oligomycins and benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone

Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-alpha, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 +/- 0.1 in healthy cells to 6.8 +/- 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 +/- 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F(0)F(1)-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 +/- 0.3, in the healthy population, to 4.8 +/- 0.3 and 5.5 +/- 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-alpha.

Topics: Amides; Amino Acid Chloromethyl Ketones; Apoptosis; Caspase 3; Caspase 8; Caspase Inhibitors; Caspases; Cathepsin D; Cell Nucleus; Cell Size; Cytosol; Detergents; Enzyme Inhibitors; Humans; Hydrogen-Ion Concentration; Intracellular Membranes; Lysosomes; Mitochondrial Proton-Translocating ATPases; Oligomycins; Permeability; Phosphatidylserines; Protein Transport; Protons; Serine; Tumor Necrosis Factor-alpha; U937 Cells

Oxidative injury is believed to be a major factor in the pathogenesis of a variety of neurodegenerative diseases. Additionally, the mode of cell death in oxidant-stressed cells can vary. The present study was conducted to evaluate the use of a primary neuronal cell-based bioassay in which different modes of oxidant-induced cell death could be studied and in which putative neuroprotective agents could be screened. Addition of 50 microM H(2)O(2) to primary cortical neuronal cultures for 1 h under normal ATP conditions resulted in approximately 40% cell death, almost exclusively of an apoptotic nature. In this condition, cell death was effectively blocked by GM1 ganglioside, the semi-synthetic ganglioside derivative LIGA20, the dopamine receptor agonist pramipexole (PPX) and the caspase inhibitor Z-VAD-FMK but not by the poly (ADP-ribose) polymerase (PARP) inhibitor 3-aminobenzamide (3-AB). Pretreatment of cells with 0.01 microM oligomycin for 45 min prior to addition of 50 microM H(2)O(2) caused significant ATP depletion and approximately the same amount of cell death as H(2)O(2) alone. However, under these conditions, cell death was primarily non-apoptotic in nature and GM1, LIGA20 and Z-VAD-FMK had no protective effects. In contrast, AB and PPX effectively blocked cell death. These results suggest that cellular ATP plays a critical role in determining the mode of cell death in primary neurons and that these types of in vitro models may provide a useful system for screening putative neuroprotective agents.

Topics: Adenosine Triphosphate; Amino Acid Chloromethyl Ketones; Animals; Annexin A5; Benzimidazoles; Caspases; Cell Death; Cell Survival; Cells, Cultured; Cerebral Cortex; Dose-Response Relationship, Drug; Drug Interactions; Embryo, Mammalian; Enzyme Inhibitors; Hydrogen Peroxide; Neurons; Neuroprotective Agents; Oligomycins; Rats; Rats, Sprague-Dawley; Thienamycins

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

Bax causes apoptosis by associating with mitochondria and triggering cytochrome c release, which activates the caspase cascade. Bax can also kill some cells independently of caspases, but the requirements for such killing are poorly understood. Here we describe an inducible fibroblast line that expresses Bax when tetracycline is withdrawn; the resulting apoptosis can be blocked by the caspase inhibitor zVAD-fmk. Even when caspases are inhibited, however, treating the Bax-expressing cells with the mitochondrial toxin oligomycin efficiently triggers death with features resembling apoptosis. Bax mutants lacking the BH3 domain remain able to cause cytochrome c release and caspase-mediated death, but cannot support this caspase-independent killing. Mutating specific BH3 residues needed for binding Bcl2 does not prevent synergy with oligomycin, implying that no such binding is required. These findings illuminate a caspase-independent pathway of death that depends on the Bax BH3 domain and on effectors emanating from mitochondria.

Topics: Amino Acid Chloromethyl Ketones; Amino Acid Sequence; Apoptosis; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cell Line; Cysteine Proteinase Inhibitors; Humans; Mitochondria; Oligomycins; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Recombinant Proteins; Transfection

Apoptosis is a programmed form of cell death characterized by biochemical and morphological changes affecting the nucleus, cytoplasm, and plasma membrane. These changes in various cellular compartments are widely regarded as mechanistically linked events in a single "program" in which activation of caspases and proteolysis of intracellular substrates represent a final common pathway leading to cell death. To date there has been very limited exploration of the linkage of this program to the plasma membrane changes, which bring about swift recognition, uptake, and safe degradation of apoptotic cells by phagocytes. Using the mitochondrial inhibitors antimycin A and oligomycin in human monocytic THP.1 cells triggered into apoptosis, we report the uncoupling of plasma membrane changes from other features of apoptosis. These inhibitors blocked increased plasma membrane permeability, externalization of phosphatidylserine, and recognition by two classes of phagocytes but not activation of caspase-3, cleavage of poly(ADP-ribose) polymerase and DNA fragmentation. Externalization of phosphatidylserine in apoptotic human leukemic U937 cells was also dissociated from caspase activation. Thus changes governing safe clearance of apoptotic cells may be regulated by an independent pathway to those bringing about caspase activation. This finding could have important consequences for attempts to manipulate cell death for therapeutic gain in vivo.

Topics: Amino Acid Chloromethyl Ketones; Antimycin A; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA Fragmentation; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Oligomycins; Phagocytes; Phosphatidylserines; Surface Properties; Tumor Cells, Cultured