When neutrophils are activated by contact with bacteria, foreign particles, and various chemicals such as phorbol esters, they generate a large quantity of superoxide by means of the NADPH oxidase and its associated electron transport chain. Most of this superoxide is converted to hydrogen peroxide, and most of the peroxide is converted to hypochlorous acid (HOC1) by the action of myeloperoxidase. In the presence of the spin-trapping agent 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), activated neutrophils yield the nitroxide adducts of both superoxide (DMPO-OOH) and hydroxyl radical (DMPO-OH). The origin of DMPO- OOH is controversial, and many workers believe that it arises from breakdown of DMPO-OOH. In addition, the principal investigator recently showed that HOC1 catalyzes the incorporation of ?17O!H2O into ?17O!DMPO-OH, thus suggesting an alternative origin of DMPO-OH in neutrophils. In these model studies, the DMPO-OH was further oxidized by HIC1 to 55-dimethyl 1-2 pyrrolidone-N-oxyl (DMPOX). The latter product has not been seen with activated neutrophils. However, the principal investigator has found that DMPOX is readily converted by NADPH into an EPR-silent species, presumably the hydroxylamine. The proposed research will test the hypothesis that activated neutrophils form DMPO-OH and DMPOX from HOC1 but subsequently reduce the DMPOX with NADPH generated by the respiratory burst. Synthetic DMPOX will be incubated with activated neutrophils and its reduction monitored by electron paramagnetic resonance (EPR). The role of NADPH as a source of reducing equivalents will be examined with site-specific inhibitors, chemical agents that oxide NADPH, and a recycling system for regenerating NADPH. The reaction of DMPOX with NADPH will be quantitated and the reaction product identified. In addition, activated neutrophils will be incubated with DMPO in the presence of 17O-labeled O2 and H2O to determine the origin of the DMPO-OH produced. Enzymic reactions that reduce nitroxides and oxidize the corresponding hydroxylamines may be responsible, in part, for the conflicting observations of radicals formed by neutrophils using spin-trapping techniques . The proposed research will help clarify the basis of this inconsistency.
