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.
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