Extracellular superoxide dismutase interaction with endothelial nitric oxide synthase in neonatal rat lung does not occur within the caveolin

Extracellular Superoxide Dismutase (ECSOD), the only extracellular scavenger of the free radical superoxide (O2·- ) is developmentally regulated. ECSOD is intracellular therefore inactive in fetal and neonatal rabbit lung. Secretion of active ECSOD to the extracellular compartment occurs with age after birth[1]. ECSOD is believed to protect nitric oxide (NO·) and preserve its function at the cellular and tissue level in newborn lung[2, 3]. ECSOD is modulated by oxygen tension in a similar fashion as endothelial NOS (eNOS) in neonatal lung. ECSOD co-localizes with eNOS in the endothelium in adult ferret heart[4]. We co-localized ECSOD and eNOS in rat lung tissues by immunohistochemistry (IHC) and immunohistofluorescent (IF) techniques. ECSOD localizes to epithelial cells in neonatal and two day old rat lung and the staining for eNOS is the same. By IF double labeling, co-localization of ECSOD and eNOS was strongly present in neonatal rat lung, less apparent in 2day rat lung and showed limited co-localization in adult rat lung.; Endothelial NOS is a membrane bound protein located in caveolae that binds with caveolin-1 when inactive[5]. We studied co-localization of ECSOD with caveolin protein in rat lung. There was no co-localization of ECSOD with caveolin protein in rat lung tissue by IF. For further studies of ECSOD and eNOS proteins, we immunoprecipitated ECSOD with eNOS using whole lung fresh tissue, ECSOD does not co-precipitate with eNOS. The converse was also found; in ECSOD immunoprecipitated protein, eNOS was not detected. According to the data, these two proteins may not be in physical bond to each other. In Western blot analysis of sucrose gradient caveolin fractions, ECSOD was not detectable in the caveolin fractions where inactive eNOS is located. The fact that ECSOD was not present in caveolae, where eNOS is located, may imply that ECSOD co-localizes with active (phosphorylated) eNOS and not with inactive (non-phosphorylated) eNOS. We suggest that ECSOD may act as an intracellular carrier protein of NO· before its secretion to the extracellular compartment. The Interaction of ECSOD with eNOS in the phosphorylated state or via other mediators associated with eNOS, and its product. NO· requires further study.