Zn(2+) induces permeability transition pore opening and release of pro-apoptotic peptides from neuronal mitochondria.
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Rapid entry of Ca(2+) or Zn(2+) kills neurons. Mitochondria are major sites of Ca(2+)-dependent toxicity. This study examines Zn(2+)-initiated mitochondrial cell death signaling. 10 nm Zn(2+) induced acute swelling of isolated mitochondria, which was much greater than that induced by higher Ca(2+) levels. Zn(2+) entry into mitochondria was dependent upon the Ca(2+) uniporter, and the consequent swelling resulted from opening of the mitochondrial permeability transition pore. Confocal imaging of intact neurons revealed entry of Zn(2+) (with Ca(2+)) to cause pronounced mitochondrial swelling, which was far greater than that induced by Ca(2+) entry alone. Further experiments compared the abilities of Zn(2+) and Ca(2+) to induce mitochondrial release of cytochrome c (Cyt-c) or apoptosis-inducing factor. In isolated mitochondria, 10 nm Zn(2+) exposures induced Cyt-c release. Induction of Zn(2+) entry into cortical neurons resulted in distinct increases in cytosolic Cyt-c immunolabeling and in cytosolic and nuclear apoptosis-inducing factor labeling within 60 min. In comparison, higher absolute [Ca(2+)](i) rises were less effective in inducing release of these factors. Addition of the mitochondrial permeability transition pore inhibitors cyclosporin A and bongkrekic acid decreased Zn(2+)-dependent release of the factors and attenuated neuronal cell death as assessed by trypan blue staining 5-6 h after the exposures. (+info)
Involvement of caspases and of mitochondria in Fas ligation-induced eosinophil apoptosis: modulation by interleukin-5 and interferon-gamma.
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In this study, we examined the relative importance of caspases and mitochondria in Fas-mediated eosinophil apoptosis. Stimulation of human peripheral blood eosinophils with an agonistic anti-human Fas monoclonal antibody, but not with control IgM, induced a time-dependent increase in their apoptosis, which was associated with a loss in mitochondrial transmembrane potential (DeltaPsi(m)) and with caspase-8 and caspase-3 activation. Interleukin (IL)-5 and interferon (IFN)-gamma, two cytokines known to prolong eosinophil survival, inhibited Fas-mediated apoptosis and caspase activation but poorly affected the decrease in DeltaPsi(m). Eosinophil incubation with bongkrekic acid, an inhibitor of the mitochondrial permeability transition pore (MPTP) opening, failed to modify Fas-mediated loss in DeltaPsi(m), caspase activation, and apoptosis. In contrast, caspase inhibitors markedly reduced eosinophil apoptosis without significantly affecting DeltaPsi(m) dissipation. We conclude that caspase-8 and caspase-3 activation, but not MPTP opening, mediate Fas-induced eosinophil apoptosis and are the main targets for the protective effect of IL-5 and IFN-gamma. (+info)
Mitochondrial ATP-sensitive potassium channels attenuate matrix Ca(2+) overload during simulated ischemia and reperfusion: possible mechanism of cardioprotection.
