Modulation of mitochondrial Ca(2+) homeostasis by Bcl-2

We have investigated the role of mitochondrial Ca(2+) (Ca(m)) homeostasis in cell survival. Disruption of Ca(m) homeostasis via depletion of the mitochondrial Ca(2+) store was the earliest event that occurred during staurosporine-induced apoptosis in neuroblastoma cells (SH-SY5Y). The decrease of Ca(m) preceded activation of the caspase cascade and DNA fragmentation. Overexpression of the anti-apoptosis protein Bcl-2 led to increased Ca(m) load, increased mitochondrial membrane potential (DeltaPsi(m)), and inhibition of staurosporine-induced apoptosis. On the other hand, ectopic expression of the pro-apoptotic protein Bik led to decreased Ca(m) load and decreased DeltaPsi(m). Inhibition of calcium uptake into mitochondria by ruthenium red induced a dose-dependent apoptosis as determined by nuclear staining and DNA ladder assay. Similarly, reducing the Ca(m) load by lowering the extracellular calcium concentration also led to apoptosis. We suggest that the anti-apoptotic effect of Bcl-2 is related to its ability to maintain a threshold level of Ca(m) and DeltaPsi(m) while the pro-apoptotic protein Bik has the opposite effect. Furthermore, both ER and mitochondrial Ca(2+) stores are important, and the depletion of either one will result in apoptosis. Thus, our results, for the first time, provide evidence that the maintenance of Ca(m) homeostasis is essential for cell survival.     

Arachidonic acid induces both Na+ and Ca2+ entry resulting in apoptosis

Marked accumulation of arachidonic acid (AA) and intracellular Ca2+ and Na+ overloads are seen during brain ischemia. In this study, we show that, in neurons, AA induces cytosolic Na+ ([Na+](cyt)) and Ca2+ ([Ca2+](cyt)) overload via a non-selective cation conductance (NSCC), resulting in mitochondrial [Na+](m) and [Ca2+](m) overload. Another two types of free fatty acids, including oleic acid and eicosapentaenoic acid, induced a smaller increase in the [Ca2+](i) and [Na+](i). RU360, a selective inhibitor of the mitochondrial Ca2+ uniporter, inhibited the AA-induced [Ca2+](m) and [Na+](m) overload, but not the [Ca2+](cyt) and [Na+](cyt) overload. The [Na+](m) overload was also markedly inhibited by either Ca2+-free medium or CGP3715, a selective inhibitor of the mitochondrial Na+(cyt)-Ca2+(m) exchanger. Moreover, RU360, Ca2+-free medium, Na+-free medium, or cyclosporin A (CsA) largely prevented AA-induced opening of the mitochondrial permeability transition pore, cytochrome c release, and caspase 3-dependent neuronal apoptosis. Importantly, Na+-ionophore/Ca2+-free medium, which induced [Na+](m) overload, but not [Ca2+](m) overload, also caused cyclosporin A-sensitive mitochondrial permeability transition pore opening, resulting in caspase 3-dependent apoptosis, indicating that [Na+](m) overload per se induced apoptosis. Our results therefore suggest that AA-induced [Na+](m) overload, acting via activation of the NSCC, is an important upstream signal in the mitochondrial-mediated apoptotic pathway. The NSCC may therefore act as a potential neuronal death pore which is activated by AA accumulation under pathological conditions.     

Important role of energy-dependent mitochondrial pathways in cultured rat cardiac myocyte apoptosis

Recent studies have suggested that apoptosis and necrosis share common features in their signaling pathway and that apoptosis requires intracellular ATP for its mitochondrial/apoptotic protease-activating factor-1 suicide cascade. The present study was, therefore, designed to examine the role of intracellular energy levels in determining the form of cell death in cardiac myocytes. Neonatal rat cardiac myocytes were first incubated for 1 h in glucose-free medium containing oligomycin to achieve metabolic inhibition. The cells were then incubated for another 4 h in similar medium containing staurosporine and graded concentrations of glucose to manipulate intracellular ATP levels. Under ATP-depleting conditions, the cell death caused by staurosporine was primarily necrotic, as determined by creatine kinase release and nuclear staining with ethidium homodimer-1. However, under ATP-replenishing conditions, staurosporine increased the percentage of apoptotic cells, as determined by nuclear morphology and DNA fragmentation. Caspase-3 activation by staurosporine was also ATP dependent. However, loss of mitochondrial transmembrane potential (DeltaPsi(m)), Bax translocation, and cytochrome c release were observed in both apoptotic and necrotic cells. Moreover, cyclosporin A, an inhibitor of mitochondrial permeability transition, attenuated staurosporine-induced apoptosis and necrosis through the inhibition of DeltaPsi(m) reduction, cytochrome c release, and caspase-3 activation. Our data therefore suggest that staurosporine induces cell demise through a mitochondrial death signaling pathway and that the presence of intracellular ATP favors a shift from necrosis to apoptosis through caspase activation.     

