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Menadione-induced apoptosis: roles of cytosolic Ca²⁺ elevations and the mitochondrial permeability transition pore

¹ Medical Research Council Secretory Control Research Group, Physiological Laboratory, University of Liverpool L69 3BX, UK
² Department of Medicine, University of Liverpool L69 3BA, UK

View larger version (20K): [in a new window]   Fig. 1. Menadione induces cytosolic Ca2+ spikes that are similar to those elicited by ACh. Cells were loaded with Fura Red AM. (A) Addition of 20 µM menadione to cells in the presence (upper trace) and absence (lower trace) of 1 mM Ca2+ induces repetitive Ca2+ spikes. (B) Addition of 100 nM ACh to cells in medium with 1 mM Ca2+ induces repetitive Ca2+ spikes. (C) Ca2+ responses to menadione following application of 100 µM ACh in the presence (upper trace) and absence (lower trace) of extracellular Ca2+ are shown.

View larger version (29K): [in a new window]   Fig. 2. (A) Menadione, but not ACh, CCK or thapsigargin induce apoptosis in a Ca2+-dependent manner. Annexin V FITC ApoAlert Kit detects apoptosis in pancreatic acinar cells after incubation with menadione. Each column represents the intensity of Annexin V FITC fluorescence of every propidium iodide negative cell in a field of view (control cells, cells after 30 minutes incubation with 5 pM CCK or 25 nM ACh or 100 nM ACh or 1 µM thapsigargin for 30 minutes or for 3 hours, 250 µM ZVADfmk + menadione, BAPTA + menadione, 50 µM Bongkrekic acid + menadione or menadione alone for 15 minutes and for 30 minutes) measured in the same conditions using the confocal microscope. a.u, arbitrary units. (B) Pre-treatment of cells with 25 µM BAPTA AM prevents cytosolic Ca2+ spikes induced by menadione. Cells were loaded with Fura Red AM. (C) Cell death as measured by 2.5 µg/ml propidium iodide staining of the nucleus 6 hours after addition of 20 µM menadione or vehicle control. (D,E) Nuclear apoptosis in cells 3 hours after addition of 20 µM menadione (E) or controls immediately after isolation (D) is shown. Nuclei were stained with 8 µg/ml Hoechst 33342 for 15 minutes and imaged with a Leica SP2 confocal microscope. a.u., arbitrary units.

View larger version (54K): [in a new window]   Fig. 3. Menadione-induced cytosolic Ca2+ moves quickly into the nucleus. The upper part of the figure shows transmitted light pictures of experimental cells (a-d). Positions of nuclei (bright spots) are clearly visible on fluorescence pictures of cells (e-h). Linescans (i-l) of fluorescent intensity of Fura Red in pancreatic acinar cells along the line connecting secretory granule region (initiation site of Ca2+ response) and nucleus after 25 nM ACh (i), 100 nM ACh (j), 20 µM menadione (k) and after addition of 100 nM ACh together with 100 nM antimycin A (l) are shown. The lower section of the figure (m-p) shows schematic representation of the results: in physiological conditions 25 nM ACh application induce a Ca2+ spike that does not enter the nucleus (m). Higher doses of ACh (100 nM) induce a large Ca2+ wave that is substantially delayed on its way to the nucleus (n). Menadione (o) and ACh together with antimycin A (p) induce Ca2+ waves that propagate uniformly. Angles ß and represent speeds of Ca2+ waves (nuclear speed ß in comparison to speed in non-nuclear region). Lower part (q) shows average values of ratio of Ca2+ speed propagation (nuclear speed ß by non-nuclear ) induced by ACh, menadione or ACh with antimycin A.

View larger version (27K): [in a new window]   Fig. 4. Effects of menadione on mitochondrial Ca2+ and membrane potential. (A) Ca2+ concentration in mitochondria following addition of 100 nM ACh and menadione. (B,a-d). Colocalisation of TMRE and Mito Tracker Green in pancreatic acinar cell cluster. (a) TMRE fluorescence; (b) Mito Tracker fluorescence; (c) overlay of Mito Tracker Green and TMRE; (d) transmitted light image of the same cell cluster; (e) changes in mitochondrial region membrane potential (green box) and nuclear region (yellow box) of the same cell measured with TMRE during application of menadione followed by 10 µM mCCCP. (C) Changes in mitochondrial membrane potential following application of menadione alone (control cell); pre-treatment with either ZVADfmk, BAPTA or bongkrekic acid (BA). (D) Changes in mitochondrial membrane potential following application of 100 nM antimycin A to control cells (upper trace) and to bongkrekic acid treated cells (lower trace) are shown. (E-G). Menadione induces a cytosolic Ca2+ rise and mitochondrial depolarisation simultaneously. (E,F) Confocal images of a two-cell cluster loaded with TMRE (E) and Fluo-4 AM (F). (G) Changes in free Ca2+ concentration, as measured by Fluo-4 (green line) and fluorescence of TMRE (red line) after application of 100 nM ACh and menadione measured from mitochondrial region of the left cell (blue box).

View larger version (59K): [in a new window]   Fig. 5. Menadione induces PTP, which causes a loss of mitochondrial staining by calcein. (A) Transmitted light image of pancreatic acinar cell. (B) Fluorescence image of the same cell loaded with calcein in the presence of CoCl2. (C) Calcein fluorescence of the cell during exposure to menadione. (D) Calcein fluorescence during exposure to menadione following precincubation with 50 µM bongkrekic acid. (E) 100 nM antimycin A does not cause a loss of calcein fluorescence.

View larger version (35K): [in a new window]   Fig. 6. Cytochrome C release and activation of caspase 9 and 3 by menadione. (A,B) Cytochrome C release in cytosolic (A) and mitochondrial (B) fractions in cell treated with vehicle control, menadione for 2 minutes with or without preincubation with BAPTA AM (n>3). The Y-axis represents the percentage of chemiluminescence of cytochrome C antibodies normalised to maximum fluorescence. Ab and Bb represent western blots of subcellular fractions from control cells, cells treated with BAPTA followed with menadione for 2 minutes and menadione treated cells for 2 minutes, respectively. a.u., arbitrary units. (C) Caspase 9 activity after incubation of cells with menadione for 30 minutes or BAPTA-AM followed by menadione or 100 nM ACh with 100 nM antimycin A or bongkrekic acid followed by menadione. Each column represents the intensity of FAM-LEHD-FMK fluorescence of every propidium-iodide-negative cell in a field of view. (D) Activation of caspase 3 by 30 minutes incubation with menadione, treatment for 30 minutes with 5 pM CCK, 25 nM ACh or bongkrekic acid followed by menadione for 30 minutes or treatment with BAPTA AM followed by menadione application for 30 min. Each column represents the intensity of PhiPhiLux fluorescence of every propidium iodide negative cell in a field of view. a.u., arbitrary units.

View larger version (44K): [in a new window]   Fig. 7. Simplified schematic model illustrating the differences in Ca2+ homeostasis and induction of the PTP after stimulation with menadione (A), ACh (B) or stimulation with ACh plus antimycin A (C). BA, Bongkrekic acid; PS, Phosphatidylserine; ROS, Reactive oxygen species; UN, Ca2+ uniporter.