MULTISITE MEASUREMENT PO2 IN RAT BRAIN

大鼠大脑中 PO2 的多点测量

• 批准号: 6206500
• 负责人: Oleg Grinberg
• 金额: $ 1.22万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6206500
• 关键词: Mammalia; biological products; biomaterials; biomedical resource; environmental toxicology; enzymes; hematology

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.

我们在此报告使用捕集剂 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 超氧自由基）。 我们之前已经控制了文化，并且 当受到环境相关水平的刺激时水平升高 亚砷酸盐。 我们随后发现信号的很大一部分 检测到的（18%）来自细胞内部。 EPR 信号来自 通过添加钆扩大细胞外室 与留在细胞外空间的 detapac 连接。 与该代理人非常接近的激进分子的范围扩大到超出了该代理人的范围 EPR 光谱中的视场。 任何残留的 EPR 信号都可以 归因于细胞内区室。 添加类似的 硝基氧探针（TEMPOL）对类似细胞样品的浓度 导致细胞内的贡献仅占总体的 2-3% 检测到信号。 进行实验是为了确保我们的 观察结果无法用以下方面来解释：(i) 探针在各个隔室中的溶解度； (二) 不充分 去除细胞外信号（我们采用了多种技术）； (iii) 内皮细胞探针之间的还原率差异 细胞。 我们还在细胞系统中添加了预制的 DMPO-OH 并测量了 这 细胞内和细胞外区室之间的分布。 我们的 研究表明，内皮细胞产生活性氧 细胞内位置，可通过 EPR 实时观察 自旋捕获。 ROS 的产生率似乎很高 和低还原率（与其他细胞系统相比）有助于 我们有能力测量这些细胞内自由基，但这 情况可能不是一个孤立的案例和细胞内自旋捕获 在其他细胞系统中也是可能的。