Using confocal microscopy, we investigated mechanisms underlying loss of plasma membrane integrity during necrotic death of cultured hepatic sinusoidal endothelial cells exposed to 2.5 mM
potassium cyanide (chemical
hypoxia). After 2-3 h, the anionic fluorophore
calcein abruptly began to enter the cytosol, and nuclei labeled with cationic
propidium after another 2-5 min. As
calcein permeated, growth of
blebs on the plasma membrane accelerated.
Lucifer yellow, another anionic fluorophore, entered identically to
calcein, whereas high molecular weight
dextrans (40-2000 kDa) entered like
propidium.
Glycine slowed, but did not prevent
calcein entry, whereas permeation of
propidium and high molecular weight
dextrans was blocked completely by
glycine. These findings suggest that opening of a
glycine-sensitive organic
anion channel, or death channel, precipitates a metastable state characterized by rapid cell swelling and
bleb growth. This metastable state culminates in non-specific breakdown of the plasma membrane permeability barrier and irreversible cell death.