Role of PIEZO Channels in Bladder Function and Dysfunction

PIEZO 通道在膀胱功能和功能障碍中的作用

• 批准号: 9815767
• 负责人: Gerard L Apodaca
• 金额: $ 65.09万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9815767
• 关键词: Affect; Afferent Neurons; Biological Assay; Bladder; Bladder Dysfunction; Bowman' s space; Cations; Cell membrane; Cells; Communication; Cyclophosphamide; Cystitis; Defect; Epithelial; Esthesia; Event; Exocytosis; Functional disorder; Goals; Guns; Hyperreflexia; Image; Ion Transport; Knockout Mice; Lead; Lower urinary tract; Mechanics; Mediator of activation protein; Membrane; Membrane Protein Traffic; Molecular Conformation; Monitor; Nature; Neuraxis; Overactive Bladder; Pathway interactions; Pelvis; Physiological; Play; Process; Reflex action; Role; Sensory; Signal Transduction; Stimulus; Stratified Epithelium; Stratum Basale; Stretching; Surface; Testing; Time; Tissues; Urodynamics; Urothelial Cell; Urothelium; Viral; afferent nerve; allodynia; gain of function; gain of function mutation; insight; loss of function; mechanotransduction; multidisciplinary; mutant; novel; patch clamp; response; tool

Abstract: During filling and voiding, the degree of tension in the bladder wall must be sensed and then relayed to the central nervous system, otherwise bladder dysfunction ensues. While there is a general understanding of the role of sensory neurons (i.e., wall mechanoceptors) in these events, we have fewer insights into how non-neuronal tissues contribute to tension sensation and transduction in the bladder. Our studies focus on the urothelium, which forms the direct interface between the bladder wall and the urinary space. This tissue responds to changes in tension by modulating its ion transport, membrane traffic, and release of mediators, which are hypothesized to alter bladder function, in part via a local urothelial:afferent reflex. However, we still have limited insights into how tension in the plasma membrane of urothelial cells is sensed, what is the nature of the downstream pathways that are activated in response to stretch, or how do these events contribute to bladder function and dysfunction. We hypothesize that urothelial-expressed PIEZO channels act as mechanosensors that in response to bladder filling promote Ca2+ entry, mediator release, and signaling to afferent nerve processes, promoting normal bladder function. We further hypothesize that dysregulation of PIEZO-dependent mechanotransduction in the urothelium will lead to bladder dysfunction. In Specific Aim 1, we will determine if PIEZO channels act as bona fide mechanosensors by demonstrating the following: (i) that functional PIEZO channels are expressed at the surface of urothelial cells; (ii) that urothelial expressed PIEZO channels respond to physiologically relevant stimuli (i.e., bladder filling); (iii) that PIEZO channels are required for mechanically regulated events including membrane traffic and mediator release; (iv) and that expression of loss-of-function or gain-of-function PIEZO mutants will lead to altered urothelial responses. In Specific Aim 2, we seek to understand how PIEZO channels promote urothelial:afferent signaling. Using novel tools, including ex vivo bladder imaging, we will explore the mechanisms by which PIEZO channels stimulate increases in intracellular Ca2+ ([Ca2+]i). Because PIEZO channels rapidly inactivate, there is likely to be a mechanism to amplify the original signal. Thus, we will define whether Ca2+-induced Ca2+ release or membrane depolarization act downstream of PIEZO channels to increase [Ca2+]i. We will also determine if the PIEZO-triggered rise in [Ca2+]i is critical for stimulating exocytosis and mediator release in the urothelium, and if urothelial PIEZO channels modulate urothelial:afferent signaling. In Specific Aim 3, we will begin to explore whether urothelial PIEZO channels contribute to bladder function and/or dysfunction. Specifically, we will determine whether loss of PIEZO expression or function results in bladder underactivity and if PIEZO channels with gain-of-function mutations lead to bladder overactivity. Finally, we will determine whether urothelial-expressed PIEZO channels contribute to the bladder hyperreflexia and pelvic allodynia associated with cyclophosphamide-induced cystitis.

摘要：在充盈和排尿过程中，必须感知膀胱壁的张力程度，然后 传递到中枢神经系统，否则会导致膀胱功能障碍。虽然有一般 了解感觉神经元（即壁机械感受器）在这些事件中的作用，我们对这些事件的了解较少 深入了解非神经元组织如何促进膀胱的紧张感和转导。我们的 研究重点是尿路上皮，它形成膀胱壁和泌尿系统之间的直接界面 空间。该组织通过调节其离子传输、膜运输和张力来响应张力的变化。 介质的释放，据推测可以部分通过局部尿路上皮：传入神经改变膀胱功能 反射。然而，我们对尿路上皮细胞质膜张力的了解仍然有限。 感知到，响应拉伸而激活的下游通路的本质是什么，或者如何 这些事件会导致膀胱功能和功能障碍。我们假设尿路上皮表达的 PIEZO 通道充当机械传感器，响应膀胱充盈促进 Ca2+ 进入、介质释放和 向传入神经过程发出信号，促进正常膀胱功能。我们进一步假设 尿路上皮中 PIEZO 依赖性机械转导的失调将导致膀胱功能障碍。在 具体目标 1，我们将通过演示来确定 PIEZO 通道是否充当真正的机械传感器 以下：（i）功能性 PIEZO 通道在尿路上皮细胞表面表达； (ii) 尿路上皮 表达的 PIEZO 通道对生理相关刺激（即膀胱充盈）做出反应； (iii) 压电 机械调节事件需要通道，包括膜运输和介质释放； （四） 功能丧失或功能获得 PIEZO 突变体的表达将导致尿路上皮细胞的改变 回应。在具体目标 2 中，我们试图了解 PIEZO 通道如何促进尿路上皮：传入神经 发信号。使用新颖的工具，包括离体膀胱成像，我们将探索其机制 PIEZO 通道刺激细胞内 Ca2+ ([Ca2+]i) 的增加。由于 PIEZO 通道迅速失活， 可能存在一种放大原始信号的机制。因此，我们将定义 Ca2+ 是否诱导 Ca2+ 释放或膜去极化作用于 PIEZO 通道下游以增加 [Ca2+]i。我们还将 确定压电触发的 [Ca2+]i 升高是否对于刺激胞吐作用和介质释放至关重要 尿路上皮，如果尿路上皮 PIEZO 通道调节尿路上皮：传入信号传导。在具体目标 3 中，我们将 开始探索尿路上皮 PIEZO 通道是否有助于膀胱功能和/或功能障碍。 具体来说，我们将确定 PIEZO 表达或功能的丧失是否会导致膀胱活动不足 具有功能获得性突变的 PIEZO 通道是否会导致膀胱过度活动。最后，我们将确定 尿路上皮表达的 PIEZO 通道是否会导致膀胱反射亢进和盆腔异常性疼痛 与环磷酰胺诱发的膀胱炎有关。