Abstract
Here we developed a new cell-permeative lucigenin derivative, 10,10′-dimethyl-9,9′-biacridinium bis(monomethyl terephthalate) (MMT), to detect intracellular superoxide production. Both MMT and lucigenin were specific to superoxide among reactive oxygen species tested. Although lucigenin barely penetrated into cells, MMT accumulated in mitochondria in a variety of cells such as neutrophils. By employing MMT, we found that, upon activation of neutrophils with phorbol myristate acetate, superoxide was generated extracellularly as well as intramitochondrially and that such intramitochondrial superoxide production was dependent on oxidative phosphorylation. We also found that, during apoptosis, superoxide was gradually produced in mitochondria in association with phosphatidylserine exposure and that the kinetics of superoxide production was very heterogeneous at the single-cell level. Thus this study demonstrates that MMT could serve as a specific probe for intramitochondrial superoxide in either activated or apoptotic cells.
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