| 11 th Annual Pediatric Critical Care Colloquium |
| Session/Time | Poster/Thu, 4:30 - 6:30 PM |
| Title | Cytokine Induced Stimulation of Cu,Za SOD Activity with Associated Fragmentation of SOD |
| Author | R Pretzlaff, V Xue, A Salzman, MD, FCCM |
| Affiliation | Division of Critical Care, University of Cincinnati College of Medicine, Cincinnati, OH |
| Introduction | Lung injury results in the activation of the inflammatory cascade and increased cellular damage. Protection from cytokine mediated damage includes the antioxidant superoxide dismutase- (S OD). Previous reports show an increase in mitochondrial SOD activity (iMnSOD) upon stimulation with hyperoxia and cytokines, but no increase in cytosolic SOD (Cu,ZnSOD) activity. We report an increase in Cu,ZnSOD activity upon cytokine stimulation and the presence of Cu,ZnSOD fragmentation. Fra-mentation of Cu,ZnSOD has been shown to occur following - alycation of the enzyme |
| Method | A549 cells were grown to contlu'ency and treated with placebo, IL-1 (100 units/ml), or TNF-A (1000 units/rr ). SOD activit was measured by Xanthinc-Xanthine oxidase inhibition as detern-Lined ..by Cytochrome C reduction. Cu,ZnSOD activity was determined by measuring total SOD and MNSCD activity and subtracting the MNSOD activity from the total. Fracmentation of Cu,ZnSOD was determined by Western analysis with PAGE on 18% Tris-alycine gels with Cu,ZnSOD antibodies and exposure with ECL' reagent.. Also, purified Cu,ZnSOD (Sicma) was alycated by incubation with alyceraldehyde 3 phosphate. |
| Result | Total SOD activity increased in the IL- I -roup by 420% (p< .05) and in the TNFcc group by 425% (p<.05), but only 68% in controls at 72 hours. Cu,ZnSOD activity increased in the IL- I and TNFA aroups by 332% (p<.05) and 330% (NS) compared v,,ith an increase of 58% in controls at 72 hours. Similar increases 1-vere seen in all at 48 hours. This study demonstrates an increase in total and Cu, ZnSOD (Sigma) was in cytokine stimulated lung epithelial cells. |
| Conclusion | Western analysis of cytokine stimulated cells demonstrates increased fragmentation of Cu,ZnSOD comparted to controls at 48 and 72 hours. analysis of glycated putified Cu, ZnSOD demonstrates a decrease in activity with increased glycatoin consistent with SOD fragmentatoin. Clycation of CU, ZnSOD may be due to the increased glycolysis and decreased mitochodrial oxidatoin seen in cytokine stimulated cells. |
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