Purinergic Regulation of Bladder Interstitial Cells of Cajal

描述(申请人提供)：尿失禁、膀胱过度活动(OAB)和神经原性膀胱通常由不适当的膀胱平滑肌(BSM)运动引起，其潜在机制尚不清楚。起搏细胞，也被称为Cajal间质细胞(ICC)，可能在调节膀胱平滑肌功能中发挥关键作用，但这一新近定义的细胞几乎完全没有研究。出生时没有膀胱ICC(BICC)的婴儿-一种称为巨囊炎-微结肠肠道动力不足综合征(MMIHS)的致命疾病-完全缺乏自主排尿功能，并死于膀胱扩张，这突显了BICC在调节BSM中的重要作用。这项研究的长期目标，与国家泌尿学研究议程的几个既定目标一致，是充分了解神经元、BICC和BSM在调节膀胱运动中的相互作用。这一特殊应用的目的是确定在BICC中工作的嘌呤能信号通路以及它们如何发挥调节BSM运动的功能。我们假设BICC的嘌呤能信号将通过钙信号和缝隙连接传递来调节BSM的运动。本研究以新的嘌呤能受体P2X2/6和A2a在BICC上表达的初步数据为指导，观察到这些受体的激活可诱导BICC收缩/松弛，我们将通过以下四个具体目标来验证我们的假说：1)证明BICC通过激活P2X2/6异构体受体介导BSM收缩；2)确定P2X2/6激活是否导致内质网钙释放，并通过缝隙连接传递介导BSM收缩；3)确定A2a受体激活是否通过抑制钙信号而松弛BICC介导的BSM收缩；4)探讨ECN Entpd2是否通过调节三磷酸腺苷和腺苷在BICC上的供应来调节P2X2/6和A2a受体功能和膀胱运动。由于有令人信服的初步数据确定了这项计划的可行性，我们预计目标1将在项目的指导阶段完成，最终的手稿将准备好并提交出版。为实现目标2和目标3而进行的初步试验可能也已在这一初始阶段开始，从而为在独立研究阶段完成其余目标创造了重要动力。我们提出的方法是创新的和综合的。为了达到我们的目的，我们建立了一种新的方法来研究BICC介导的BSM运动。我们将使用多种转基因动物模型，结合受体的特定药物调节、实时钙成像和质谱仪，研究不同信号通路的参与情况。我们期望这项研究能从纵向上促进我们对BICC如何调节BSM运动的理解，并最终建立一个新的BICC-BSM相互作用模型，从而改变膀胱运动调节的范式。最终，这项工作可能会为膀胱疾病定义新的治疗方案和分子靶点。

DESCRIPTION (provided by applicant): Urinary incontinence, overactive bladder (OAB) and neurogenic bladder often arise from inappropriate bladder smooth muscle (BSM) motility and the underlying mechanisms are poorly understood. Pacemaker cells, also known as interstitial cells of Cajal (ICC) are likely to play a critical role in modulating bladder smooth muscle functio but this recently defined cell, is almost completely unstudied. Infants born without bladder ICC (BICC) - a lethal condition called megacystis-microcolon intestinal hypoperistalsis syndrome (MMIHS) - have a complete absence of autonomic voiding function and die with dilated bladders, underscoring a vital role for BICC in modulating BSM. The long-term goal of this research, in alignment with several stated goals of the National Urology Research Agenda, is to fully understand the interactions of neurons, BICC, and BSM in regulating bladder motility. The objective in this particular application is to identify the purinergic signaling pathways which operate in BICC and how they function to regulate BSM motility. We hypothesize that purinergic signaling to BICC will regulate BSM motility through calcium signaling and gap junction transmission. Guided by strong preliminary data demonstrating novel purinergic receptors, P2X2/6 and A2a expression on BICC and observing that activation of these receptors induces BSM contraction/relaxation, we will investigate our hypothesis through the following four specific aims: 1) to demonstrate that BICC mediates BSM contraction through activation of P2X2/6 heteromeric receptors; 2) to define whether P2X2/6 activation results in Ca2+ release from ER and mediates BSM contraction through gap junction transmission; 3) to define whether activation of A2a receptors relaxes BICC-mediated BSM contraction through inhibiting Ca2+ signaling; and 4) to determine whether ectonucleotidase Entpd2 regulates P2X2/6 and A2a receptor function and bladder motility through modulating the availability of ATP and adenosine on BICC. Due to the compelling preliminary data which defines the feasibility of this plan, we expect Aim 1 to be completed during the mentored phase of the project and the resulting manuscript prepared and submitted for publication. Preliminary experiments in pursuit of aims 2 and 3 will likely have begun in this initial phase also, thereby generating important momentum for completion of the remaining aims during the independent research phase. The approach we propose is innovative and integrative. To achieve our aims we have established a new method to study BICC-mediated BSM motility. We will use multiple genetically modified animal models to investigate the involvement of various signaling pathways using bladder muscle strip myography in conjunction with specific pharmacological modulation of receptors, real-time calcium imaging and mass spectrometry. We expect this research to vertically advance our understanding of how BSM motility is regulated by BICC, and eventually establish a novel BICC-BSM interaction model that will shift the paradigm for how bladder motility is regulated. Ultimately, this work may define new treatment solutions and molecular targets for bladder disease.