bongkrekic-acid and fluorexon

In normal pancreatic acinar cells, the oxidant menadione evokes repetitive cytosolic Ca(2+) spikes, partial mitochondrial depolarisation, cytochrome c release and apoptosis. The physiological agonists acetylcholine and cholecystokinin also evoke cytosolic Ca(2+) spikes but do not depolarise mitochondria and fail to induce apoptosis. Ca(2+) spikes induced by low agonist concentrations are confined to the apical secretory pole of the cell by the buffering action of perigranular mitochondria. Menadione prevents mitochondrial Ca(2+) uptake, which permits rapid spread of Ca(2+) throughout the cell. Menadione-induced mitochondrial depolarisation is due to induction of the permeability transition pore. Blockade of the permeability transition pore with bongkrekic acid prevents activation of caspase 9 and 3. In contrast, the combination of antimycin A and acetylcholine does not cause apoptosis but elicits a global cytosolic Ca(2+) rise and mitochondrial depolarisation without induction of the permeability transition pore. Increasing the cytosolic Ca(2+) buffering power by BAPTA prevents cytosolic Ca(2+) spiking, blocks the menadione-elicited mitochondrial depolarisation and blocks menadione-induced apoptosis. These results suggest a twin-track model in which both intracellular release of Ca(2+) and induction of the permeability transition pore are required for initiation of apoptosis.

Topics: Acetylcholine; Amino Acid Chloromethyl Ketones; Animals; Anti-Bacterial Agents; Antifibrinolytic Agents; Antimycin A; Apoptosis; Bongkrekic Acid; Calcium; Calcium Signaling; Caspase 3; Caspase 9; Caspases; Cell Nucleus; Cells, Cultured; Chelating Agents; Cysteine Proteinase Inhibitors; Egtazic Acid; Fluoresceins; Fluorescent Dyes; Membrane Potentials; Mice; Mitochondria; Pancreas; Vasodilator Agents; Vitamin K 3

Hypericin (HY) is a powerful photo-inducer of apoptosis in Jurkat cells as measured by caspase-3 activation, cell shrinkage, phosphatidylserine (PS) exposure and the appearance of hypoploid DNA. These processes are preceded by rapid Bcl-2-independent mitochondrial transmembrane depolarization and a drop in cytoplasmic pH. Pre-incubation of cells with inhibitors of the mitochondrial permeability transition pore, such as cyclosporin A or bongkrekic acid, does not protect cells from mitochondrial membrane potential (deltapsim) decrease. However, monitoring of mitochondrial entrapped calcein by confocal fluorescence imaging gives clear evidence of HY photo-induced mitochondrial permeability. This should be considered as the result of a non-specific alteration of mitochondrial membrane integrity brought about by lipid peroxidation. Nevertheless, synthesis of the anti-apoptotic protein Bcl-2 appears to delay the subsequent time course of PS exposure and to reduce caspase-3 activation and the fraction of cells which become hypoploid. We interpret this partially protective effect as the consequence of a direct interaction of Bcl-2 with cytosolic cytochrome c previously released from mitochondria upon deltapsim decrease and/or of Bcl-2 inhibition of the deleterious retro-effect of caspase-3 on the mitochondrial permeability transition pore and/or the mitochondrial membrane components.

Topics: 3T3 Cells; Animals; Anthracenes; Apoptosis; Bongkrekic Acid; Caspase 3; Caspases; Cell Line; Cell Membrane Permeability; Cyclosporine; Dose-Response Relationship, Radiation; Enzyme Activation; Fibroblasts; Flow Cytometry; Fluoresceins; Humans; Hydrogen-Ion Concentration; Intracellular Membranes; Membrane Potentials; Mice; Microscopy, Confocal; Mitochondria; Perylene; Phosphatidylserines; Photosensitivity Disorders; Proto-Oncogene Proteins c-bcl-2; Time Factors