The applicant will further pursue the observation that intracellular generation of reactive oxygen species (ROS) contributes to endotoxin-stimulated prostanoid synthesis and release from pulmonary endothelium. Two closely associated hypotheses are proposed based on preliminary data demonstrating that endotoxin causes intracellular generation of ROS, and that the antioxidant, DMSO, as well as extracellularly generated nitric oxide (NO) and peroxynitrite (ONOO) downregulate endotoxin-stimulated prostaglandin release is the result of intracellular generation of ROS, including generation and availability of intracellular NO and ONOO and (ii) antioxidants and supranormal levels of intracellular NO and ONOO inhibit endotoxin-stimulated prostaglandin synthesis through effects on ecNOS and COX-2 mRNA. Both NO and ONOO have been generally thought to mediate oxidant tissue injury, but their excessive generation has recently been reported to confer both beneficial and deleterious effects. The proposed experiments represent a comprehensive approach to three specific aims and utilize techniques of cell biology, biochemistry and molecular biology. Experiments are proposed to determine: 1) the time course of induction of ecNOS and COX-2 following exposure to endotoxin and whether this is a consequence of intracellular oxidant stress, 2) the effects of exogenously generated NO and ONOO on the endotoxin response and whether the observed beneficial effects are the result of alterations in their intracellular level and, 3) whether endothelial cells genetically engineered to hyperexpress NO are protected from endotoxin-induced prostaglandin synthesis and release. Since the preliminary data also indicate differences in iNOS activity in bovine pulmonary artery endothelial cells (BPAEC) and bovine lung microvascular cells (BMVEC), the applicant will examine 1) and 2) in both BPAEC and BMVEC; human cells cultured from those same two sites will also be examined, as necessary. Improved understanding of the mechanism(s) of endotoxin-induced endothelial changes will result in improved and novel strategies for treatment of patients with the adult respiratory distress syndrome.
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