Role of TRP channels in human detrusor function and dysfunction

TRP 通道在人类逼尿肌功能和功能障碍中的作用

• 批准号: 9122401
• 负责人: Georgi V Petkov
• 金额: $ 33.28万
• 依托单位: UNIVERSITY OF SOUTH CAROLINA AT COLUMBIA
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9122401
• 关键词: Affect; Age; American; Animal Model; Applications Grants; Biological Assay; Bladder; Bladder Diseases; Bladder Dysfunction; Cells; Clinical; Clinical Research; Collaborations; Data; Disease; Electrophysiology (science); Etiology; Functional disorder; Future; Goals; Health; Health Care Costs; Human; Image; Imaging Techniques; In Situ; In Vitro; Investigation; Ion Channel; Isometric Exercise; Knowledge; Laser Scanning Confocal Microscopy; Life; Ligation; Link; Messenger RNA; Methods; Mission; Modality; Molecular; Molecular Biology; Mus; Muscle; Muscle Tension; Muscle function; Muscle relaxation phase; Nature; Nerve; Outcome Study; Overactive Bladder; Paper; Pathologic; Patients; Pharmacotherapy; Physiological; Physiology; Play; Prevalence; Public Health; Regulation; Relaxation; Reporting; Reverse Transcriptase Polymerase Chain Reaction; Rodent; Role; Scientist; Signal Pathway; Smooth Muscle; Smooth Muscle Myocytes; Speed; Staining method; Stains; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transcript; Translational Research; Urine; Urologist; Western Blotting; Whole Organism; Work; base; bladder surgery; clinically significant; cost; drug discovery; improved; in vivo; inhibitor/antagonist; innovation; insight; member; new therapeutic target; novel; novel therapeutic intervention; novel therapeutics; patch clamp; ratiometric; receptor; research study; urinary

 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.

 描述（由申请人提供）：膀胱过度活动症（OAB）通常与逼尿肌过度活动（DO）相关，是一种使人衰弱的病理状况，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有限。迫切需要新的治疗方法，特别是那些直接针对逼尿肌平滑肌（DSM）的方法，以促进膀胱充盈和尿液储存期间的DSM松弛。我们是第一个在人DSM中鉴定功能性瞬时受体电位（TRP）melastatin-4（TRPM 4）通道的人，并且我们已经表明，与脉管系统相比，TRPM 4通道在DSM中的表达要高得多。该项目将研究新的概念，即Ca 2+激活的TRPM 4通道在人类DSM兴奋性和收缩性中具有关键作用，并且TRPM 4通道的抑制会降低DSM收缩性。我们的中心假设是TRPM 4通道在人类DSM生理学和DO/OAB的病因学中起关键作用，因此代表了DO/OAB治疗的新治疗靶点。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水平的实验方法。我们具有独特的优势，可以定期使用来自开放膀胱手术的具有临床特征的人类DSM组织来治疗对照（非OAB）和OAB患者。目的1将使用多种最先进的方法，包括单细胞RT-PCR，qPCR，Western印迹，原位邻位连接分析，共聚焦免疫组织化学和免疫细胞化学分析DSM全组织和单细胞，以确定TRPM 4通道在对照和OAB患者的DSM细胞中的表达和定位。目的2将阐明TRPM 4通道在来自对照和OAB患者的新鲜分离（未培养）的天然人DSM细胞的兴奋性中的作用及其与IP 3受体的功能联系。这一目标将使用先进的电生理方法，活细胞钙离子成像，和新的选择性TRPM 4通道抑制剂，9-菲咯。目的3将阐明TRPM 4通道在对照和OAB患者的人DSM组织以及小鼠体内的肌源性和神经诱发收缩中的功能作用。我们认为TRPM 4通道是DSM功能障碍的新靶点，其药理学调节为治疗膀胱疾病提供了一种以前未探索的机制。用选择性抑制剂靶向TRPM 4通道具有减少DO和缓解OAB的潜力。因此，本研究具有重要的临床意义。这种转化研究的高度创新性通过基础科学家和泌尿科医生之间的合作得到加强，允许分子（目标1），细胞（目标2），组织和体内（目标3发现）的联系。本项目完成后，将揭示TRPM 4通道在生理和病理生理条件下对人类DSM的调控。从该项目中获得的知识将促进我们对调节人类DSM功能和功能障碍的机制的理解，并验证TRPM 4通道作为OAB的新治疗方式。