Metabolic inhibition potentiates oxidant-mediated injury of the endothelial cytoskeleton.
The aim of this study was to determine if exposure of cells to oxidants and metabolic inhibition, conditions which are present during ischemia-reperfusion, act synergistically to produce cytoskeletal disruption. Adherent bovine pulmonary artery endothelial cells were subjected to metabolic inhibition by incubating the cells in glucose-free buffer containing 650 nM oligomycin for 2 hr. Cells were rescued from metabolic inhibition by washing the cells with buffer containing 5.5 mM glucose and were simultaneously exposed to 0, 25, 100, or 5000 microM H2O2. At various time points during recovery from metabolic inhibition the microfilaments and microtubules were stained for microscopic evaluation. Intracellular ATP levels were determined by the luciferin/luciferase assay. Cells that were not metabolically inhibited showed minimal microfilament disruption at lower doses of H2O2. Cells that were subjected to metabolic inhibition but not exposed to H2O2 showed microfilament disruption after 2 hr of metabolic inhibition, but normal microfilament architecture was seen in over 95% of the cells by 1 hr after recovery from metabolic inhibition. Cells that were metabolically inhibited and then exposed to doses of H2O2 as low as 25 microM showed marked microfilament disruption at 1 and 2 hr after the metabolic inhibition was relieved. The microtubules were distorted, but did not depolymerize except when exposed to concentrations of H2O2 > or = 5000 microM. Metabolic inhibition appeared to selectively potentiate the effect of subsequent oxidant exposure and the potentiation largely affected microfilament architecture with secondary effects on microtubule morphology and endothelial cell shape.
Delius
R E
RE
Department of Surgery, Ann Arbor Veterans Affairs Medical Center, Michigan, USA.
Dezan
J
J
Omann
G M
GM
Hinshaw
D B
DB
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000648:ultrastructure*; D004357:Drug Synergism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology*; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008856:Microscopy, Fluorescence; D009840:Oligomycins / Q000494:pharmacology*; D011651:Pulmonary Artery / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology
D009840:Oligomycins; D000255:Adenosine Triphosphate; D006861:Hydrogen Peroxide