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Mitochondrial ATP-sensitive potassium (mitoK(ATP)) channels play a key role in ischemic preconditioning of the heart. However, the mechanism of cardioprotection remains controversial. We measured rhod-2 fluorescence in adult rabbit ventricular cardiomyocytes as an index of mitochondrial matrix Ca(2+) concentration ([Ca(2+)](m)), using time-lapse confocal microscopy. To simulate ischemia and reperfusion (I/R), cells were exposed to metabolic inhibition (50 minutes) followed by washout with control solution. Rhod-2 fluorescence gradually increased during simulated ischemia and rose even further with reperfusion. The mitoK(ATP) channel opener diazoxide attenuated the accumulation of [Ca(2+)](m) during simulated I/R (EC(50)=18 micromol/L). These effects of diazoxide were blocked by the mitoK(ATP) channel antagonist 5-hydroxydecanoate (5HD). In contrast, inhibitors of the mitochondrial permeability transition (MPT), cyclosporin A and bongkrekic acid, did not alter [Ca(2+)](m) accumulation during ischemia, but markedly suppressed the surge in rhod-2 fluorescence during reperfusion. Measurements of mitochondrial membrane potential, DeltaPsi(m), in permeabilized myocytes revealed that diazoxide depolarized DeltaPsi(m) (by 12% at 10 micromol/L, P<0.01) in a 5HD-inhibitable manner. Our data support the hypothesis that attenuation of mitochondrial Ca(2+) overload, as a consequence of partial mitochondrial membrane depolarization by mitoK(ATP) channels, underlies cardioprotection. Furthermore, mitoK(ATP) channels and the MPT differentially affect mitochondrial calcium homeostasis: mitoK(ATP) channels suppress calcium accumulation during I/R, while the MPT comes into play only upon reperfusion. (+info)
The binding of atractylate and carboxy-atractylate to mitochondria.
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35S-labelled atractylate and carboxy-atractylate are produced biosynthetically and used for studying the binding of these specific ligands to the ADP, ATP carrier in beef heart mitochondria. The following results are obtained. 1. Inhibition of translocation activity goes parallel to the increase of binding by [35S]atractylate. No additional binding is observed after full inhibition of translocation is reached giving evidence that atractylate binds exclusively to the carrier. 2. The maximum number of binding sites of both atractylates is about 1.6 mumol/g protein in beef heart mitochondria and decreases on treatment of the membrane by Pi, freezing, ageing, etc. The dissociation constants of the binding are approximately for atractylate Kd = 5-10(-8) M and for carboxy-atractylate Kd = 10(-8) M. The mass action plots of the concentration dependence for the binding are nonlinear-convex in particular with carboxy-atractylate and more linear with atractylate. Nonlinearity appears to be caused by some retardation of equilibration in the case of very high affinity binding. 3. The binding of atractylate and carboxy-atractylate is relatively fast in intact mitochondria and slower in aged membranes. There is a slower and a faster binding portion. 4. The atractylates remove ADP in a nearly 1:1 stoichiometry from untreated mitochondria. In aged and Pi-treated membranes the ratio deltaADP/deltaatractylate approaches 0. Obviously binding of carrier sites to ADP is more sensitive to alterations than that of the atractylates. The assumption is maintained that the binding site for atractylate is identical with that for ADP and ATP. 5. Bongkrekate prevents binding of both atractylates. However, when added after, it only removes atractylate but not the carboxy compound because of its different tight binding. The removal of atractylate depends on the synergistic effect of bongkrekate with ADP. 6. The binding studies with [35S]atractylate and in particular the interaction with bongkrekate support the reorienting carrier model in which atractylate as an impermeable ligand fixes the binding site of the carrier outside while with bongkrekate the carrier site is turned to the inside. (+info)
Activation of caspase-8 in 3-deazaadenosine-induced apoptosis of U-937 cells occurs downstream of caspase-3 and caspase-9 without Fas receptor-ligand interaction.
