Intracellular generation of reactive oxygen species in endothelial cells exposed to anoxia-reoxygenation.
Reactive oxygen species (ROS) play an important role in the pathogenesis of ischemia-reperfusion injury. Extracellular H2O2 generation from bovine pulmonary artery endothelial cells (EC) is known to increase in response to anoxia-reoxygenation (A-R). To determine potential sources of intracellular ROS formation in EC in response to A-R, a fluorometric assay based on the oxidation of 2',7'-dichlorofluorescin was used. Intracellular ROS production declined 40% during 6 h of anoxia (P < 0.05). After A-R, the rates of intracellular ROS formation increased to 148 +/- 9% (P < 0.001) that of normoxic EC (100 +/- 3%). In EC exposed to A-R, allopurinol and NG-methyl-L-arginine (L-NMMA), inhibitors of xanthine oxidase (XO) and nitric oxide synthase (NOS), respectively, reduced intracellular ROS formation by 25 +/- 1% (P < 0.001) and 36 +/- 4% (P < 0.01). Furthermore, at low doses (i.e., 20 microM), deferoxamine and diethylenetriaminepentaacetic acid (DTPA) significantly inhibited intracellular ROS formation. However, at 100 microM, only deferoxamine caused further reduction in DCF fluorescence. In summary, EC respond to A-R by generating increased amounts of XO- and NOS-derived intracellular ROS. The inhibition, to a similar extent, caused by allopurinol and L-NMMA, as well as the effect of deferoxamine and DTPA suggest that the ROS detected is peroxynitrite. Based on these findings and previous work, we conclude that EC generate ROS in response to A-R from at least two different sources: a plasma membrane-bound NADPH oxidase-like enzyme that releases H2O2 extracellularly and XO, which generates intracellular O2-, which in turn may react with nitric oxide to form peroxynitrite.
Zulueta
J J
JJ
Pulmonary and Critical Care Division, Tupper Research Institute, New England Medical Center, Tufts University School of Medicine, Boston, Massachusetts 02111, USA.
Sawhney
R
R
Yu
F S
FS
Cote
C C
CC
Hassoun
P M
PM
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D000860:Hypoxia / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism*; D007502:Iron Chelating Agents / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D010100:Oxygen / Q000494:pharmacology*; D017382:Reactive Oxygen Species / Q000378:metabolism*; D014812:Vitamin K / Q000494:pharmacology; D019323:omega-N-Methylarginine / Q000494:pharmacology
D004791:Enzyme Inhibitors; D007502:Iron Chelating Agents; D017382:Reactive Oxygen Species; D014812:Vitamin K; D019323:omega-N-Methylarginine; D019001:Nitric Oxide Synthase; D010100:Oxygen