EFFECTS OF CA2+ DEREGULATION ON MITOCHONDRIAL-MEMBRANE POTENTIAL AND CELL VIABILITY IN NUCLEATED CELLS FOLLOWING LYRIC COMPLEMENT ATTACK

We have previously shown [Papadimitriou JC. Ramm LE. Drachenberg CB. Trump BF. Shin ML. (1991) J. Immunol., 147, 212-217] that formation of lytic C5b-9 channels on Ehrlich ascites tumor cells induced rapid depletion of adenine nucleotides associated with prelytic leakage preceding cell death. Extracellular Ca2+ concentration ([Ca2+](e)) reduction by chelation markedly delayed the onset of cell death, although the adenine nucleotide leakage was enhanced. In the present study, we examined the temporal relationships between ionized cytosolic Ca2+ ([Ca2+](i)), mitochondrial membrane potential (Delta Psi(m)) and cell death in individual cells by digital imaging fluorescence microscopy (DIFM), during the earliest phase of C5b-9 attack. The results showed an immediate, > 20-fold rise in [Ca2+](i), rapidly followed by dissipation of Delta Psi(m) and subsequent acute cell death. These events were markedly delayed by chelation of Ca-e(2+), but not by nominally Ca2+ free medium. Differing from previous reports indicating propidium iodide labeling of viable cells bearing C5b-9 channels, with DIFM we observed nuclear fluorescence with that marker only in association with cell death. These findings indicate that Ca2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca2+](i), as well as for the rapid loss of Delta Psi(m), followed by acute cell death. When this [Ca2+](i) increase is prevented, the cell death is probably related to metabolic depletion.