Mechanistic Role of Peroxynitrite in Gallbladder Disease

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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.