Role of PIEZO Channels in Bladder Function and Dysfunction

文摘：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。当有一位将军 对于感觉神经元(即壁式机械感受器)在这些事件中的作用，我们了解的较少 洞察非神经组织如何对膀胱的紧张感和转导起作用。我们的 研究的重点是构成膀胱壁和尿路之间的直接界面的尿路上皮。 太空。这种组织通过调节其离子运输、膜运输和 介质的释放，这些介质被认为可以改变膀胱功能，部分是通过局部尿路上皮：传入 条件反射。然而，我们对尿路上皮细胞质膜张力的了解仍然有限。 感觉到，响应拉伸而激活的下游通路的性质是什么，或者是如何 这些事件导致了膀胱功能和功能障碍。我们假设尿路上皮细胞表达的压电波 通道起机械传感器的作用，对膀胱充盈作出反应，促进钙离子进入，介质释放，和 向传入神经突起发出信号，促进正常的膀胱功能。我们进一步假设 尿路上皮细胞的压电性机械转导功能失调将导致膀胱功能障碍。在……里面 具体目标1，我们将通过演示压电通道是否真正起到机械传感器的作用 如下：(I)功能性压电通道在尿路上皮细胞表面表达；(Ii)尿路上皮细胞 表达的压电波通道对生理相关刺激(即，膀胱充盈)作出反应；(Iii)该压电波 机械调节事件需要通道，包括膜交通和介质释放；(4) 而功能缺失或功能获得突变的表达将导致尿路上皮的改变 回应。在具体目标2中，我们试图了解压电通道如何促进尿路上皮：传入 发信号。使用新的工具，包括体外膀胱成像，我们将探索其机制 压电通道刺激细胞内钙([Ca~(2+)]_i)升高。因为压电通道会迅速失活， 很可能会有一种机制来放大原始信号。因此，我们将定义钙诱导的钙离子是否 释放或膜去极化作用于压电通道的下游以增加[钙]i。我们还将 确定压电引起的细胞内[钙]i升高是否对刺激胞吐和细胞内介质释放起关键作用。 尿路上皮，如果尿路上皮压电通道调节尿路上皮：传入信号。在具体目标3中，我们将 开始探索尿路上皮压电通道是否与膀胱功能和/或功能障碍有关。 具体地说，我们将确定是否压电表达或功能的丧失会导致膀胱活动不足 如果具有功能增益突变的压电波通道导致膀胱过度活动。最后，我们将确定 尿路上皮细胞表达的压电波通道是否与膀胱反射亢进和盆腔痛有关 与环磷酰胺引起的膀胱炎有关。

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.