Superoxide: a major factor for stress protein induction in reoxygenation injury in the intestinal cell line Caco-2.
OBJECTIVE
Acute intestinal ischemia is followed by cellular destruction and loss of mucosal barrier function. Posthypoxic injury of cellular proteins leads to the synthesis of heat shock proteins. The role of oxygen radicals in this process, however, is not fully established.


METHODS
In the present study, using the intestinal cell line Caco-2, we investigated the relationship between the synthesis of the heat shock protein HSP70, detected by Western blot and oxygen radicals as well as lactate dehydrogenase (LDH) release, as measured in photometrical tests.


RESULTS
Various periods of hypoxia and 30 min of reoxygenation resulted in an increased generation of superoxide as measured by the tetrazolium base 3-(4, 5-dimethylthiazol-2-yl)2,5-diphenyltetrazoliumbromide. The inhibitor of superoxide dismutase (SOD), diethyldithiocarbamate (DDC) increased and addition of SOD decreased intracellular superoxide levels. HSP70 synthesis was detectable after 2 h of hypoxia. Similar to superoxide production, DDC increased and SOD reduced the HSP70 synthesis. In contrast, the increased LDH release from the cells observed after hypoxia was not significantly altered by DDC and SOD.


CONCLUSIONS
The production of superoxide correlates with HSP70 induction, but not with LDH release. We conclude that hypoxia/reoxygenation induces heat shock protein production, a result of protein damage, by increased superoxide generation, whereas superoxide does not correlate with membrane damage in Caco-2 cells.
Gebhardt
B R
BR
Department of Pediatrics, 2nd Medical Department, Johann Wolfgang Goethe University, Frankfurt, Germany.
Ries
J
J
Caspary
W F
WF
Boehles
H
H
Stein
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism; D015153:Blotting, Western; D018938:Caco-2 Cells / Q000378:metabolism* / Q000473:pathology; D002851:Chromatography, High Pressure Liquid; D004050:Ditiocarb / Q000494:pharmacology; D018840:HSP70 Heat-Shock Proteins / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D006358:Hot Temperature; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism / Q000473:pathology; D007424:Intracellular Fluid / Q000378:metabolism; D018384:Oxidative Stress / Q000502:physiology; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D013482:Superoxide Dismutase / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D013481:Superoxides / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D013481:Superoxides; D000255:Adenosine Triphosphate; D004050:Ditiocarb; D013482:Superoxide Dismutase