Hydroperoxides (ROOH) and alpha,Beta-unsaturated aldehydes (2-enals) are products of lipid peroxidation. Exposure to ROOH or 2-enals inhibits subsequent stimulation of the alveolar macrophage respiratory burst (O2 production by an NADPH oxidase) mimicking the effects of hyperoxia or NO2 NADPH oxidase is a multicomponent complex composed of membrane and cytosolic proteins and cofactors, and is not active in unstimulated cells. Signal transduction leading to expression o activity involves membrane depolarization, mobilization of intracellular Ca2+ through phosphoinositide turnover and plasma membrane channels, and protein phosphorylation. We hypothesize that the principal inhibitory effect of ROOH is upon signal transduction rather than directly upon the oxidase. We also hypothesize that 2-enals both inactivate NADPH oxidase directly and interfere with signal transduction. The hypothesis that 2-enals and ROOH are responsible for effects of NO2 and hyperoxia will be tested by identifying and quantitating NO2 and hyperoxia-induced lipid peroxidation products and by comparing the effect of No2 and hyperoxic pre-exposure on signal transduction and NADPH oxidase activity in the presence and absence of inhibitors of lipid peroxidation. We will examine the effects of 2-enals and ROOH upon plasma and mitochondrial membrane potentials, intracellular Ca2+ movements, inositol phosphate turnover, and protein phosphorylation. We will also test whether 2-enals and ROOH alone affect second messengers and/or whether they alter signal transduction pathways in response to stimuli of the respiratory burst. These effects will be compared with each other and with inhibition of the respiratory burst for both temporal and concentration dependence. We will examine the potential for direct alteration of the NADPH oxidase using cell-free preparations. Cells will be exposed to 2-enals and ROOH and then disrupted. Activation of the NADPH oxidase will be achieved using detergents or phospholipids. Reconstitution will be done by mixing cytosolic or membrane fractions from exposed cells with the complementary fraction from control cells. The KM for NADPH and potential reversibility of inactivation by dithiothreitol will be examined. The methodology includes dual wavelength spectrophotometry and fluorimetry, cell fractionation, chromatography and mass spectrometry. Our long-term objective is the understanding of the underlying mechanisms for loss of cell function due to oxidant exposure.
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