Nitric Oxide in Bladder Neural-Epithelial Signaling

描述（由申请人提供）：传统上，膀胱中的感觉信号被认为是膀胱传入神经的直接激活。新的研究结果强调了膀胱尿路上皮细胞在感觉事件转导中的关键作用。我们已经证明，尿路上皮细胞表现出许多“神经元样”特性（包括表达传感器分子，使它们能够对化学/热/机械刺激做出反应并释放化学介质），这可能使尿路上皮细胞“直接”与膀胱神经或通过尿路上皮细胞间的相互作用间接交流。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尚不清楚。机械力被认为在尿路上皮中“启动”一系列信号事件（钙波）。机械刺激诱导的化学因子随之释放，可激活尿路上皮细胞表面受体，放大尿路上皮细胞内及周围的信号。尽管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，对感觉转导机制或细胞间通信模式知之甚少。采用多学科方法，包括药理学，siRNA，递质释放，膜片钳和新型成像技术，我们的目标是评估尿路上皮细胞如何接受和整合多种刺激。特异性目标1将描述尿路上皮细胞机械传感器及其机械转导途径。有证据表明，TRPV1对尿路上皮机械诱导的嘌呤能信号传导至关重要，但尿路上皮细胞对机械力的反应机制尚不明确。这个目的部分将检查TRP通道在尿路上皮机械转导中的参与。特异性目的2将评估各种递质从尿路上皮细胞释放的机制。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囊泡的运动和释放递质。特异性目的3将阐明机械刺激在偶联和邻近细胞靶标中引起的反应。该目标将利用日益复杂的条件（单细胞内的信号；尿路上皮-神经元共培养和完整组织）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。这些研究的结果将帮助我们了解尿路上皮细胞如何接收和整合多种刺激，从而为从膀胱到神经系统的信息传递提供重要的“链接”。了解这些机制可能为未来临床治疗膀胱功能障碍的新靶点的识别提供重要的见解。

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.