Effects of Ca 2+ deregulation on mitochondrial membrane potential and cell viability in nucleated cells following lytic complement attack

Abstract

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 Ca 2+ concentration ([Ca 2+] 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 Ca 2+ ([Ca 2+] i), mitochondrial membrane potential (Δψ 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 [Ca 2+] i, rapidly followed by dissipation of Δψ m and subsequent acute cell death. These events were markedly delayed by chelation of Ca 2+ e, but not by nominally Ca 2+ 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 Ca 2+ influx through lytic C5b-9 channels is responsible for the massive increase in [Ca 2+] i, as well as for the rapid loss of Δψ m, followed by acute cell death. When this [Ca 2+] i increase is prevented, the cell death is probably related to metabolic depletion.