Calcium Dynamics in Interstitial Cells of Cajal

描述（由申请人提供）：胃肠（GI）运动的控制需要几种细胞类型（如平滑肌和肠神经）之间的复杂相互作用。近年来，我们在这个复杂的系统中加入了Cajal间质细胞（ICC）。ICC调节平滑肌功能，ICC的丧失与几种运动障碍有关。国际刑事法院网络不断更替，网络的维护需要对国际刑事法院的扩散进行严格管制。Ano-1是一种Ca ~（2+）激活的Cl ~-通道，最近发现其选择性地表达于肠道肌层以及包括胃肠道间质瘤在内的几种肿瘤中的ICC上。本提案的总体目标是确定Ano 1在ICC功能中的作用。该提议的中心假设是Ano-1在ICC生理学中具有双重的新颖作用。它是调控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得到检验。具体目标1将检验Ano 1调节ICC增殖的假设，具体目标2将检验Ano 1是慢波复极化所需的假设。第一个具体的目的是支持的初步数据表明，Ano 1的图尼卡肌层中只表达在ICC上，Ano 1可用于检测运动障碍的ICC的损失，缺乏Ano 1与ICC的增殖减少，并增加Ano-1的表达与ICC增殖的增加。初步数据还提供了证据，从生物信息学分析的微阵列数据比较敲除野生型小鼠，与控制增殖相关的关键分子，包括CDK 1/cdc 2，下调时Ano 1是缺席。第二个具体目标得到初步数据的支持，这些数据表明Cl-通道阻滞剂改变了慢波平台期的持续时间，当Ano-1表达降低时慢波延长，并且在Ano-1表达后慢波恢复正常。PI将通过结合创新技术来检验中心假设，包括免疫组化数据的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、敲除小鼠、器官型和单细胞培养、微阵列数据的生物信息学分析、蛋白质组学、Ca 2+和Cl-成像、微电极记录和膜片钳技术。成功完成拟定研究具有基础意义和临床影响。我们对新发现的蛋白质Ano 1的研究将为ICC网络的维持和慢波的调节提供机制信息。具有直接临床意义的是，我们的工作将告知临床医生和病理学家使用Ano 1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。我们的工作也有广泛的影响，超越了胃肠道。由于Ano-1在许多器官和肿瘤中表达，包括胃肠道间质瘤，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： 胃肠动力障碍是常见的，并与相当大的发病率和死亡率。最近的一项发现是，一种关键的细胞类型，Cajal间质细胞（ICC）是正常胃肠运动所必需的，并且在这些疾病中的几种中丢失。该提案将研究一种新发现的蛋白Ano 1在控制ICC功能中的作用，包括控制ICC数量，这可能会导致新的策略来取代丢失或受损的ICC并恢复正常的胃肠动力。

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.