MULTISITE MEASUREMENT PO2 IN RAT BRAIN

我们在这里报告使用捕捉剂DMPO获得的结果 检测内皮细胞产生的自由基(DMPO 连接到羟基和 超氧阴离子自由基)。我们以前曾控制过文化，而且 在与环境相关的环境污染水平刺激下水平升高 亚砷酸盐。我们随后发现，信号中的很大一部分 检测到的(18%)起源于细胞内。EPR信号来自 细胞外隔室通过加入Gd而变宽 与保留在细胞外空间的地达帕克有关。 与该试剂密切接近的自由基变宽，超出 EPR频谱中的视场。任何剩余EPR信号都可以是 归因于细胞内的隔间。添加类似的 氮氧化物探针(Temol)对相似细胞样品的浓度 导致细胞内部对整体的贡献仅为2%-3% 检测到信号。我们进行了实验，以确保我们的 观察结果不能用以下方面来解释：(一)不同于 探头在不同隔间中的溶解度；(2)不足 去除细胞外信号(我们采用了几种技术)； (3)内皮细胞探针之间的减少率差异 细胞。我们还向细胞系统中加入了预制的DMPO-OH，并测量了 这个 细胞内室和胞外室之间的分布。我们的 研究表明，内皮细胞在 细胞内位置，可由EPR实时观察 自旋陷阱。看来，ROS的高产量很可能 以及较低的减灭率(与其他细胞系统相比) 我们测量这些细胞内自由基的能力，但这 这种情况可能不是孤立的，细胞内的自旋捕获 可能在其他蜂窝系统中也是可能的。

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.