Mechanistic Role of Peroxynitrite in Gallbladder Disease

描述(由申请人提供)： 胆囊炎影响着2000多万美国人，随着年龄的增长，女性、少数族裔和肥胖症的发病率都在增加。超氧化物(O2-)迅速氧化胆囊中的一氧化氮形成过氧亚硝酸盐(ONOO-)，过氧亚硝酸盐又可以质子化形成细胞毒性自由基。硝基酪氨酸是一种稳定的ONOO氧化终产物，在胆囊炎时会在人的胆囊炎中增加。我们的长期目标是了解如何调节胆囊炎以达到预防和治疗的目的。这项应用的目的是确定ONOO-如何在机制上促进胆囊损伤和功能障碍。应用的中心假设是，ONOO-通过诱导O2-的产生和直接的细胞毒作用导致平滑肌细胞损伤和细胞死亡而促进胆囊炎。我们的假设是建立在强有力的初步数据基础上的，这些数据表明，在胆囊炎期间，含有酪氨酸的蛋白质硝化增加，ONOO诱导O2-的产生，以及ONOO诱导的氧化应激。这项拟议研究的基本原理是，一旦知道过氧亚硝酸盐是如何导致胆囊炎的，它的产生和影响就可以用新的创新方法来预防和治疗各种疾病。我们特别准备好进行这项拟议的研究，因为我们拥有确定自由基产生的生化技术，确定细胞损伤和细胞死亡的分子技术，以及防止自由基损伤的细胞和分子生物学技术。我们计划通过以下三个具体目标来验证我们的假设并实现这一应用的目标：1)确定在胆囊炎过程中存在的含有酪氨酸的硝化蛋白的增加；2)确定ONOO是否诱导胆囊肌中O2-的产生；3)确定ONOO是否诱导胆囊细胞损伤和死亡。这项拟议的工作是创新的，因为它研究了一种特定的活性氧物种(ONOO-)，在影响人类胆囊的条件下。此外，它还利用了自由基生物学中的技术，这些技术在我们的实验室随时都可以获得。我们期望，由此产生的方法将确定ONOO诱导胆囊肌细胞损伤和死亡的机制。这些结果将具有重大意义，因为预计它们将为该国越来越多的胆囊病患者提供预防和治疗干预的新目标。此外，预计这一结果将从根本上推进自由基生物学和胆囊生理学领域的发展。

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.