Thom S R, Ischiropoulos H
University of Pennsylvania Medical Center, Philadelphia, PA, USA.
Res Rep Health Eff Inst. 1997 Dec(80):1-19; discussion 21-7.
The purpose of this study was to determine whether platelets and vascular endothelial cells would liberate nitric oxide free radical (NO)* and NO-derived oxidant species after exposure to carbon monoxide (CO) at concentrations up to 100 parts per million (ppm). We hypothesized that exposure to environmentally relevant concentrations of CO would increase production of agents that may be involved in human pathological processes, such as atherosclerosis. Platelets obtained from rats released NO when incubated with CO, but CO did not increase platelet nitric oxide synthase activity. Platelets released comparable NO levels when they were exposed to CO in vitro and when taken from rats that had been exposed to CO. Partial pressures of CO as low as 10 ppm could successfully compete with NO for intraplatelet binding sites in in vitro studies. We conclude that CO enhanced the release of NO from platelets because it inhibited NO sequestration by intraplatelet binding sites, and that this phenomenon can occur with exposure to CO concentrations found in the environment. Bovine pulmonary artery endothelial cells released NO in response to CO exposure. Carbon monoxide did not affect the transport of L-arginine across the plasma membrane or nitric oxide synthase activity; therefore, the mechanism appeared to be based on a disturbance of intracellular NO sequestration. Cells incubated with CO also released into the surrounding medium peroxynitrite, an NO-derived oxidant, based on oxidation of dihydrorhodamine 123 and p-hydroxyphenylacetic acid. Peroxynitrite-mediated oxidative stress to endothelial cells was identified as increased concentrations of nitrotyrosine in cell lysates, and by measuring the release of radioactive chromium. Carbon monoxide caused an acute injury when cells were continuously exposed for 4 hours, and a delayed injury when cells were exposed for 2 hours. Delayed injury was documented by leakage of radioactive chromium and by uptake of a vital fluorescent stain, ethidium homodimer-1, between 6 and 20 hours after CO exposure. Oxidative stress caused by CO exhibited several unique aspects because CO exposure did not alter the cellular content of reduced sulfhydryls nor did CO augment oxidative stress caused by superoxide, hydrogen peroxide, or a flux of NO. We concluded that concentrations of CO achieved in vivo when humans are exposed to CO concentrations found in the environment can cause endothelial cells to liberate NO and NO-derived oxidants, and that these products can adversely affect cell physiology.
本研究的目的是确定血小板和血管内皮细胞在暴露于浓度高达百万分之一百（ppm）的一氧化碳（CO）后是否会释放一氧化氮自由基（NO）*和NO衍生的氧化物种。我们假设，暴露于环境相关浓度的CO会增加可能参与人类病理过程（如动脉粥样硬化）的物质的产生。从大鼠获得的血小板在与CO孵育时会释放NO，但CO不会增加血小板一氧化氮合酶的活性。当血小板在体外暴露于CO时以及从暴露于CO的大鼠体内获取时，它们释放的NO水平相当。在体外研究中，低至10 ppm的CO分压能够成功与NO竞争血小板内的结合位点。我们得出结论，CO增强了血小板中NO的释放，因为它抑制了血小板内结合位点对NO的隔离，并且这种现象在暴露于环境中发现的CO浓度时就会发生。牛肺动脉内皮细胞在暴露于CO时会释放NO。一氧化碳不影响L-精氨酸跨质膜的转运或一氧化氮合酶的活性；因此，其机制似乎基于细胞内NO隔离的紊乱。基于二氢罗丹明123和对羟基苯乙酸的氧化，与CO孵育的细胞还会向周围培养基中释放过氧亚硝酸盐，一种NO衍生的氧化剂。过氧亚硝酸盐介导的对内皮细胞的氧化应激表现为细胞裂解物中硝基酪氨酸浓度的增加，以及通过测量放射性铬的释放来确定。当细胞连续暴露4小时时，一氧化碳会导致急性损伤，而当细胞暴露2小时时会导致延迟性损伤。延迟性损伤通过放射性铬的泄漏以及在CO暴露后6至20小时之间对一种活性荧光染料乙锭同二聚体-1的摄取来记录。由CO引起的氧化应激表现出几个独特的方面，因为CO暴露不会改变细胞内还原巯基的含量，CO也不会增强由超氧化物、过氧化氢或NO通量引起的氧化应激。我们得出结论，当人类暴露于环境中发现的CO浓度时，体内达到的CO浓度会导致内皮细胞释放NO和NO衍生的氧化剂，并且这些产物会对细胞生理产生不利影响。
