Oxidant injury is involved in many pathologic conditions including hyaline membrane disease and bronchopulmonary dysplasia. As part of the physiologic response to injury, activated endothelium produces a potent phospholipid, platelet-activating factor (PAF), that induces neutrophil adhesion and activation. When endothelium is exposed to oxidant injury it releases lipids with PAF-like activity that may contribute to further tissue damage. A mechanism for regulating the accumulation of PAF and PAF-like lipids is the activity or abundance of the enzyme that degrades them, platelet-activating factor acetylhydrolase (PAF-AH). PAF-AH is found in plasma where it is secreted from macrophages and intracellularly in a variety of tissues and cell types.

We determined the effect of oxidant exposure on the activity of PAF-AH. Recombinant PAF-AH (rPAF-AH) was incubated in the presence or absence of oxidants and its activity was measured by determining the rate of conversion of 3H-PAF to lyso-PAF and 3H-acetate. SIN-1 spontaneously releases the oxidants nitric oxide (NO) and superoxide (O2) that can combine to form another potent oxidant peroxynitrite (OONO). NO and O2- are produced endogenously by many cell types including endothelium, neutrophils and macrophages. SIN-1 inhibits the activity of rPAF-AH in a dose-dependent and time-dependent manner. Exposure to 10mM SIN-1 for 4 hrs results in a 63% reduction in PAF-AH activity compared to control(100 vs 37.4 ± 17.7%, p<0.05, n=5). Similar results were obtained by measuring PAF-AH activity in cultured human macrophage cell homogenates, macrophage conditioned media, and the LDL fraction of human plasma. In order to determine the mechanism by which SIN-1 inhibits PAF-AH activity immunoblot analysis was performed using an antibody specific for PAF-AH and an antibody which recognizes nitro-tyrosine residues. Nitro-tyrosine residues result from exposure to OONO. Immunoblot analysis reveals that exposure to SIN-1 results in decreased immunoreactive PAF-AH and nitrosylation of tyrosine residues in the PAF-AH protein. Nitrosylation and protein degradation may be involved in the inhibition of PAF-AH by oxidants.