Reactive oxidizing species produced near the plasma membrane induce apoptosis in bovine aorta endothelial cells

Many cytotoxic agents initiate apoptosis by generating reactive oxidizing s pecies (ROS). The goal of this study was to determine whether apoptosis cou ld be induced by initial reactions of ROS near the plasma membrane. Bovine aorta endothelial cells (BAEC) were illuminated with evanescent wave visibl e radiation, which has limited penetration into the basal surface of cells, or by trans-radiation. Imaging of fluorescent dyes localizing in the plasm a membrane, mitochondria, or nucleus confirmed that evanescent wave radiati on excited only dyes in and near the plasma membrane. Singlet oxygen, an RO S generated by photosensitization, has a very short lifetime, ensuring that it oxidizes molecules residing in or very close to the plasma membrane whe n evanescent wave radiation is used. Cells with condensed nuclei were consi dered apoptotic and were quantified after treatment with varying doses of l ight. Annexin V staining without propidium iodide staining confirmed that t hese cells were apoptotic. The doses required to induce apoptosis using eva nescent wave radiation were 10-fold greater than those needed for trans-irr adiation. Quantitative analysis of the evanescent wave penetration into cel ls supports a mechanism in which the singlet oxygen created near the plasma membrane, rather than at intracellular sites, was responsible for initiati on of apoptosis. (C) 2000 Academic Press.