Role of TRP channels in human detrusor function and dysfunction

 描述(申请人提供)：膀胱过度活动(OAB)，通常与逼尿肌过度活动(DO)有关，是一种使人衰弱的病理疾病，影响超过3400万人 美国人，仅在美国每年的成本就超过650亿美元。目前的OAB疗法在疗效和缓解方面都是有限的。迫切需要新的治疗方法，特别是那些直接靶向逼尿肌(DSM)的治疗方法，以促进DSM在膀胱充盈和储尿过程中的松弛。我们首次在人的DSM中鉴定了功能性瞬时受体电位(Trp)-Melastatin-4(TRPM4)通道，并且我们发现与血管系统相比，TRPM4通道在DSM中的表达要高得多。这个项目将检验一个新的概念，即钙激活的TRPM4通道在人类DSM的兴奋性和收缩能力中起关键作用，而抑制TRPM4通道会降低DSM的收缩能力。我们的中心假设是TRPM4通道在人类DSM生理学和DO/OAB的病因中起关键作用，因此是DO/OAB治疗的一个新的治疗靶点。我们的策略是在分子、细胞、组织和整个生物体水平上使用多层次的实验方法。我们有独特的优势，定期使用来自开放膀胱手术的临床特征的人类DSM组织作为对照(非OAB)和OAB患者。目的1采用单细胞逆转录聚合酶链式反应、定量聚合酶链式反应、Western印迹杂交、原位邻近连接分析、免疫组织化学和免疫组织化学共聚焦分析等多种方法，对正常对照和OAB患者的DSM细胞中TRPM4通道的表达和定位进行研究。目的2阐明TRPM4通道在正常对照和OAB患者新鲜分离(未培养)的人DSM细胞兴奋性中的作用及其与IP3受体的功能联系。这一目标将使用先进的电生理学方法、活细胞钙成像和新型选择性TRPM4通道抑制剂9-菲罗啉。目的3阐明TRPM4通道在正常人、OAB患者和小鼠体内人DSM组织肌源性和神经诱发收缩中的功能作用。我们认为，TRPM4通道是治疗DSM功能障碍的新靶点，它们的药理调节为治疗膀胱疾病提供了一种以前未知的机制。靶向TRPM4通道的选择性抑制剂有可能减少DO和缓解OAB。因此，本研究具有重要的临床意义。基础科学家和泌尿科医生之间的合作加强了这项转化研究的高度创新性，允许将分子(目标1)、细胞(目标2)、组织和体内(目标3的发现)联系起来。该项目完成后，将揭示TRPM4通道在生理和病理生理条件下对人DSM的调节。从这个项目中获得的知识将促进我们对人类DSM功能和功能障碍调控机制的理解，并验证TRPM4通道作为OAB的一种新的治疗方式。

 DESCRIPTION (provided by applicant): Overactive bladder (OAB), often associated with detrusor overactivity (DO), is a debilitating pathological condition affecting more than 34 million Americans, at an annual cost of more than $65 billion in the US alone. Current OAB therapies are limited in efficacy and relief. Novel therapeutic approaches are urgently needed, especially those that directly target detrusor smooth muscle (DSM), in order to facilitate DSM relaxation during bladder filling and urinary storage. We are the first to identify functional Transient Receptor Potential (TRP) melastatin-4 (TRPM4) channels in human DSM and we have shown that TRPM4 channel expression is much higher in DSM as compared to the vasculature. This project will examine the novel concept that the Ca2+- activated TRPM4 channels have a key role in human DSM excitability and contractility and that inhibition of TRPM4 channels decreases DSM contractility. Our central hypothesis is that TRPM4 channels play critical roles in human DSM physiology and the etiology of DO/OAB, and therefore represent a novel therapeutic target for DO/OAB treatment. Our strategy is to use a multi-level experimental approach at molecular, cellular, tissue, and whole organism levels. We have the unique advantage of regularly using clinically-characterized human DSM tissues from open bladder surgeries on control (non-OAB) and OAB patients. Aim 1 will use multiple state-of-the-art approaches including single-cell RT-PCR, qPCR, Western blot, in situ proximity ligation assay, confocal immunohistochemical and immunocytochemical analyses of DSM whole tissue and single cells to identify the TRPM4 channel expression and localization in human DSM cells from control and OAB patients. Aim 2 will elucidate the role of TRPM4 channels in the excitability of freshly- isolated (not cultured) native human DSM cells from control and OAB patients and their functional link with IP3 receptors. This aim will use advanced electrophysiological approaches, live-cell Ca2+ imaging, and the novel selective TRPM4 channel inhibitor, 9-phenanthrol. Aim 3 will elucidate the functional roles of TRPM4 channels in myogenic and nerve-evoked contractions of human DSM tissues from control and OAB patients, and in vivo in mice. We propose that TRPM4 channels are novel targets for DSM dysfunction and their pharmacological modulation offers a previously unexplored mechanism for treating bladder disorders. Targeting TRPM4 channels with selective inhibitors has the potential to reduce DO and alleviate OAB. Therefore, this investigation has important clinical significance. The highly innovative nature of this translational research is strengthened by the collaboration between basic scientists and urologists, allowing the linking of molecular (Aim 1), cellular (Aim 2), tissue, and in vivo (Aim 3 findings. Upon completion of this project, the regulation of human DSM by TRPM4 channels under physiological and pathophysiological conditions will be revealed. The knowledge gained from this project will advance our understanding of the mechanisms regulating human DSM function and dysfunction and validate TRPM4 channels as a novel therapeutic modality for OAB.