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3-Deazaadenosine (DZA), a cellular methylation blocker was reported to induce the caspase-3-like activities-dependent apoptosis in U-937 cells. In this study, we analyzed the activation pathway of the caspase cascade involved in the DZA-induced apoptosis using specific inhibitors of caspases. In the U-937 cells treated with DZA, cytochrome c release from mitochondria and subsequent activation of caspase-9, -8 and -3 were observed before the induction of apoptosis. zDEVD-Fmk, a specific inhibitor of caspase-3, and zLEHD-Fmk, a specific inhibitor of caspase-9, prevented the activation of caspase-8 but neither caspase-3 nor caspase-9, indicating that caspase-8 is downstream of both caspase-3 and caspase-9, which are activated by independent pathways. zVAD-Fmk, a universal inhibitor of caspases, kept the caspase-3 from being activated but not caspase-9. Moreover, ZB4, an antagonistic Fas-antibody, exerted no effect on the activation of caspase-8 and induction of apoptosis by DZA. In addition, zVAD-Fmk and mitochondrial permeability transition pore (MPTP) inhibitors such as cyclosporin A (CsA) and bongkrekic acid (BA) did not block the release of cytochrome c from mitochondria. Taken together, these results suggest that in the DZA-induced apoptosis, caspase-8 may serve as an executioner caspase and be activated downstream of both caspase-3 and caspase-9, independently of Fas receptor-ligand interaction. And caspase-3 seems to be activated by other caspses including IETDase-like enzyme and caspse-9 seems to be activated by cytochrome c released from mitochondria without the involvement of caspases and CsA- and BA- inhibitory MPTP. (+info)
WK175, a novel antitumor agent, decreases the intracellular nicotinamide adenine dinucleotide concentration and induces the apoptotic cascade in human leukemia cells.
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We recently developed a class of novel antitumor agents that elicit a potent growth-inhibitory response in many tumor cells cultured in vitro. WK175, a member of this class, was chosen as a model compound that showed strong in vitro efficacy. WK175 interferes with the intracellular steady-state level of NAD(+), resulting in a decreased cellular NAD(+) concentration. We found that WK175 induces apoptotic cell death without any DNA-damaging effect. The apoptotic death signaling pathway initiated by WK175 was examined in detail: mitochondrial membrane potential, cytochrome c release, caspase 3 activation, caspase 3 and poly(ADP-ribose) polymerase cleavage, and the appearance of a sub-G(1) cell cycle population were determined in time course studies in THP-1 (a human monocytic leukemia cell line) cells. We found activation of this cascade after 24 h of treatment with 10 nM WK175. Induction of apoptosis was prevented by bongkrekic acid, Z-Asp-Glu-Val-Asp-fluoromethylketone, and Z-Leu-Glu-His-Asp-fluoromethylketone, inhibitors of the mitochondrial permeability transition and of caspase 3 and 9, respectively, but not by Ac-Tyr-Val-Ala-Asp-CHO, a specific caspase 1 inhibitor, suggesting the involvement of the permeability transition pore, caspase 3, and caspase 9 in the WK175-induced apoptotic cascade. These results imply that decreased NAD(+) concentration initiates the apoptotic cascade, resulting in the antitumor effect of WK175. (+info)
Menadione-induced apoptosis: roles of cytosolic Ca(2+) elevations and the mitochondrial permeability transition pore.
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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. (+info)
Close location of the first loop to the third loop of the mitochondrial ADP/ATP carrier deduced from cross-linking catalyzed by copper-o-phenanthroline of the solubilized carrier with Triton X-100.
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Effects of the cross-linking catalyst copper-o-phenanthroline [Cu(OP)2] on the bovine heart mitochondrial ADP/ATP carrier solubilized with Triton X-100 were studied under various conditions. Without detergent treatment, Cu(OP)2 specifically catalyzed the formation of intermolecular disulfide bridges in submitochondrial particles between two Cys56 residues in the first loop facing the matrix space of the dimeric carrier [Majima, E., Ikawa, K., Takeda, M., Hashimoto, M., Shinohara, Y., and Terada, H. (1995) J. Biol. Chem. 270, 29548-29554]. However, an intramolecular disulfide bridge between Cys56 and Cys256 in the third loop was formed in the solubilized carrier. Proteolytic digestion of the carrier with lysylendopeptidase showed that it first cleaves the Lys42-Gln43 bond and then the Lys48-Gln49 bond of the first loop in the membrane-bound carrier, but it cleaves both sites almost simultaneously in the solubilized carrier. These features were observed only with the m-state carrier; the c-state carrier was not subject to any cross-linking or proteolytic digestion. It is suggested that the protruding first loop is located close to the third loop, which could be exposed to a certain degree. (+info)