Inflammatory states in the gastrointestinal tract (GI) have been described to be associated with oxygen and nitrogen radicals. It is generally accepted that oxygen radicals (from xanthine oxidase and neutrophils) are involved in GI mucosal injury. Constitutive nitric oxide regulates GI motility, leukocyte adherance and mucosal permeability. However, it is unknown if and how functional roles of nitric oxide are altered by oxygen radicals produced during GI inflammation. Our objective is to investigate interactions between oxygen radicals and nitric oxide, and mechanistically elucidate functions of nitric oxide.
Four specific aims are designed to test the central hypothesis that during early events of inflammatory states where oxygen radical production is significantly elevated, constitutively produced nitric oxide potentiates the injury (through the formation of strong oxidants), and excessively produced nitric oxide ameliorates free radical-induced GI injury. Two classes of oxygen radical generators, namely, organic peroxyl/alkoxyl radicals and superoxide radical are chosen to produce oxidative injury in the GI tract. These free radicals are endogenously produced in gastrointestinal cells through the metabolic breakdown of organic (or lipid) hydroperoxides and trinitrobenzene sulfonic acid (TNBS) to, respectively, form peroxyl/alkoxyl radicals, and superoxide radical. These test compounds are relevant since lipid hydroperoxides are present in dietary fats, and TNBS is a chemical irritant commonly used to produce inflammatory responses in chronic colitis models. To specifically study reactions of oxygen radicals and nitric oxide, electron paramagnetic resonance (EPR) spectroscopy will be used.
Specific aim #1 will test the hypothesis that nitric oxide reacts with oxygen radicals by a free radical-free radical reaction. Thus, the depletion of hydroperoxide-derived peroxyl/alkoxyl radical as well as nitric oxide will be tested in enzymatic systems.
Specific aim #2 will test the hypothesis that the production of oxygen radicals by isolated GI cells and intestinal epithelial cells-6, IEC-6, and biochemical markers of oxidative injury are correlated. Effects of nitric oxide donors on these parameters will give insights into the conditions which nitric oxide is protective or damaging.
Specific aim #3 focus on in vivo and in situ intestinal segment experimentation to test the hypothesis that free radical-induced oxidative injury is ameliorated when constitutive nitric oxide is removed or when excessive nitric oxide is present.
Specific aim #4 is designed to characterize the novel TNBS-nitro radical anion and superoxiide radical by the GI cells and IEC-6 as well as in situ colonic segments. Effects of NO donors and nitric oxide synthase inhibitors will also be investigated. Our overall studies will provide novel understanding of basic molecular mechanisms of how and the outcome of the interactions of oxygen radicals and nitric oxide in modulating the GI oxidative injury. Insights on possible therapeutic interventions can be derived to alleviate morbidity associated with human GI diseases.
