Urothelial Purinergic Signaling During Bladder Filling

项目摘要/摘要 膀胱在充盈过程中必须避免过早收缩，当膀胱容量达到临界值时必须排空。 压力也达到了。异常的大小便或排尿常常与异常有关 膀胱充盈过程中逼尿肌的兴奋性。目前的治疗方法主要是 效果不佳，而且常常会产生无法忍受的副作用。迫切需要更好地理解 充盈过程中膀胱功能调节的基本机制可能会产生更多的新想法 有效控制膀胱兴奋性。增加DSM收缩能力的兴奋性嘌呤(如三磷酸腺苷和二磷酸腺苷) 并释放降低DSM收缩能力的抑制性嘌呤(例如，NAD、ADP-核糖、AMP和腺苷) 从尿路上皮开始，在膀胱壁深处形成“调节性嘌呤池”。这个的相对构成 池(例如，抑制性与兴奋性)可能在膀胱充盈过程中发生变化，以实现足够的DSM兴奋性。 然而，在充盈过程中，嘌呤介导的尿路上皮和DSM之间的信号传递的局部机制不是 明白了。这个项目将研究1)几种机制--释放、新陈代谢和跨上皮 转运--决定尿下层可利用的嘌呤介体的类型和相对数量 充盈过程中的(Subu)/固有层(Lp)及2)细胞外嘌呤对非神经细胞类型的影响 调节DSM兴奋性的膀胱壁。具体目标1将检验不对称的假设 在储藏期间，嘌呤的可用性导致Subu/LP中的抑制性/兴奋性嘌呤的比率更高 在高容量和高压力的情况下减少这一比例有利于排尿。ATP、ADP、 在充盈过程中，Suu/LP和腔内将同时检测NAD、ADP-核糖、AMP和腺苷。 特定目标2将验证嘌呤的新陈代谢和跨上皮运输调节适当的假说 膀胱充盈期间Subu/LP中的嘌呤利用率。《特指目标3》将检验尿路上皮 嘌呤通过影响尿路上皮细胞在充盈过程中对膀胱兴奋性的内在控制作用， 粘膜下PDGFR+细胞和DSM细胞。要获得对Subu/LP的直接访问权限，我们将使用分散(例如 去除DSM的膀胱模型，我们将进行体内和体外膀胱壁的微透析。 我们将使用分析化学、电生理学、分子生物学、蛋白质生物化学以及功能和 钙离子成像方法，包括光遗传感受器在膀胱中特定细胞类型中的表达 墙。研究将使用转基因小鼠，如PDGFREGFP/+，smMHC-GCaMP6f，PDGFR-GCaMP6， Trpv4eGFP、AQP3-GCaMP6m小鼠和特异性基因缺失小鼠。关键机制将在以下方面进行验证 食蟹猴(猕猴)的膀胱，以确定如何在小鼠身上获得知识 膀胱相当于灵长类动物的膀胱。在项目期结束时，我们将了解生物 尿路上皮嘌呤能信号在尿路上皮与DSM机械敏感性连接中的意义 我们可能会发现治疗异常膀胱兴奋性的新机制靶点。

PROJECT SUMMARY/ABSTRACT The bladder must refrain from premature contraction during filling and empty when critical intravesical volume and pressure are reached. Abnormal continence or voiding of urine are frequently associated with abnormal excitability of the detrusor smooth muscle (DSM) in the course of bladder filling. Current therapies are largely ineffective and frequently have intolerable side effects. There is a pressing need to better understand the fundamental mechanisms of regulation of bladder function during filling that may yield novel ideas for more efficient control of bladder excitability. Excitatory purines that increase DSM contractility (e.g., ATP and ADP) and inhibitory purines that decrease DSM contractility (e.g., NAD, ADP-ribose, AMP and adenosine) are released from the urothelium and form “a regulatory purine pool” deep in the bladder wall. The relative composition of this pool (e.g., inhibitory vs. excitatory) might be changing during bladder filling to enable adequate DSM excitability. However, purine-mediated local mechanisms of signaling between the urothelium and DSM during filling are not understood. This project will investigate 1) several mechanisms - release, metabolism and transurothelial transport - that determine the type and relative amount of purine mediators available in suburothelium (SubU)/lamina propria (LP) during filling and 2) influences of extracellular purines on non-neural types of cells in the bladder wall that regulate DSM excitability. Specific Aim 1 will test the hypothesis that asymmetrical availability of purines leads to a higher ratio of inhibitory/excitatory purines in SubU/LP during the storage phase of bladder filling whereas reduction of this ratio at high volume and pressure facilitates micturition. ATP, ADP, NAD, ADP-ribose, AMP and adenosine will be examined simultaneously in SubU/LP and in lumen during filling. Specific Aim 2 will test the hypothesis that metabolism and transurothelial transport of purines regulate adequate purine availability in the SubU/LP during bladder filling. Specific Aim 3 will test the hypothesis that urothelial purines contribute to the intrinsic control of bladder excitability during filling by affecting urothelial cells, submucosal PDGFR+ cells and DSM cells. To obtain direct access to SubU/LP, we will use a decentralized (ex vivo) bladder model with DSM removed and we will perform in vivo and ex vivo microdialysis of the bladder wall. We will use analytical chemistry, electrophysiology, molecular biology, protein biochemistry, and functional and Ca2+ imaging methodologies, including expression of optogenetic sensors in selected cell types in the bladder wall. Studies will employ transgenic mice such as Pdgfregfp/+, smMHC-GCaMP6f, PDGFR-GCaMP6, Trpv4eGFP, AQP3-GCaMP6m mice and mice with specific gene deletions. Key mechanisms will be validated in bladders from Cynomolgus monkeys (Macaca fascicularis) to determine how knowledge obtained in mouse bladder translates to the primate bladder. At the end of the project period, we will understand the biological significance of urothelial purinergic signaling for mechanosensitive connectivity between the urothelium and DSM and we may identify novel mechanistic targets for the treatment of anomalous bladder excitability.