digitonin and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

In the present work, Jurkat cells undergoing anti-Fas antibody (anti-Fas)-triggered apoptosis exhibited in increasing proportion a massive release of cytochrome c from mitochondria, as revealed by double-labeling confocal immunofluorescence microscopy. The cytochrome c release was followed by a progressive reduction in the respiratory activity of the last respiratory enzyme, cytochrome c oxidase (COX), and with a little delay, by a decrease in overall endogenous respiration rate, as measured in vivo in the whole cell population. Furthermore, in vivo titration experiments showed that an approximately 30% excess of COX capacity over that required to support endogenous respiration, found in naive cells, was maintained in anti-Fas-treated cells having lost approximately 40% of their COX respiratory activity. This observation strongly suggested that only a subpopulation of anti-Fas-treated cells, which maintained the excess of COX capacity, respired. Fractionation of cells on annexin V-coated paramagnetic beads did indeed separate a subpopulation of annexin V-binding apoptotic cells with fully released cytochrome c and completely lacking respiration, and a nonbound cell subpopulation exhibiting nearly intact respiration and in their great majority preserving the mitochondrial cytochrome c localization. The above findings showed a cellular mosaicism in cytochrome c release and respiration loss, and revealed the occurrence of a rate-limiting step preceding cytochrome c release in the apoptotic cascade. Furthermore, the striking observation that controlled digitonin treatment caused a massive and very rapid release of cytochrome c and complete loss of respiration in the still respiring anti-Fas-treated cells, but not in naive cells, indicated that the cells responding to digitonin had already been primed for apoptosis, and that this treatment bypassed or accelerated the rate-limiting step most probably at the level of the mitochondrial outer membrane.

Topics: Amino Acid Chloromethyl Ketones; Antibodies; Apoptosis; Cell Membrane Permeability; Cell Respiration; Cell Separation; Cytochrome c Group; Digitonin; Electron Transport Complex IV; fas Receptor; Fluorescent Antibody Technique; Humans; Jurkat Cells; Kinetics; Mitochondria; Mosaicism; Oxidoreductases, N-Demethylating; Oxygen; Succinic Acid

Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c-GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity - characteristics of apoptotic cells. Once initiated, the release of cytochrome- c-GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 degrees C to 37 degrees C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Digitonin; Electron Transport; Enzyme Activation; Enzyme Inhibitors; Green Fluorescent Proteins; HeLa Cells; Humans; Image Processing, Computer-Assisted; Intracellular Membranes; Luminescent Proteins; Membrane Potentials; Mitochondria; Oligomycins; Phosphatidylserines; Recombinant Fusion Proteins; Sodium Azide; Temperature; Ultraviolet Rays