Role of Pannexins in Urinary Bladder Pathology

 描述（由申请人提供）：我们最近确定，ATP可以通过泛连接蛋白通道从膀胱上皮（尿道上皮）释放，以响应拉伸和化学刺激。由于嘌呤能信号传导被认为在膀胱病理学的发展中起关键作用，因此泛连接蛋白通道可能是治疗膀胱病症如间质性膀胱炎/膀胱疼痛综合征（IC/PBS）的重要靶标。因此，我们提出的研究旨在进一步研究泛连接蛋白通道在尿路上皮嘌呤能信号传导中的作用。 在我们的第一个目标中，我们建议检查负责泛连接蛋白通道本身的激活或失活的途径，如亚硝基化或磷酸化。虽然以前的研究已经阐明了各种各样的刺激导致ATP释放，迄今为止还没有研究检查连接尿路上皮细胞刺激泛连接蛋白通道开放的途径。因此，我们将试图确定参与激活/失活泛连接蛋白通道在尿路上皮的途径。由于我们先前已经证明用α 3或α 7烟碱受体激动剂模拟尿路上皮细胞可以分别激活或抑制ATP释放，我们将在培养的大鼠尿路上皮细胞中使用这些化合物作为我们的实验刺激。我们希望这些实验 将大大增加我们对连接尿路上皮刺激和ATP释放的机制的理解。由于ATP与许多病理性膀胱疾病（如膀胱过度活动症）密切相关，我们认为这项研究有可能阐明治疗膀胱病理的新靶点。 因为已经发现泛连接蛋白通道对于各种组织中炎性小体的激活是重要的，并且炎症是膀胱病理学的重要组成部分，所以我们提出的研究的第二部分将研究泛连接蛋白通道对于膀胱炎症的诱导或维持是如何重要的。为了研究这个问题，我们建议通过注射细菌脂多糖或环磷酰胺诱导膀胱炎症，并研究将pannexin介导的ATP释放与膀胱病理学中观察到的排尿频率增加联系起来的信号通路。这些信号传导途径包括尿路上皮细胞表面嘌呤能受体的激活、NLRP 3/NLRC 4炎性体的寡聚化以及IL-1 β受体刺激下游事件的激活，如细胞旁通透性增加。我们希望这方面的实验将阐明 涉及炎症的病理性膀胱疾病如间质性膀胱炎的药理学或遗传学治疗的新靶点。

 DESCRIPTION (provided by applicant): We have recently determined that ATP can be released from the urinary bladder epithelium (urothelium) in response to stretch and chemical stimulation through pannexin channels. As purinergic signaling is thought to play a critical role i the development of bladder pathology, pannexin channels may be an important target in the treatment of disorders of the urinary bladder such as interstitial cystitis/painful bladder syndrom (IC/PBS). Therefore our proposed research aims to further examine the role of pannexin channels in purinergic signaling in the urothelium. In our first aim, we propose to examine the pathways responsible for the activation or inactivation of the pannexin channel itself, such as nitrosylation or phosphorylation. While previous studies have elucidated a wide variety of stimuli responsible for causing ATP release, no studies to date have examined the pathways that connect urothelial cell stimulation to pannexin channel opening. Therefore we will attempt to determine the pathways involved in activating/deactivating pannexin channels in the urothelium. As we have previously demonstrated that simulation of urothelial cells with a3 or a7 nicotinic receptor agonists can activate or inhibit ATP release respectively, we will use these compounds in cultured rat urothelial cells as our experimental stimuli. It is our hope that these experiments will greatly increase our understanding of the mechanisms linking urothelial stimulation to ATP release. As ATP has been strongly implicated in a number of pathological bladder disorders, such as overactive bladder, we believe that this research has the potential to elucidate new targets for the treatment of bladder pathology. Because pannexin channels have been found to be important for the activation of the inflammasome in a wide variety of tissues, and inflammation is an important part of bladder pathology, the second half of our proposed study will examine how pannexin channels are important to the induction or maintenance of inflammation in the urinary bladder. To examine this question, we propose to induce bladder inflammation by injection of either bacterial lipopolysaccharides or cyclophosphamide and study the signaling pathways that connect pannexin-mediated ATP release to the increased frequency of urination observed in bladder pathology. These signaling pathways include activation of purinergic receptors on the surface of urothelial cells, the oligomerization of NLRP3/NLRC4 inflammasomes, and the activation of events downstream of IL-1ß receptor stimulation, such as increased paracellular permeability. It is our hope that the experiments in this aim will elucidate a new target for pharmacological or genetic treatment of pathological bladder disorders involving inflammation, such as interstitial cystitis.