Mitochondrial membrane potential modulates regulation of mitochondrial Ca2+ in rat ventricular myocytes

Although recent studies focused on the contribution of mitochondrial Ca2+ to the mechanisms of ischemia-reperfusion injury, the regulation of mitochondrial Ca2+ under pathophysiological conditions remains largely unclear. By using saponin-permeabilized rat myocytes, we measured mitochondrial membrane potential (DeltaPsi(m)) and mitochondrial Ca2+ concentration ([Ca2+](m)) at the physiological range of cytosolic Ca2+ concentration ([Ca2+](c); 300 nM) and investigated the regulation of [Ca2+](m) during both normal and dissipated DeltaPsi(m). When DeltaPsi(m) was partially depolarized by carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP, 0.01-0.1 microM), there were dose-dependent decreases in [Ca2+](m). When complete DeltaPsi(m) dissipation was achieved by FCCP (0.3-1 microM), [Ca2+](m) remained at one-half of the control level despite no Ca2+ influx via the Ca2+ uniporter. The DeltaPsi(m) dissipation by FCCP accelerated calcein leakage from mitochondria in a cyclosporin A (CsA)-sensitive manner, which indicates that DeltaPsi(m) dissipation opened the mitochondrial permeability transition pore (mPTP). After FCCP addition, inhibition of the mPTP by CsA caused further [Ca2+](m) reduction; however, inhibition of mitochondrial Na+/Ca2+ exchange (mitoNCX) by a Na+-free solution abolished this [Ca2+](m) reduction. Cytosolic Na(+) concentrations that yielded one-half maximal activity levels for mitoNCX were 3.6 mM at normal DeltaPsi(m) and 7.6 mM at DeltaPsi(m) dissipation. We conclude that 1) the mitochondrial Ca2+ uniporter accumulates Ca2+ in a manner that is dependent on DeltaPsi(m) at the physiological range of [Ca2+](c); 2) DeltaPsi(m) dissipation opens the mPTP and results in Ca2+ influx to mitochondria; and 3) although mitoNCX activity is impaired, mitoNCX extrudes Ca2+ from the matrix even after DeltaPsi(m) dissipation.     

Effect of Bcl-x(L) overexpression on reactive oxygen species,intracellular calcium,and mitochondrial membrane potential following injury in astrocytes

Many studies have demonstrated the protective effects of Bcl-x(L) against both apoptotic and necrotic cell death, but the mode of action of Bcl-x(L) remains unclear. This work analyzed effects of Bcl-x(L) overexpression on cellular levels of reactive oxygen species (ROS), intracellular calcium ([Ca(2+)](i)), and mitochondrial membrane potential (DeltaPsi(m)) in cultured mouse primary astrocytes after exposure to glucose deprivation (GD) or hydrogen peroxide (H(2)O(2)). Upon exposure to GD or H(2)O(2), uninfected and Lac-Z-expressing astrocytes showed an immediate, rapid increase in ROS accumulation that was slowed and or reduced by Bcl-x(L). Changes in DeltaPsi(m) in response to the two insults differed. H(2)O(2) induced a decrease in DeltaPsi(m) that was initially greater in Bcl-x(L) cells, but then held stable. DeltaPsi(m) in control and Lac-Z-expressing cells initially declined more slowly, but after about 20 min showed rapid deterioration. Five hours of GD caused mitochondrial membrane hyperpolarization followed by a decrease in DeltaPsi(m,) which was not observed with Bcl-x(L) overexpression. Bcl-x(L) failed to inhibit the calcium dysregulation seen in control cells exposed to 400 microM H(2)O(2), but still improved cell survival. There was no increase in [Ca(2+)](i) with 5 h of GD. These data thus dissociate the effect of Bcl-x(L) on calcium homeostasis from effects on ROS, DeltaPsi(m,) and for H(2)O(2) exposure, cell survival.     

