Nitrogen dioxide, (NO2), an environmental pollutant, is toxic to lung cells including pulmonary endothelial cells. One of the major mechanisms of NO2- induced pulmonary injury is peroxidation of membrane lipids. Peroxidative cleavage of membrane lipid can alter the fluidity and the dynamics of the plasma membrane lipid bilayer. It can also impair membrane-dependent transport and metabolic functions. Since abnormalities in membrane fluidity are implicated in the pathogenesis of disease, the specific aims of this study are: (I) identifying NO2 concentration- and time- dependent alterations of fluidity in physically distinct domains in the plasma membrane of endothelial cells, (II) identifying and quantitating the individual classes of lipids affected during NO2- induced oxidant stress, (III) establishing the relationship between the changes in fluidity and lipid composition with a alterations in lipid peroxidation, 5-hydroxytryptamine (5-HT) uptake and metabolism, angiotensin-converting enzyme (ACE) activity, and insulin receptor binding, and (IV) endeavoring to modify NO2- induced oxidant injury with the biological antioxidant alpha- tocopherol and its analogue tocopherol succinate. To achieve these aims, we will use fluorescence spectroscopy to evaluate the effect of NO2 on different regions of the lipid bilayer of the plasma membrane by using specific fluorescent probes. To identify the individual classes of lipids affected, we will extract, separate, and quantitate the lipids of control and NO2 exposed endothelial cell membranes. The functional ability of control and NO2 exposed cells will be assessed by measuring 5-HT uptake, ACE activity, insulin receptor binding as well as lipid peroxide formation and lactate dehydrogenase (LDH) release as indices of injury before and after modulation of membrane fluidity by regidizers (cholesterol), fluidizers (cis-vacennic acid, anesthetics, and anionic drugs), and injury initiators (linoleic acid hydroperoxide). Finally, the protective effect of biological antioxidants will be evaluated by using alpha-tocopherol and its analogue tocopherol succinate for their preferential partitioning into lipid domains and for characterizing the physical interactions of alpha-tocopherol with membrane lipids using fluorescence polarization and differential scanning calorimetry (DSC). Elucidation of the fundamental mechanisms of cell membrane injury will provide ways to prevent the manifestations of NO2- induced cell and lung injury.
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