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 Ca(2+) overload-induced toxicity, reflects three sequential changes in plasmalemmal permeability. MTX initially activates Ca(2+)-permeable, nonselective cation channels (CaNSC) and causes a massive increase in cytosolic free Ca(2+) concentration ([Ca(2+)](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 Ca(2+)-binding proteins for Ca(2+) and Sr(2+) vs. Ba(2+) 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 Ca(2+) with either Sr(2+) or Ba(2+) 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 Ba(2+) 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 Ca(2+)-containing solutions, were poorly defined and greatly reduced in number in the presence of Ba(2+). Taken together, these results suggest that intracellular high-affinity Ca(2+)-binding proteins are involved in the MTX-induced changes in plasmalemmal permeability that are responsible for cell demise.
Wisnoskey
Brian J
BJ
Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, OH 44109-1998, USA.
Estacion
Mark
M
Schilling
William P
WP
D000818:Animals; D001011:Aorta / Q000166:cytology; D001464:Barium / Q000493:pharmacokinetics; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000493:pharmacokinetics; D002413:Cations, Divalent / Q000378:metabolism; D002417:Cattle; D016923:Cell Death / Q000187:drug effects*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D008387:Marine Toxins / Q000494:pharmacology*; D018715:Microscopy, Video; D009336:Necrosis; D035964:Oxocins / Q000494:pharmacology*; D013324:Strontium / Q000493:pharmacokinetics; D000095702:Polyether Toxins
D001464:Barium; D002118:Calcium; D002413:Cations, Divalent; D008387:Marine Toxins; D035964:Oxocins; D013324:Strontium; C020394:maitotoxin; D000095702:Polyether Toxins