Nitric Oxide in Bladder Neural-Epithelial Signaling

描述（由申请人提供）：传统上，膀胱中的感觉信号传导归因于膀胱传入神经的直接激活。新的发现强调了膀胱尿路上皮细胞作为感觉事件转导的关键参与者。我们已经表明，尿路上皮细胞表现出许多“神经元样”的特性（包括传感器分子的表达，使他们能够响应化学/热/机械刺激，并释放化学介质），使它可能是尿路上皮细胞沟通“直接”与膀胱神经或间接通过尿路上皮细胞-细胞相互作用。尿路上皮细胞不断暴露于各种形式的机械刺激，但这些细胞的反应机制还没有很好地定义。机械力被认为是“启动”一系列信号事件（钙波）在整个尿路。反过来，伴随机械刺激诱导的化学因子的释放可以激活尿路上皮细胞表面受体，并放大尿路上皮细胞内和附近的信号。虽然这些数据表明，尿路上皮细胞在细胞间信号传导中起着重要作用，但这是一个相对未开发的领域，关于感觉转导机制或细胞间通讯模式的信息很少。使用涉及药理学，siRNA，递质释放，膜片钳和新型成像技术的多学科方法，我们的目标是评估尿路上皮细胞如何接收和整合多种刺激。具体目标1将表征尿路上皮细胞机械传感器及其机械转导途径。有证据表明，TRPV 1是必不可少的机械诱发的嘌呤信号由尿路上皮细胞，但机制，尿路上皮细胞响应机械力还没有得到很好的定义。这一目标的一部分将检查参与TRP通道在尿路上皮力学转导。具体目标2将评估各种递质从尿路上皮细胞释放的机制。其目的是利用荧光染料成像来研究化学和物理刺激如何刺激尿路上皮细胞中囊泡的运动和释放递质。具体目标3将阐明耦合和相邻细胞靶点中机械刺激引起的反应。这一目标将利用日益复杂的条件（单细胞内的信号传导;尿路上皮-神经元共培养物和完整组织）来检查细胞-细胞相互作用的机制。这些成像方法将使我们能够评估膀胱不同区域之间以及尿路上皮层内的尿路上皮细胞信号传导。这些研究的结果将有助于我们了解尿路上皮细胞如何接收和整合多种刺激，从而提供了一个重要的“链接”的信息从膀胱到神经系统的传输。了解这些机制可能会提供重要的洞察力，为未来的膀胱功能障碍的临床管理的新目标的识别。

DESCRIPTION (Provided by Applicant): Traditionally sensory signaling in the urinary bladder has been attributed to direct activation of bladder afferents. New findings have highlighted urinary bladder urothelial cells as key players in the transduction of sensory events. We have shown that urothelial cells exhibit a number of "neuron-like" properties (including expression of sensor molecules that allow them to respond to chemical/thermal/mechanical stimuli and to release chemical mediators) making it likely that urothelial cells communicate "directly" with bladder nerves or indirectly via urothelial cell-cell interactions. Urothelial cells are constantly exposed to various forms of mechanical stimuli, yet the mechanisms by which these cells respond are not well defined. Mechanical forces are thought to "initiate" a series of signaling events (calcium waves) throughout the urothelium. In turn, the accompanying release of chemical factors induced by mechanical stimuli could activate urothelial cell surface receptors and amplify the signal within and adjacent to the urothelial cell. Although these data suggest that urothelial cells play an important role in cell-cell signaling, this is a relatively unexplored area with little information known about the mechanism for sensory transduction or the mode of communication between cells. Using a multidisciplinary approach involving pharmacology, siRNA, transmitter release, patch clamp and novel imaging techniques, our goals are to evaluate how urothelial cells receive and integrate multiple stimuli. Specific Aim 1 will characterize urothelial cell mechanosensors and their mechano-transduction pathways. Evidence has shown that TRPV1 is essential for mechanically-evoked purinergic signaling by the urothelium, yet the mechanisms by which urothelial cells respond to mechanical forces are not well defined. This aim in part will examine the involvement of TRP channels in urothelial mechanotransduction. Specific Aim 2 will evaluate the mechanism by which various transmitters are released from urothelial cells. The aim will utilize imaging with fluorescent dyes to study how chemical and physical stimuli stimulate movement and release of transmitters from vesicles in urothelial cells. Specific Aim 3 will elucidate the responses evoked by mechano-stimulation in coupled and adjacent cellular targets. This aim will utilize conditions of increasing complexity (signaling within a single cell; urothelial-neuron co-cultures and intact tissue) to examine mechanisms for cell-cell interactions. These imaging methods will enable us to evaluate urothelial-cell signaling between different regions of the bladder as well as within the urothelial layers. Results from these studies will help us to understand how urothelial cells receive and integrate multiple stimuli thus providing an important "link" in the transfer of information from the urinary bladder to the nervous system. Understanding these mechanisms may provide important insight for the identification of novel targets for the future clinical management of bladder dysfunctions.