Calcium dependence of bleb formation and cell death in hepatocytes

Calcium dependence of bleb formation and cell death was evaluated in rat hepatocytes following ATP depletion by metabolic inhibition with KCN and iodoacetate ('chemical hypoxia'). Cytosolic free Ca²⁺ was measured in single cells by ratio imaging of Fura-2 fluorescence using multiparameter digitized video microscopy. Cells formed surface blebs within 10 to 20 minutes after chemical hypoxia and most cells lost viability within an hour. An increase of cytosolic free Ca²⁺ was not required for bleb formation to occur. One to a few minutes prior to the onset of cell death, free Ca²⁺ increased rapidly in high Ca²⁺ buffer (1.2 mM) but not in low Ca²⁺ buffer (<1 μM). In either buffer, the rate of cell killing was the same. As the onset of cell death was approached in both high and low Ca²⁺ buffers, Fura-2 began to leak from the cells at an accelerating rate indicating rapidly increasing plasma membrane permeability. In high Ca²⁺ buffer, cytosolic free Ca²⁺ increased in parallel with dye leakage. No regional changes in cytosolic free Ca²⁺ were observed during this metastable period of increased membrane permeability. In many experiments, actual rupture of cell surface blebs could be observed which led to micron-size discontinuities of the cell surface and cell death. We conclude that a metastable period characterized by increasing plasma membrane permeability marked the onset of cell death in cultured hepatocytes which culminated in rupture of a cell surface bleb. An increase of cytosolic free Ca²⁺ was not required for the metastable state to develop or cell death to occur.