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 (M_w 314 Da), YO-PRO-1 (M_w 375 Da), and POPO-3 (M_w 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.