Role of Ectonucleotidase in Voiding Function and Dysfunction

摘要： 下尿路症状（LUTS）影响了很大比例的人口， 社会成本和用于管理LUTS的有效药物有限。嘌呤能信号是一个突出的 失调的途径，这在LUTS中一直有报道。虽然嘌呤能收缩性很小 在正常人膀胱中，它在人类患者的膀胱中显著增加， 记录了啮齿动物的生理学然而，导致这些功能通路的潜在机制 变化是未知的。为了研究这一点，我们已经确定了多种新的功能性嘌呤能受体， 膀胱中的调节剂。我们已经证明，嘌呤能信号不仅对心肌收缩很重要， 膀胱壁，由ATP/ADP激活的P2 X1/P2 Y12信号转导介导，但对膀胱也很重要。 由腺苷激活的A2 b受体信号传导介导的舒张。此外，A2 b受体介导 松弛信号可以完全抑制P2 X1/P2 Y12介导的收缩。通过比较研究， 定义了一个关键基因-ENTPD 1（也称为CD 39）-在人类和小鼠中差异表达 膀胱（正常人膀胱比小鼠高约8倍）。ENTPD 1是一种限速酶 依次转化ATP/ADP/AMP。ENTPD 1控制激动剂级联反应的动力学 (ATP>ADP>腺苷），因此在嘌呤能受体表达的控制中起关键作用， 功能有趣的是，已经在自身免疫性疾病中鉴定了人ENTPD 1突变， 在某些实施方案中，LUTS是神经退行性疾病的一种，并且LUTS是这些疾病中的突出症状。与此相一致， 在LUTS患者的膀胱组织中已经报道了受损的ATP水解。因此，我们假设 ENTPD 1是膀胱功能的关键调节器。作为支持，我们有来自全球Entpd 1的初步数据- /-小鼠，表明Entpd 1的缺失导致具有异常排尿表型的扩张膀胱。我们 我们将通过以下目的来验证我们的假设：（1）我们将产生内脏平滑肌特异性的 Entpd 1基因敲除小鼠模型证明ENTPD 1在调节正常逼尿肌中的关键重要性 （2）我们将使用两种ENTPD 1突变小鼠模型，分别具有高和低的收缩力和膀胱功能。 ENTPD 1表达水平以模拟正常（高）和患病（低）的人膀胱，然后 检查排尿功能/功能障碍;（3）通过国家疾病研究交流中心（NDRI） 网络，我们将获得人膀胱组织，并研究ENTPD 1在膀胱中的表达和功能。 两种性别的膀胱我们将解决为什么嘌呤能收缩的差异， 人类和啮齿动物膀胱之间，男性和女性人类膀胱之间，以及正常和患病之间 人类膀胱我们希望这项研究能够大大推进我们对膀胱收缩如何 并且松弛由ENTPD 1及其相关的嘌呤能信号控制。这些研究具有很高的 有可能为人类LUTS患者引入新的治疗方法，因为有多种靶向选择。

Abstract: Lower urinary tract symptoms (LUTS) impact a large percentage of the population with enormous financial and social costs, and effective drugs for management of LUTS are limited. Purinergic signaling is a prominent dysregulated pathway, which has been consistently reported in LUTS. While purinergic contractility is minimal in normal human bladder, it increases significantly in bladders of human patients, to more closely resembles documented rodent physiology. However, the underlying mechanism which results in these functional pathway alterations is not known. To study this, we have identified multiple novel functional purinergic receptors and modulators in bladder. We have demonstrated that purinergic signaling is not only important for contraction in bladder wall, as mediated by ATP/ADP activated P2X1/P2Y12 signaling, but is also important for bladder relaxation mediated by adenosine activated A2b receptor signaling. Furthermore, A2b receptor mediated relaxation signaling can completely inhibit P2X1/P2Y12 mediated contractions. By comparative studies, we have defined a key gene - ENTPD1 (also known as CD39) – that is differentially expressed in human and mouse bladders (~ 8-fold higher in normal human bladders than in mouse). ENTPD1 is a rate-limiting enzyme converting ATP/ADP/AMP sequentially. ENTPD1 controls the kinetics of an agonist cascade (ATP>ADP>adenosine), and hence plays a critical role in the control of purinergic receptor expression and function. Interestingly, human ENTPD1 mutations have been identified in autoimmune diseases and neurodegenerative disorders, and LUTS is a prominent symptom in these diseases. Consistent with this, impaired ATP hydrolysis has been reported in bladder tissues from LUTS patients. Therefore, we hypothesize that ENTPD1 is a key regulator in bladder function. In support, we have preliminary data from a global Entpd1- /- mouse, indicating that deletion of Entpd1 results in a dilated bladder with abnormal voiding phenotype. We will test our hypothesis through the following aims: (1) We will generate a visceral smooth muscle specific Entpd1 knockout mouse model to demonstrate the critical importance of ENTPD1 in regulating normal detrusor contractility and bladder function; (2) We will use two ENTPD1 mutant mouse models with high and low ENTPD1 expression levels to mimic human bladders that are normal (high) and diseased (low), and then examine voiding function/dysfunction; (3) Through the National Disease Research Interchange (NDRI) network, we will procure human bladder tissues, and study the expression and function of ENTPD1 in the bladders of both sexes. We will solve the puzzles of why there are purinergic contractile differences between human and rodent bladders, between male and female human bladders, and between normal and diseased human bladders. We expect this study to substantially advance our understanding on how bladder contraction and relaxation is controlled by ENTPD1 and its associated purinergic signaling. These studies have high potential to introduce novel therapies for human LUTS patients as there are multiple targetable options.