METABOLIC INHIBITION POTENTIATES OXIDANT-MEDIATED INJURY OF THE ENDOTHELIAL CYTOSKELETON

The aim of this study was to determine if exposure of cells to oxidant s and metabolic inhibition, conditions which are present during ischem ia-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 c ontaining 650 nM oligomycin for 2 hr. Cells were rescued from metaboli c inhibition by washing the cells with buffer containing 5.5 mM glucos e and were simultaneously exposed to 0, 25, 100, or 5000 mu M H2O2. At various time points during recovery from metabolic inhibition the mic rofilaments and microtubules were stained for microscopic evaluation. Intracellular ATP levels were determined by the luciferin/luciferase a ssay. Cells that were not metabolically inhibited showed minimal micro filament disruption at lower doses of H2O2. Cells that were subjected to metabolic inhibition but not exposed to H2O2 showed microfilament d isruption 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 an d then exposed to doses of H2O2 as low as 25 mu M showed marked microf ilament disruption at 1 and 2 hr after the metabolic inhibition was re lieved, The microtubules were distorted, but did not depolymerize exce pt when exposed to concentrations of H2O2 greater than or equal to 500 0 mu M. Metabolic inhibition appeared to selectively potentiate the ef fect of subsequent oxidant exposure and the potentiation largely affec ted microfilament architecture with secondary effects on microtubule m orphology and endothelial cell shape. (C) 1996 Academic Press, Inc.