Calcium dependence of phalloidin-induced liver cell death

The role of Ca2+ in toxic liver cell death was studied with primary cultures of adult rat hepatocytes. Within 1 hr of exposure to phalloidin, a bicyclic heptapeptide isolated from the mushroom Amanita pahlloides, at 50 micrograms/ml, 60--70% of the cells were dead (trypan blue stainable). There was no loss of viability of the same cells exposed to phalloidin in culture medium devoid of Ca2+. A marked structural alteration of the surface of the phalloidin-treated hepatocytes characterized by innumerable evaginations seen by scanning electron microscopy occurred in the presence or absence of Ca2+. Pretreatment of the cells with cytochalasin B at 10 micrograms/ml prevented the surface alteration and the death of the cells in Ca2+ medium. Exposure of the cells to phalloidin in the absence of Ca2+ followed by exposure to cytochalasin B and then to Ca2+ also prevented the cell death. These results suggest a two-step mechanism by which phalloidin causes liver cell death. Initially phalloidin interacts in a Ca2+-independent process with cell membrane-associated actin. The second step is a Ca2+-dependent process that most likely represents an increased influx of Ca2+ across a compromised cell membrane permeability barrier and down the steep concentration gradient that exists between the outside and inside of the cell. These results strengthen the hypothesis that disturbances in Ca2+ homeostasis induced in vivo by a variety of hepatotoxins are causally related to liver cell death.