MITOCHONDRIAL-MEMBRANE POTENTIAL AND NUCLEAR-CHANGES IN APOPTOSIS CAUSED BY SERUM AND NERVE GROWTH-FACTOR WITHDRAWAL - TIME-COURSE AND MODIFICATION BY (-)-DEPRENYL

Studies in non-neural cells have suggested that a fall in mitochondria l membrane potential (Delta Psi(M)) is one of the earliest events in a poptosis. It is not known whether neural apoptosis caused by nerve gro wth factor (NGF) and serum withdrawal involves a decrease in Delta Psi (M). We used epifluorescence and laser confocal microscopy with the mi tochondrial potentiometric dyes chloromethyl-tetramethyl rosamine meth yl ester and ',6,6'-tetrachloro-1,1',3,3'-tetraethybenzimidazol carboc yanine iodide to estimate Delta Psi(M). PC12 cells were differentiated in media containing serum and NGF for 6 d before withdrawal of trophi c support. After washing, the cells were incubated with media containi ng serum and NGF (M/S+N), media without serum and NGF, or media with t he ''trophic-like'' monoamine oxidase B inhibitor, (-)-deprenyl. Mitoc hondria in cells without trophic support underwent a progressive shift to lower Delta Psi(M), values that was significant by 3 hr after wash ing. The percentages of cells with nuclear chromatin condensation or n uclear DNA fragmentation were not significantly increased above those for cells in M/S+N until 6 hr after washing. Replacement of cells into M/S+N or treatment with (-)-deprenyl markedly reduced the proportion of mitochondria with decreased Delta Psi(M). Measurements of cytoplasm ic peroxyl radical levels with 2',7'-dihydrodichlorofluorescein fluore scence and intramitochondrial Ca2+ with dihydro-rhodamine-2-acetylmeth yl ester indicated that cytoplasmic peroxyl radical levels were not in creased until after 6 hr, whereas increases in intramitochondrial Ca2 paralleled the decreases in Delta Psi(M). (-)-Deprenyl appeared to al ter the relationship between intramitochondrial Ca2+ levels and Delta Psi(M), possibly through its reported capacity to increase the synthes is of proteins such as BCL-2.