copper-bis(3-5-diisopropylsalicylate) and 3-nitrotyrosine

Opposing effects have been ascribed to nitric oxide (NO) on retinal microvascular survival. We investigated whether changes in the redox state may contribute to explain apparent conflicting actions of NO in a model of oxygen-induced retinal vasoobliteration. Retinal microvascular obliteration was induced by exposing 7-day-old rat pups (P7) for 2 or 5 days to 80% O(2). The redox state of the retina was assessed by measuring reduced glutathione and oxidative and nitrosative products malondialdehyde and nitrotyrosine. The role of NO on vasoobliteration was evaluated by treating animals with nitric oxide synthase (NOS) inhibitors (N-nitro-l-arginine; L-NA) and by determining NOS isoform expression and activity; the contribution of nitrosative stress was also determined in animals treated with the degradation catalyst of peroxynitrite FeTPPS or with the superoxide dismutase mimetic CuDIPS. eNOS, but not nNOS or iNOS, expression and activity were increased throughout the exposure to hyperoxia. These changes were associated with an early (2 days hyperoxia) decrease in reduced glutathione and increases in malondialdehyde and nitrotyrosine. CuDIPS, FeTPPS, and L-NA treatments for these 2 days of hyperoxia nearly abolished the vasoobliteration. In contrast, during 5 days exposure to hyperoxia when the redox state rebalanced, L-NA treatment aggravated the vasoobliteration. Interestingly, VEGFR-2 expression was respectively increased by NOS inhibition after short-term (2 days) exposure to hyperoxia and decreased during the longer hyperoxia exposure. Data disclose that the dual effects of NO on newborn retinal microvascular integrity in response to hyperoxia in vivo depend on the redox state and seem mediated at least in part by VEGFR-2.

Topics: Animals; Animals, Newborn; Antioxidants; Glutathione; Isoenzymes; Malondialdehyde; Metalloporphyrins; Microcirculation; Nitric Oxide; Nitric Oxide Synthase; Nitroarginine; Oxidation-Reduction; Oxidative Stress; Oxygen; Rats; Rats, Sprague-Dawley; Retina; Retinal Diseases; Retinal Vessels; Salicylates; Tyrosine; Vascular Endothelial Growth Factor Receptor-2

Previous work from our laboratory indicated that the bile salt sodium deoxycholate (NaDOC) induced apoptosis in cultured cells and in normal goblet cells of the colonic mucosa. We also reported that the normal-appearing flat mucosa of patients with colon cancer exhibited apoptosis resistance. Using immunofluorescence in conjunction with confocal microscopy, we now report that high physiological concentrations (0.5 mM) of NaDOC result in the formation of nitrotyrosine residues, a footprint for the formation of reactive nitrogen species, including peroxynitrite, in plasma membrane-associated proteins of HT-29 cells. Because peroxynitrite is formed from the reaction between nitric oxide and superoxide anion, we specifically looked at the role of nitric oxide and superoxide anion in NaDOC-induced apoptosis. Pretreatment of cells with the inhibitor/antioxidants, N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase, copper (II) 3,5-diisopropyl salicylate hydrate, a superoxide dismutase mimetic compound, and Trolox, a water-soluble analog of alpha-tocopherol, alone or in combination, sensitized cells to apoptosis induced by 0.5 mM NaDOC. These results suggest that nitric oxide may be part of a signaling pathway that is responsible for apoptosis resistance. The results also indicate that nitric oxide does not appear to protect cells against NaDOC-induced apoptosis by scavenging superoxide anion.

Topics: Apoptosis; Bile Acids and Salts; Colonic Neoplasms; Deoxycholic Acid; Enzyme Inhibitors; Fluorescent Antibody Technique; Free Radical Scavengers; Humans; Microscopy, Confocal; NG-Nitroarginine Methyl Ester; Nitrates; Nitric Oxide; Nitric Oxide Synthase; Oxidative Stress; Salicylates; Superoxides; Tumor Cells, Cultured; Tyrosine