义和临床影响。我们在新发现的蛋白质ANO1上的工作将提供有关ICC网络如何维持和慢波调节的机械信息。具有直接的临床意义，我们的工作将为临床医生和病理学家提供使用与ANO1抗体评估运动障碍中ICC的抗体，并为将来的治疗干预提供目标。我们的作品还具有超越胃肠道的广泛含义。由于ANO-1在包括胃肠道肿瘤在内的许多器官和肿瘤中都表达，因此我们的发现可能适用于GI区域以外的其他几个器官。 公共卫生相关性： 胃肠道运动障碍很常见，并且与大量发病率和死亡率有关。最近的发现是，正常胃肠道运动需要一种关键细胞类型，即Cajal（ICC）的间隙细胞（ICC），并且在其中几种疾病中丢失。该建议将研究新发现的蛋白ANO1在ICC功能控制中的作用，包括控制ICC数字，这可能会导致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。