Maitotoxin-induced membrane blebbing and cell death in bovine aortic endothelial cells.
BACKGROUND
Maitotoxin, a potent cytolytic agent, causes an increase in cytosolic free Ca2+ concentration ([Ca2+]i) via activation of Ca2+-permeable, non-selective cation channels (CaNSC). Channel activation is followed by formation of large endogenous pores that allow ethidium and propidium-based vital dyes to enter the cell. Although activation of these cytolytic/oncotic pores, or COP, precedes release of lactate dehydrogenase, an indication of oncotic cell death, the relationship between CaNSC, COP, membrane lysis, and the associated changes in cell morphology has not been clearly defined. In the present study, the effect maitotoxin on [Ca2+]i, vital dye uptake, lactate dehydrogenase release, and membrane blebbing was examined in bovine aortic endothelial cells.


RESULTS
Maitotoxin produced a concentration-dependent increase in [Ca2+]i followed by a biphasic uptake of ethidium. Comparison of ethidium (Mw 314 Da), YO-PRO-1 (Mw 375 Da), and POPO-3 (Mw 715 Da) showed that the rate of dye uptake during the first phase was inversely proportional to molecular weight, whereas the second phase appeared to be all-or-nothing. The second phase of dye uptake correlated in time with the release of lactate dehydrogenase. Uptake of vital dyes at the single cell level, determined by time-lapse videomicroscopy, was also biphasic. The first phase was associated with formation of small membrane blebs, whereas the second phase was associated with dramatic bleb dilation.


CONCLUSIONS
These results suggest that maitotoxin-induced Ca2+ influx in bovine aortic endothelial cells is followed by activation of COP. COP formation is associated with controlled membrane blebbing which ultimately gives rise to uncontrolled bleb dilation, lactate dehydrogenase release, and oncotic cell death.
Rammelkamp Center for Education and Research, Metrohealth Medical Center, and Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio, USA. mestacion@research.metrohealth.org
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D000818:Animals; D001011:Aorta / Q000166:cytology; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism; D002417:Cattle; D016923:Cell Death; D002462:Cell Membrane / Q000187:drug effects* / Q000648:ultrastructure; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D045022:Lactate Dehydrogenases / Q000378:metabolism; D008387:Marine Toxins / Q000633:toxicity*; D035964:Oxocins / Q000633:toxicity*; D000095702:Polyether Toxins
D002118:Calcium; D005456:Fluorescent Dyes; D045022:Lactate Dehydrogenases; D008387:Marine Toxins; D035964:Oxocins; C020394:maitotoxin; D000095702:Polyether Toxins