Urothelial Purinergic Signaling During Bladder Filling

膀胱充盈期间的尿路上皮嘌呤能信号传导

• 批准号: 9886930
• 负责人: Violeta N. Mutafova-Yambolieva
• 金额: $ 58.35万
• 依托单位: UNIVERSITY OF NEVADA RENO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-01-09 至 2023-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9886930
• 关键词: Adenosine; Affect; Afferent Neurons; Analytical Chemistry; Attention; Biological; Biology; Bladder; Bladder Control; Bladder Dysfunction; Bladder Urothelium; Bladder mucosa; Cell physiology; Cells; Communication; Complex; Data; Decentralization; Diphosphates; Electrophysiology (science); Gene Deletion; Image; Inflammation; Investigation; Knowledge; Lamina Propria; Macaca fascicularis; Measures; Mechanics; Mediating; Mediator of activation protein; Metabolic; Metabolism; Methodology; Microdialysis; Modeling; Molecular Biology; Mucous Membrane; Mus; Nerve; Neural Pathways; Nicotinamide adenine dinucleotide; Oryctolagus cuniculus; Outcomes Research; Overactive Bladder; PDGFRB gene; Phase; Physiological; Platelet-Derived Growth Factor Receptor; Preparation; Primates; Process; Protein Biochemistry; Purines; Quality of life; Regulation; Ribose; Role; Seminal; Signal Transduction; Smooth Muscle; Smooth Muscle Myocytes; Testing; Transgenic Mice; Translating; Urination; Urine; Urothelial Cell; Urothelium; Validation; afferent nerve; aquaporin 3; cell type; chemical release; clinical investigation; detrusor muscle; extracellular; in vivo; insight; interstitial cell; intravesical; irritation; mechanotransduction; neuroregulation; new therapeutic target; nonhuman primate; novel; novel strategies; operation; optogenetics; pre-clinical; premature; pressure; relating to nervous system; response; sensor; side effect; tripolyphosphate; wasting

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.

项目摘要/摘要 膀胱在填充过程中必须避免过早收缩，在关键的静脉内填充时必须排空 达到压力。尿液的异常连续性或空隙通常与异常有关 在膀胱填充过程中，迫切肌肉（DSM）的兴奋性。当前的疗法主要是 无效且经常具有可抗副作用。迫切需要更好地了解 填充过程中膀胱功能调节调节的基本机制，可能会产生新颖的想法 有效控制膀胱兴奋性。增加DSM收缩性的兴奋性购买（例如，ATP和ADP） 降低DSM收缩力（例如NAD，ADP-核糖，AMP和腺苷）的抑制性王子被释放 从尿路上皮，形成“膀胱壁”深处的“调节性纯池”。此的相对组成 池（例如，抑制性与兴奋性）在膀胱填充过程中可能正在发生变化，以实现足够的DSM兴奋性。 然而，在填充过程中，尿皮上皮和DSM之间的纯介导的局部信号传导不是 理解齿。该项目将调查1）几种机制 - 释放，代谢和跨性皮物质 运输 - 确定沿层皮内皮可用的纯介体的类型和相对量 （subu）/lamina prepria（LP）在填充过程中和2）影响细胞外嘌呤对非神经类型的细胞的影响 调节DSM令人兴奋的膀胱墙。具体目标1将检验不对称的假设 购买的可用性导致在存储阶段的Subu/Lp中抑制性/兴奋性嘌呤的比例更高 膀胱填充的填充物，而在高体积和压​​力下的降低却促进了排尿。 ATP，ADP， NAD，ADP-核糖，AMP和腺苷将在填充过程中仅在Subu/LP和Lumen中检查。 特定目标2将检验以下假设，即采购的代谢和跨皮物质运输适应足够 膀胱填充期间的subu/lp中的嘌呤可用性。特定目标3将检验尿路上皮的假设 嘌呤通过影响尿路上皮细胞的填充过程中膀胱兴奋性的内在控制， 粘膜粘膜PDGFR+细胞和DSM细胞。为了获得直接访问Subu/lp，我们将使用分散的 取消了带有DSM的Vivo）膀胱模型，我们将在膀胱壁的体内和离体微透析中执行。 我们将使用分析化学，电生理学，分子生物学，蛋白质生物化学和功能和功能 Ca2+成像方法，包括在膀胱中选定的细胞类型中的光遗传传感器的表达 墙。研究将采用转基因小鼠，例如PDGFREGFP/+，SMMHC-GCAMP6F，PDGFR-GCAMP6， TRPV4EGFP，AQP3-GCAMP6M小鼠和具有特定基因缺失的小鼠。关键机制将在 来自cynomolgus猴子（Macaca fascicularis）的膀胱，以确定在小鼠中获得的知识 膀胱转化为主要膀胱。在项目期结束时，我们将了解生物学 尿路上皮嘌呤能信号传导对尿路上皮和DSM之间的机械连通性的重要性 我们可能会确定用于治疗异常膀胱刺激的新型机械靶标。