Underlying mechanisms controlling urothelial ATP release and their contributions to urinary bladder physiology and pathophysiology

项目摘要 ATP首先被发现是从膀胱上皮（尿道上皮）释放出来的， 1997年的膀胱扩张从那以后的几年里，许多研究已经确定了额外的刺激， ATP释放，然而，相对较少的研究已经检查了这种释放的机制。因为它是 通常认为尿ATP在控制排尿的感觉通路中起着重要作用， 更深入地了解尿路上皮ATP释放对于了解膀胱至关重要 生理学/病理学。 我们最近发现了两种不同的机制控制ATP释放的尿路上皮。的 首先涉及泛连接蛋白半通道，一种可渗透ATP的大孔离子通道。我们认为这 生理机制在排尿的生理控制中起重要作用，如药理学抑制或 泛连接蛋白通道的遗传敲低导致大鼠反射性膀胱活动的显著抑制。的 第二种机制涉及分泌性溶酶体。我们有证据表明，这种机制在 出现膀胱炎症，因为细菌内毒素可刺激溶酶体胞吐。这 这使我们假设，通过不同机制释放的ATP可能具有不同的机制， 生理效应。 充分了解ATP在膀胱生理学和病理学中的作用的下一步是充分 表征负责激活或抑制任一释放的细胞内信号传导途径 机制为此，我们建议使用已知的泛连接蛋白介导的释放（膀胱炎）的活化剂。 扩张，α3烟碱受体刺激和P2 Y 6嘌呤能受体刺激）或溶酶体介导的 释放（toll样受体刺激），以检查可能控制 release.例如，已知泛连接蛋白介导的释放依赖于细胞内钙离子， RhoA/ROCK通路的信号传导和活化。我们目前的研究旨在确定是否每种刺激 （机械拉伸，离子通道的激活和代谢型受体的激活）进入这些细胞 已知的细胞内途径。我们还将研究NAADP后溶酶体钙的释放 溶酶体pH的信号传导或改变，已知调节其他组织中溶酶体胞吐作用的途径， 改变溶酶体从尿道分泌ATP。最后，我们将描述两种ATP释放的特征， 机制相互作用，因为我们以前已经证明，抑制泛连接蛋白介导的释放， 增强溶酶体释放，表明释放机制之间的串扰。我们希望本次 该项目将导致更全面地了解控制嘌呤信号的机制， 膀胱和导致更有效的治疗膀胱病理。

Project Summary ATP was first discovered to be released from the urinary bladder epithelium (urothelium) in response to bladder distension in 1997. In the years since, many studies have determined additional stimuli that result in ATP release, however relatively few studies have examined the mechanism(s) of this release. As it is commonly thought that urinary ATP plays a significant role in the sensory pathways that control micturition, a deeper understanding of urothelial ATP release is essential for understanding urinary bladder physiology/pathology. We have recently discovered two distinct mechanisms controlling ATP release from the urothelium. The first involved pannexin hemi-channels, a type of large-pore ion channel permeable to ATP. We believe that this mechanism plays a significant role in the physiological control of micturition, as pharmacological inhibition or genetic knockdown of pannexin channels causes a marked inhibition of reflex bladder activity in the rat. The second mechanism involves secretory lysosomes. We have evidence that this mechanism plays a role in the emergence of bladder inflammation, as lysosomal exocytosis can be stimulated by bacterial endotoxins. This has led us to hypothesize that the ATP released through different mechanisms may have separate physiological effects. The next step into fully understanding ATP's role in bladder physiology and pathology is to fully characterize the intracellular signaling pathways responsible for activating or inhibiting either release mechanism. To that end, we propose to use known activators of either pannexin mediated release (bladder distension, α3 nicotinic receptor stimulation and P2Y6 purinergic receptor stimulation) or lysosomal mediated release (toll-like receptor stimulation) to examine common intracellular signaling pathways that may control release. For example, it is already known that pannexin mediated release is dependent on intracellular calcium signaling and activation of the RhoA/ROCK pathway. Our current research aims to determine if each stimulus (mechanical stretch, activation of an ion channel and activation of a metabotropic receptor) feeds into these known intracellular pathways. We will also examine how release of lysosomal calcium following NAADP signaling or alterations in lysosomal pH, pathways known to modulate lysosomal exocytosis in other tissues, alter lysosomal release of ATP from the urothelium. Finally, we will characterize how the two ATP release mechanisms interact, as we have previously demonstrated that inhibition of pannexin-mediated release potentiated lysosomal release, suggesting a crosstalk between release mechanisms. It is our hope that this project will lead to a more complete understanding of the mechanisms controlling purinergic signaling in the urinary bladder and lead to more effective treatments for bladder pathology.