Source of oxygen free radicals produced by rat hepatocytes during postanoxic reoxygenation.
The aim of this study was to determine the cellular source of oxygen free radicals generated by isolated hepatocytes during post-anoxic reoxygenation. Superoxide anions (O2.-) were detected by lucigenin chemiluminescence. Cell damage was assessed by LDH release. During anoxia, the chemiluminescence decreased to background levels while LDH release increased 8-fold. During reoxygenation, O2.- formation increased 15-fold within 15 min then declined towards control levels. LDH release increased from 161 to 285 mU/min in the first 30 min of reoxygenation, then declined toward the control rate. Allopurinol, an inhibitor of the xanthine-xanthine oxidase system, did not inhibit O2.- formation nor LDH release. Antimycin, a mitochondrial complex III inhibitor that does not block O2.- formation, increased both O2.- generation and LDH release 82 and 133% respectively. Diphenyleneiodonium (DPI), a mitochondrial and microsomal NADPH oxidase inhibitor, reduced O2.- and LDH release 60-70%. SOD, which catalyzes the dismutation of O2.- to H2O2, was without effect on O2.- and LDH release, but TEMPO, a stable nitroxide which mimics SOD and easily penetrates the cell membrane, decreased O2.-86% without affecting LDH. These results suggest that mitochondria or microsomes are the principal sites of O2.- production during reoxygenation of isolated hepatocytes, whereas the cytosolic xanthine/xanthine oxidase system is apparently not involved.
Caraceni
P
P
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, PA 15261, USA.
Ryu
H S
HS
van Thiel
D H
DH
Borle
A B
AB
D000493:Allopurinol / Q000494:pharmacology; D000818:Animals; D000968:Antimycin A / Q000494:pharmacology*; D002478:Cells, Cultured; D003497:Cyclic N-Oxides / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008099:Liver / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000378:metabolism; D008930:Mitochondria, Liver / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013482:Superoxide Dismutase / Q000494:pharmacology; D013481:Superoxides / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000037:antagonists & inhibitors
D003497:Cyclic N-Oxides; D013481:Superoxides; D000493:Allopurinol; D000968:Antimycin A; D007770:L-Lactate Dehydrogenase; D013482:Superoxide Dismutase; D014969:Xanthine Oxidase; D010100:Oxygen; C003959:TEMPO