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

项目摘要 三磷酸腺苷首先被发现从膀胱上皮(尿路上皮)释放，以响应 1997年的膀胱肿胀。在那之后的几年里，许多研究确定了额外的刺激，导致 三磷酸腺苷的释放，然而，相对较少的研究探讨了这种释放的机制(S)。就像现在这样 人们普遍认为尿液中的三磷酸腺苷在控制排尿的感觉通路中起着重要作用。 深入了解尿路上皮细胞三磷酸腺苷的释放是了解膀胱的关键 生理学/病理学。 我们最近发现了两种控制尿路上皮释放三磷酸腺苷的不同机制。这个 首先涉及pannin半通道，这是一种对ATP具有通透性的大孔离子通道。我们相信这一点 机制在排尿的生理控制中起着重要作用，如药物抑制或 PAnnexin通道的基因敲除导致大鼠反射膀胱活动的显著抑制。这个 第二种机制涉及分泌型溶酶体。我们有证据表明，这种机制在 出现膀胱炎，因为细菌内毒素可刺激溶酶体胞吐。这 使我们假设通过不同机制释放的三磷酸腺苷可能有不同的 生理效应。 要全面了解ATP在膀胱生理和病理中的作用，下一步是充分 描述负责激活或抑制释放的细胞内信号通路 机制。为此，我们建议使用已知的PAnnexin介导性释放(膀胱)激活剂 扩张、α3烟碱受体刺激和P2Y6嘌呤能受体刺激)或溶酶体介导 释放(Toll样受体刺激)以检测可能控制的常见细胞内信号通路 放手。例如，已知pAnnexin介导的释放依赖于细胞内钙离子。 RhoA/ROCK通路的信号和激活。我们目前的研究旨在确定每一种刺激 (机械拉伸，激活离子通道和激活代谢性受体)馈入这些 已知的细胞内途径。我们还将研究NAADP后溶酶体钙的释放 信号或溶酶体pH的变化，已知调节其他组织中溶酶体胞吐的途径， 改变尿路上皮细胞三磷酸腺苷的溶酶体释放。最后，我们将描述两个ATP是如何释放的 相互作用的机制，正如我们之前已经证明的那样，抑制pAnnexin介导的释放 促进溶酶体的释放，表明释放机制之间存在串扰。我们希望这件事 该项目将导致对控制嘌呤能信号转导机制的更全面的理解。 并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.