MECHANISMS OF OXIDANT PRODUCTION IN RESPIRATORY FAILURE

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We have previously demonstrated that highly reactive oxygen species are generated by the diaphragm during conditions of intense muscle stimulation. Furthermore, in conditions designed to simulate respiratory failure in an animal model, there is evidence of increased free radicals in the diaphragm and in the blood. The primary purpose of this study is to determine the source of the free radical signals in conditions of respiratory failure. All experiments will be carried out in a well- developed model of acute respiratory failure in the rat, induced by inspiratory resistive loading. Our specific aims are: 1) To test the hypotheses that hydroxyl radical (OH.) is produced as a consequence of respiratory failure. To address this specific aim, we will use new sophisticated intracellular and extra cellular spin trapping techniques and electron spin resonance (ESR) spectroscopy to capture OH. and other radicals. We will also define the kinetics of free radical production during the process of developing failure and determine if conditions related to collapse of the gas exchange system, such as hypoxemia, potentiate the free radical response. 2) We will test the hypothesis that acute respiratory failure causes alterations in diaphragm mitochondrial function which favor free radical production. In this experiment, we will measure the respiratory capacity of isolated mitochondria from animals in respiratory failure and determine the capacity of the mitochondria to generate free radicals during electron transport. 3) We will test the hypothesis that free radical production in acute respiratory failure occurs via activation of non-mitochondrial oxygenase pathways. Two potentially important candidates for non-mitochondrial free radical production are the cyclooxygenase pathway of arachidonic acid metabolism and the xanthine oxidase pathway of superoxide production. In this aim, we will block these enzyme systems and observe the effect on free radical production. Our long term objectives are to identify the relevant cellular mechanisms underlying respiratory failure and, in particular, the loss of diaphragm contractile function which is often associated with it. Furthermore, we hope to eventually understand the role of free radicals in muscle function and determine if there is a direct link between excess free radical production, muscle fatigue and contractile state.