pH-dependent oxidant production following inhibition of the mitochondrial electron transport chain in pulmonary endothelial cells.
We investigated the effect of changes in intracellular pH (pHi) and Na/H antiport activity on intracellular oxidant production in human pulmonary artery endothelial cells (HPAEC) following disruption of cellular metabolism. Oxidant production was measured with oxidant-sensitive probes (2',7'-dichlorofluorescein diacetate [H2DCF], dihydroethidium [DHE]) following treatment with inhibitors of mitochondrial electron transport and glycolysis (antimycin/2-deoxyglucose, A/D). A/D treatment increased oxidant production in a dose-dependent fashion over 2 hours. Omission of 2-deoxyglucose did not alter the magnitude of oxidant production. Inhibition at more proximal sites in the mitochondrial electron transport chain inhibited oxidant production. These data suggested that the mitochondrial electron transport chain was the source of oxidant production. Fluorescent imaging experiments confirmed the mitochondrial origin of the increased oxidant production under these conditions. Maneuvers that reduced pHi and inhibited Na/H exchange (acidosis, specific Na/H exchange inhibitors) attenuated oxidant production, whereas maneuvers that raised pHi (monensin) potentiated oxidant production. The results with the pH-insensitive probe (DHE) confirmed that oxidant production was pH-dependent. Oxidant production preceded significant loss of cell viability at 6 h following A/D treatment. These results demonstrate that oxidant production following inhibition of mitochondrial electron transport in HPAEC is pH-dependent and may contribute to endothelial cell injury by increasing endogenous oxidative stress.
Pulmonary Disease Division, University of Pennsylvania School of Medicine, VA Medical Center, Philadelphia, Pennsylvania, USA. michael.cutaia@med.va.gov
M
M
Cutaia
J
J
Kroczynski
K
K
Tollefson
D000968:Antimycin A / Q000031:analogs & derivatives* / Q000494:pharmacology; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000494:pharmacology; D004579:Electron Transport / Q000187:drug effects; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D005456:Fluorescent Dyes; D006019:Glycolysis / Q000187:drug effects; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D016877:Oxidants / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000037:antagonists & inhibitors / Q000378:metabolism
D005456:Fluorescent Dyes; D016877:Oxidants; D017923:Sodium-Hydrogen Exchangers; C032456:antimycin; D000968:Antimycin A; D003847:Deoxyglucose