Purinergic Regulation of Bladder Interstitial Cells of Cajal

描述（由申请人提供）：尿失禁、膀胱过动症（OAB）和神经源性膀胱通常由膀胱平滑肌（BSM）运动不适当引起，其潜在机制尚不清楚。起搏器细胞，也被称为Cajal间质细胞（ICC），可能在调节膀胱平滑肌功能中起关键作用，但这种最近定义的细胞几乎完全没有被研究过。出生时没有膀胱ICC （BICC）的婴儿（一种致命的疾病，称为巨囊-微结肠肠蠕动不足综合征（MMIHS））完全缺乏自主排尿功能，死亡时膀胱扩张，强调了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。在这个特殊的应用目的是确定嘌呤能信号通路在BICC中运作，以及它们如何调节BSM运动。我们假设向BICC传递嘌呤能信号将通过钙信号和间隙连接传递调节BSM运动。基于大量的前期数据，我们证实了新型嘌呤能受体P2X2/6和A2a在BICC上的表达，并观察到这些受体的激活可诱导BSM收缩/舒张，我们将通过以下四个具体目标来验证我们的假设：1)证明BICC通过激活P2X2/6异质受体介导BSM收缩；2)确定P2X2/6激活是否导致内质网Ca2+释放，并通过隙结传递介导BSM收缩；3)确定A2a受体的激活是否通过抑制Ca2+信号传导来放松bcc介导的BSM收缩；4)确定外核苷酶Entpd2是否通过调节BICC上ATP和腺苷的可用性来调节P2X2/6和A2a受体功能和膀胱运动。由于确定该计划可行性的令人信服的初步数据，我们预计Aim 1将在项目的指导阶段完成，并准备好最终的手稿并提交发表。为实现目标2和目标3而进行的初步实验也可能在这个初始阶段开始，从而为在独立研究阶段完成其余目标产生重要的动力。我们提出的方法是创新和综合的。为了达到我们的目的，我们建立了一种新的方法来研究bcc介导的BSM运动。我们将使用多种转基因动物模型，利用膀胱肌肉条形肌图结合受体的特定药理调节、实时钙成像和质谱，研究各种信号通路的参与。我们期望这项研究能够垂直推进我们对BSM运动如何受BICC调节的理解，并最终建立一个新的BICC-BSM相互作用模型，从而改变膀胱运动如何被调节的范式。最终，这项工作可能为膀胱疾病确定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.