Delius R E, Dezan J, Omann G M, Hinshaw D B
Department of Surgery, Ann Arbor Veterans Affairs Medical Center, Michigan, USA.
J Surg Res. 1996 Jul 15;64(1):79-88. doi: 10.1006/jsre.1996.0310.
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.
本研究的目的是确定细胞暴露于氧化剂和代谢抑制（缺血再灌注期间存在的情况）是否会协同作用导致细胞骨架破坏。将贴壁的牛肺动脉内皮细胞置于含有650 nM寡霉素的无葡萄糖缓冲液中孵育2小时，使其受到代谢抑制。通过用含有5.5 mM葡萄糖的缓冲液洗涤细胞，使细胞从代谢抑制中恢复，同时将细胞暴露于0、25、100或5000 μM的过氧化氢中。在从代谢抑制恢复的不同时间点，对微丝和微管进行染色以进行显微镜评估。通过荧光素/荧光素酶测定法测定细胞内ATP水平。未受到代谢抑制的细胞在较低剂量的过氧化氢作用下微丝破坏最小。受到代谢抑制但未暴露于过氧化氢的细胞在代谢抑制2小时后出现微丝破坏，但从代谢抑制恢复1小时后，超过95%的细胞微丝结构正常。受到代谢抑制然后暴露于低至25 μM过氧化氢剂量的细胞在代谢抑制解除后1小时和2小时显示出明显的微丝破坏。微管发生扭曲，但除非暴露于≥5000 μM的过氧化氢浓度下，否则不会解聚。代谢抑制似乎选择性地增强了随后氧化剂暴露的作用，这种增强主要影响微丝结构，对微管形态和内皮细胞形状有次要影响。
