Mechanistic Role of Peroxynitrite in Gallbladder Disease

描述（由申请人提供）： 胆囊炎影响超过2000万美国人，随着年龄的增长，女性，少数民族和肥胖症的发病率不断增加。超氧化物（O2-）迅速氧化胆囊中的一氧化氮，形成过氧亚硝酸根（ONOO-），后者又可被质子化，形成细胞毒性自由基。硝基酪氨酸是ONOO-氧化的稳定终产物，在胆囊炎时在人胆囊中增加。我们的长期目标是了解如何调节胆囊炎症以达到预防和治疗的目的。本申请的目的是确定ONOO-如何在机制上促进胆囊损伤和功能障碍。该申请的中心假设是ONOO-通过诱导O2-的产生和直接细胞毒性作用导致平滑肌细胞损伤和细胞死亡而导致胆囊炎症。我们的假设已经制定了强有力的初步数据的基础上，证明增加硝化的蛋白质含有酪氨酸在胆囊炎，ONOO-诱导产生的O2-，ONOO-诱导的氧化应激。这项研究的基本原理是，一旦知道过氧亚硝酸盐是如何导致胆囊炎症的，它的产生及其影响就可以通过新的创新方法来控制，以预防和治疗各种疾病。我们特别准备进行这项拟议的研究，因为我们有生化技术，以确定自由基的生产，分子技术，以确定细胞损伤和细胞死亡，细胞和分子生物学技术，以防止自由基损伤。我们计划通过追求以下三个具体目标来测试我们的假设并实现本申请的目的：1）鉴定胆囊炎症期间存在的增加的含酪氨酸的硝化蛋白; 2）确定ONOO-是否诱导胆囊平滑肌中O2-的产生; 3）确定ONOO-是否诱导胆囊细胞损伤和死亡。这项工作是创新的，因为它研究了影响人类胆囊的条件下的特定活性氧（ONOO-）。此外，它还利用了自由基生物学技术，这些技术在我们的实验室中很容易获得。我们期望所得到的方法将确定ONOO-诱导胆囊平滑肌细胞损伤和死亡的机制。这些结果将是重要的，因为它们有望为该国越来越多的胆囊疾病患者提供新的预防和治疗干预目标。此外，预计该结果将从根本上推进自由基生物学和胆囊生理学领域。

DESCRIPTION (provided by applicant): Cholecystitis affects over 20 million Americans, with an increasing incidence with advancing age, in females, in minorities, and with obesity. Superoxide (O2-) rapidly oxidizes nitric oxide in the gallbladder to form peroxynitrite (ONOO-), which in turn can be protonated to form cytotoxic radicals. Nitrotyrosine, a stable end product of ONOO- oxidation, is increased in human gallbladders during cholecystitis. Our long-range goal is to understand how gallbladder inflammation can be modulated for preventive and therapeutic purposes. The objective of this application is to determine how ONOO-contributes mechanistically to gallbladder injury and dysfunction. The central hypothesis of the application is that ONOO- contributes to gallbladder inflammation by inducing generation of O2- and by direct cytotoxic effects leading to smooth muscle cell injury and cell death. Our hypothesis has been formulated on the basis of strong preliminary data demonstrating increased nitration of proteins containing tyrosine during cholecystitis, ONOO--induced generation of O2-, and ONOO--induced oxidative stress. The rationale for the proposed research is that once it is known how peroxynitrite contributes to gallbladder inflammation, its production and its effects can be manipulated with new and innovative approaches to the prevention and treatment of a variety of diseases. We are particularly well prepared to undertake this proposed research because we have the biochemical techniques to determine free radical production, the molecular techniques to determine cell injury and cell death, and the cell and molecular biology techniques to prevent free radical injury. We plan to test our hypothesis and accomplish the objective of this application by pursuing the following three specific aims: 1) Identify the increased nitrated proteins containing tyrosine present during gallbladder inflammation; 2) Determine if ONOO- induces generation of O2- in gallbladder smooth muscle; 3) Determine if ONOO- induces gallbladder cellular injury and death. The proposed work is innovative because it investigates a specific reactive oxygen species (ONOO-) in conditions that affect the human gallbladder. Additionally, it takes advantage of techniques in free radical biology, which are readily available in our laboratory. It is our expectation that the resultant approach will identify the mechanisms by which ONOO- induces cellular injury and death in gallbladder smooth muscle. These results will be significant because they are expected to provide new targets for preventive and therapeutic interventions for the growing number of persons in this country who have gallbladder disease. In addition, it is expected that the results will fundamentally advance the field of free radical biology and gallbladder physiology.