Quantitative Analysis of Adenine Nucleotides During the Prelytic Phase of Cell Death Mediated by C5b-9

The nucleated cell death mediated by C5b-9 depends on the extent of C fixation and parameters that affect the ability of the cell to eliminate C5b-9. When C5b-9 formation exceeds elimination, cell death can be initiated. High Ca2+ in the medium accelerates Ehrlich ascites cell death induced by a large number of C5b-9, whereas osmotic prevention of cell swelling has little effect in protecting Ehrlich cells from killing by C5b-9. In the present study, we investigated the interrelationship between intracellular Ca2+, intra- and extracellular adenine nucleotides, and mitochondrial membrane potential, to understand the mechanism of acute cell death induced by C5b-9. When Ehrlich cells carrying C5b-8 were exposed to C9, rapid and profound ATP depletion in the cell was observed before cell death. Leakage of the adenine nucleotides ATP, ADP, and AMP also began during the prelytic phase. Studies using digital imaging fluorescence microscopy showed that loss of mitochondrial membrane potential was noted immediately after C9 addition but before nuclear staining with propidium iodide. These findings suggest that an increase in intracellular Ca2+ through C5b-9 channels and loss of mitochondrial membrane potential may initiate rapid cell death. The prelytic leakage of ATP precursors may also contribute to cell death by decreasing nucleotide pools, because recovery of ATP production was observed after a similar degree of ATP loss in cells exposed to sublethal doses of KCN, in which ADP and AMP leakage was not present.