Urothelial Purinergic Signaling During Bladder Filling

项目总结/摘要 膀胱必须避免过早收缩期间填充和排空时，临界膀胱内容量 压力达到。异常排尿或排尿通常与异常 逼尿肌平滑肌（DSM）在膀胱充盈过程中的兴奋性。目前的治疗方法主要是 无效且经常具有无法忍受的副作用。迫切需要更好地了解 在充盈过程中调节膀胱功能的基本机制，可能会产生更多的新想法 有效控制膀胱兴奋性。增加DSM收缩性的兴奋性嘌呤（例如，ATP和ADP） 和降低DSM收缩性的抑制性嘌呤（例如，NAD、ADP-核糖、AMP和腺苷）被释放 并在膀胱壁深处形成“调节嘌呤池”。这其中的相对成分 池（例如，抑制性对兴奋性）可能在膀胱充盈期间发生变化，以实现足够的DSM兴奋性。 然而，嘌呤介导的局部机制，信号之间的尿路上皮和DSM在填补不 明白本项目将研究1）几种机制-释放，代谢和跨上皮 转运--决定了亚磷酸盐中嘌呤介质的类型和相对量 （SubU）/固有层（LP）在填充过程中和2）细胞外嘌呤对非神经型细胞的影响， 调节DSM兴奋性的膀胱壁。具体目标1将检验不对称 嘌呤的可用性导致SubU/LP中抑制性/兴奋性嘌呤的比例在储存阶段较高 而在高容量和压力下降低该比率有利于排尿。ATP，ADP， 填充期间，将同时检查SubU/LP和管腔中的NAD、ADP-核糖、AMP和腺苷。 具体目标2将检验嘌呤的代谢和跨上皮转运充分调节 膀胱充盈期间SubU/LP中的嘌呤可用性。具体目标3将检验以下假设： 嘌呤通过影响尿路上皮细胞而在充盈期间有助于膀胱兴奋性的内在控制， 粘膜下PDGFR+细胞和DSM细胞。为了获得对SubU/LP的直接访问，我们将使用分散的（例如， 体内）膀胱模型，并且我们将进行膀胱壁的体内和离体微透析。 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，功能和 Ca 2+成像方法，包括在膀胱中选定细胞类型中的光遗传学传感器的表达 墙研究将使用转基因小鼠，如Pdgfr-EGFP/+、smMHC-GCaMP 6 f、PDGFR-GCaMP 6、 Trpv 4 eGFP、AQP 3-GCaMP 6 m小鼠和具有特定基因缺失的小鼠。关键机制将在 食蟹猴（Macaca fascicularis）的膀胱，以确定小鼠如何获得知识 膀胱翻译成灵长类动物的膀胱在项目结束时，我们将了解生物学 尿路上皮嘌呤能信号对尿路上皮和DSM之间机械敏感性连接的意义 并且我们可以确定治疗异常膀胱兴奋性的新的机制靶点。

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.