digitonin and Necrosis

Among the many unsolved problems of calcium signalling, the role of calcium elevations in apoptotic and necrotic cell death has been a focus of research in recent years. Evidence has been presented that calcium oscillations can effectively trigger apoptosis under certain conditions and that dysregulation of calcium signalling is a common cause of cell death. These effects are regularly mediated through calcium signal propagation to the mitochondria and the ensuing mitochondrial membrane permeabilization and release of pro-apoptotic factors from mitochondria to the cytoplasm. The progress in this area depended on the development of (1) fluorescent/luminescent probes, including fluorescent proteins that can be genetically targeted to different intracellular locations and (2) the digital imaging technology, fluorescence-activated cell sorting and fluorescent high throughput approaches, which allowed dynamic measurements of both [Ca2+] in the intracellular compartments of interest and the downstream processes. Fluorescence single cell imaging has been the only possible approach to resolve the cell-to-cell heterogeneity and the complex subcellular spatiotemporal organization of the cytoplasmic and mitochondrial calcium signals and downstream events. We outline here fluorometric and fluorescence imaging protocols that we set up for the study of calcium in the context of apoptosis.

Topics: Animals; Apoptosis; Calcium; Calcium Signaling; Caspases; Cell Death; Cell Line; Digitonin; Fluorescent Dyes; Green Fluorescent Proteins; Humans; Microscopy, Fluorescence; Mitochondria; Mitochondrial Membrane Transport Proteins; Mitochondrial Membranes; Mitochondrial Permeability Transition Pore; Necrosis; Permeability; Rats

Incubation of T. cruzi epimastigotes with the lectin Cramoll 1,4 in Ca(2+) containing medium led to agglutination and inhibition of cell proliferation. The lectin (50 microg/ml) induced plasma membrane permeabilization followed by Ca(2+) influx and mitochondrial Ca(2+) accumulation, a result that resembles the classical effect of digitonin. Cramoll 1,4 stimulated (five-fold) mitochondrial reactive oxygen species (ROS) production, significantly decreased the electrical mitochondrial membrane potential (Delta Psi(m)) and impaired ADP phosphorylation. The rate of uncoupled respiration in epimastigotes was not affected by Cramoll 1,4 plus Ca(2+) treatment, but oligomycin-induced resting respiration was 65% higher in treated cells than in controls. Experiments using T. cruzi mitochondrial fractions showed that, in contrast to digitonin, the lectin significantly decreased Delta Psi(m) by a mechanism sensitive to EGTA. In agreement with the results showing plasma membrane permeabilization and impairment of oxidative phosphorylation by the lectin, fluorescence microscopy experiments using propidium iodide revealed that Cramoll 1,4 induced epimastigotes death by necrosis.

Topics: Animals; Calcium Signaling; Cell Membrane Permeability; Cell Proliferation; Cell Survival; Digitonin; Fabaceae; Glycoconjugates; Humans; Keratinocytes; Membrane Potential, Mitochondrial; Necrosis; Oxidative Phosphorylation; Plant Lectins; Reactive Oxygen Species; Trypanosoma cruzi

Topics: Annexin A5; Apoptosis; Caspase Inhibitors; Caspases; Cell Separation; Cell Survival; Digitonin; DNA Fragmentation; Enzyme Activation; fas Receptor; Flow Cytometry; Humans; Jurkat Cells; Necrosis; Phosphatidylserines; Signal Transduction; Time Factors; Tubulin