MULTISITE MEASUREMENT PO2 IN RAT BRAIN

我们在这里报告使用捕集剂DMPO获得的结果 为了检测由内皮细胞产生的自由基（DMPO 与羟基连接， 超氧自由基）。 我们以前有对照培养， 当受到环境相关水平的刺激时， 亚砷酸盐。 我们随后发现， 检测到（18%）来自细胞内。 EPR信号来自 细胞外室通过添加钆而扩大 与detapac相连，detapac留在细胞外空间。 靠近这种试剂的自由基变得更宽， EPR谱中的视场。 任何残留的EPR信号都可以 归因于细胞内区室。 添加类似物 氮氧化物探针（TEMPOL）浓度与相似细胞样品 导致细胞内对整体的贡献仅为2-3%， 检测到信号。 进行实验是为了确保我们的 观察结果不能用以下方面来解释：（一） 探针在各个隔室中的溶解度;（ii）不充分 去除细胞外信号（我们采用了几种技术）; (iii)内皮细胞的探针之间的降低率差异 细胞 我们还将预形成的DMPO-OH添加到细胞系统中，并测量 的 细胞内和细胞外区室之间的分布。 我们 研究表明，内皮细胞产生活性氧， 细胞内的位置，这可以通过EPR在真实的时间观察到 自旋捕获 很有可能高速率的活性氧产生 和低还原率（与其他细胞系统相比） 我们测量这些细胞内自由基的能力， 条件可能不是一个孤立的情况下，细胞内自旋捕获 在其他细胞系统中也是可能的。

We report here on results obtained using the trapping agent DMPO to detect free radicals produced by endothelial cells (both DMPO linked to hydroxyl and superoxide radicals). We have previously control cultures, and elevated levels when stimulated by environmentally relevant levels of arsenite. We subsequently found that a large component of the signals detected (18%) arose from within the cells. EPR signals from the extracellular compartment were broadened by addition of gadolinium linked to detapac, which remained in the extracellular space. Radicals in close proximity to this agent become broadened beyond the field of view in the EPR spectrum. Any residual EPR signal can be attributed to the intracellular compartment. Addition of similar concentrations of a nitroxide probe (TEMPOL) to similar cells samples resulted in only a 2-3% contribution from within cells to the overall signal detected. Experiments were undertaken to ensure that our observations could not be explained in terms of (i) differences on solubility of the probes in the various compartments; (ii) inadequate removal of the extracellular signal (we employed several techniques); (iii) differences in the reduction rate between probes by endothelial cells. We also added pre-formed DMPO-OH to cell systems and measured the distribution between intra- and extra-cellular compartments. Our studies show that endothelial cells produce reactive oxygen species at intracellular locations, which can be observed in real time by EPR spin-trapping. It seems likely that a high rate of production of ROS and low reduction rate (compared to other cells sytstems) contributed to our ability to measure these intracellular radicals, but this condition may not be an isolated case and intracellular spin-trapping may be possible in other cell systems.