Maitotoxin-induced cell death cascade in bovine aortic endothelial cells: divalent cation specificity and selectivity

The maitotoxin (MTX)-induced cell death cascade in bovine aortic endothelial cells (BAECs), a model for Ca²⁺ overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca²⁺-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_i). This is followed by the opening of large endogenous cytolytic/oncotic pores (COP) that allow molecules <800 Da to enter the cell. The cells then lyse not by rupture of the plasmalemma but through the activation of a "death" channel that lets large proteins (e.g., 140–160 kDa) leave the cell. These changes in permeability are accompanied by the formation of membrane blebs. In this study, we took advantage of the well-known differences in affinity of various Ca²⁺-binding proteins for Ca²⁺ and Sr²⁺ vs. Ba²⁺ to probe their involvement in each phase of the cell death cascade. Using fluorescence techniques at the cell population level (cuvette-based) and at the single-cell level (time-lapse videomicroscopy), we found that the replacement of Ca²⁺ with either Sr²⁺ or Ba²⁺ delayed both MTX-induced activation of COP, as indicated by the uptake of ethidium bromide, and subsequent cell lysis, as indicated by the uptake of propidium iodide or the release of cell-associated green fluorescent protein. MTX-induced responses were mimicked by ionomycin and were significantly delayed in BAPTA-loaded cells. Experiments at the single-cell level revealed that Ba²⁺ not only delayed the time to cell lysis but also caused desynchronization of the lytic phase. Last, membrane blebs, which were numerous and spherical in Ca²⁺-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba²⁺. Taken together, these results suggest that intracellular high-affinity Ca²⁺-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise. necrosis; vital dyes; membrane blebs; time-lapse videomicroscopy; fura 2