Reactive oxygen species in reoxygenation injury of rat brain capillary endothelial cells.
OBJECTIVE
To clarify the mechanism of anoxia/reoxygenation (A/R) injury of rat brain capillary endothelial cells (BCEC).


METHODS
BCEC isolated from Sprague-Dawley rats by enzymatic treatment and centrifugation were subjected to anoxia (95% N2, 5% CO2) for 20 minutes and then to reoxygenation (95% air, 5% CO2) for 3 hours. Enzyme inhibitors, including oxypurinol, indomethacin, and N(G)-nitro-L-arginine methyl ester, or specific free-radical scavengers, such as superoxide dismutase, catalase, and the ferric iron chelator deferoxamine, were added before A/R injury. The BCEC were incubated in a range of Ca2+ concentrations from 1 to 0.01 mmol/L during A/R injury. Cytotoxicity was assayed by release of intracellular lactate dehydrogenase (LDH).


RESULTS
With A/R injury, LDH release from the control group (no protective agents) significantly increased (44.8 +/- 3.3%), compared with a small increase in a normoxic group. BCEC treated with oxypurinol, indomethacin, or N(G)-nitro-L-arginine methyl ester showed suppression of LDH release. LDH release was almost totally suppressed by superoxide dismutase and partially by catalase or deferoxamine. The LDH release was partly dependent on calcium concentration.


CONCLUSIONS
BCEC subjected to A/R become potent generators of free radicals, especially superoxide anion. Free radical production depends on both xanthine oxidase and cyclooxygenase pathways. Peroxynitrite and extracellular Ca2+ both contribute importantly to reoxygenation injury of BCEC.
Wu
S
S
Department of Neurosurgery, Kobe University School of Medicine, Japan.
Tamaki
N
N
Nagashima
T
T
Yamaguchi
M
M
D000818:Animals; D002118:Calcium / Q000502:physiology; D002196:Capillaries / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D002374:Catalase / Q000494:pharmacology; D002478:Cells, Cultured; D002560:Cerebrovascular Circulation / Q000187:drug effects / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D003676:Deferoxamine / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016166:Free Radical Scavengers / Q000494:pharmacology; D007213:Indomethacin / Q000494:pharmacology; D008297:Male; D019331:NG-Nitroarginine Methyl Ester / Q000494:pharmacology; D010101:Oxygen Consumption / Q000502:physiology; D010117:Oxypurinol / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D013482:Superoxide Dismutase / Q000494:pharmacology
D002614:Chelating Agents; D004791:Enzyme Inhibitors; D016166:Free Radical Scavengers; D017382:Reactive Oxygen Species; D002374:Catalase; D013482:Superoxide Dismutase; D010117:Oxypurinol; D003676:Deferoxamine; D002118:Calcium; D019331:NG-Nitroarginine Methyl Ester; D007213:Indomethacin