Calcium influx through receptor-operated channel induces mitochondria-triggered paraptotic cell death

We address the specific role of cytoplasmic Ca(2+) overload as a cell death trigger by expressing a receptor-operated specific Ca(2+) channel, vanilloid receptor subtype 1 (VR1), in Jurkat cells. Ca(2+) uptake through the VR1 channel, but not capacitative Ca(2+) influx stimulated by the muscarinic type 1 receptor, induced sustained intracellular [Ca(2+)] rises, exposure of phosphatidylserine, and cell death. Ca(2+) influx was necessary and sufficient to induce mitochondrial damage, as assessed by opening of the permeability transition pore and collapse of the mitochondrial membrane potential. Ca(2+)-induced cell death was inhibited by ruthenium red, protonophore carbonyl cyanide m-chlorophenylhydrazone, or cyclosporin A treatment, as well as by Bcl-2 expression, indicating that this process requires mitochondrial calcium uptake and permeability transition pore opening. Cell death occurred without caspase activation, oligonucleosomal/50-kilobase pair DNA cleavage, or release of cytochrome c or apoptosis inducer factor from mitochondria, but it required oxidative/nitrative stress. Thus, Ca(2+) influx triggers a distinct program of mitochondrial dysfunction leading to paraptotic cell death, which does not fulfill the criteria for either apoptosis or necrosis.     

Bcl-2 protects against FCCP-induced apoptosis and mitochondrial membrane potential depolarization in PC12 cells.

This report addresses the relation between Bcl-2 and mitochondrial membrane potential (DeltaPsi(m)) in apoptotic cell death. Rat pheochromocytoma (PC12) cells are differentiated into neuron-like cells with nerve growth factor (NGF). It is known that Bcl-2 can attenuate apoptosis induced by deprivation of neurotrophic factor. The protective effect of Bcl-2 has been correlated with preservation of DeltaPsi(m). Protonophores, such as carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), collapse the proton gradient across the mitochondrial inner membrane, resulting in a complete abolition of the mitochondrial membrane potential. Based on the analysis of morphology, of phosphatidylserine exposure and of nuclear fragmentation we conclude that FCCP induces apoptosis in PC12 cells, which can be prevented by overexpression of Bcl-2. To determine whether the cytoprotective effect of Bcl-2 is due to stabilization of DeltaPsi(m), we investigated the effect of Bcl-2 on changes in DeltaPsi(m), induced by FCCP in PC12 cells. We showed that treatment with FCCP induced a reduction in DeltaPsi(m), as assessed with the lipophilic cationic membrane potential-sensitive dye JC-1, and that Bcl-2 protects against FCCP-induced changes in NGF differentiated PC12 cells. Our data indicate that Bcl-2 protects against FCCP-induced cell death by stabilizing DeltaPsi(m).     

Na+/H+ exchanger inhibitor cariporide attenuates the mitochondrial Ca2+ overload and PTP opening

The Na(+)/H(+) exchanger (NHE) inhibitor cariporide has a cardioprotective effect in various animal models of myocardial ischemia-reperfusion. Recent studies have suggested that cariporide interacts with mitochondrial Ca(2+) overload and the mitochondrial permeability transition (MPT); however, the precise mechanisms remain unclear. Therefore, we examined whether cariporide affects mitochondrial Ca(2+) overload and MPT. Isolated adult rat ventricular myocytes were used to study the effects of cariporide on hypercontracture induced by ouabain or phenylarsine oxide (PAO). Mitochondrial Ca(2+) concentration ([Ca(2+)](m)) and the mitochondrial membrane potential (DeltaPsi(m)) were measured by loading myocytes with rhod-2 and JC-1, respectively. We also examined the effect of cariporide on the MPT using tetramethylrhodamine methyl ester (TMRM) and oxidative stress generated by laser illumination. Cariporide (1 microM) prevented ouabain-induced hypercontracture (from 40 +/- 2 to 24 +/- 2%, P < 0.05) and significantly attenuated ouabain-induced [Ca(2+)](m) overload (from 149 +/- 6 to 121 +/- 5% of the baseline value, P < 0.05) but did not affect DeltaPsi(m). These results indicate that cariporide attenuates the [Ca(2+)](m) overload without the accompanying depolarization of DeltaPsi(m). Moreover, cariporide increased the time taken to induce the MPT (from 79 +/- 11 to 137 +/- 20 s, P < 0.05) and also attenuated PAO-induced hypercontracture (from 59 +/- 3 to 50 +/- 4%, P < 0.05). Our data indicate that cariporide attenuates [Ca(2+)](m) overload and MPT. Thus these effects might potentially contribute to the mechanisms of cardioprotection afforded by NHE inhibitors.     

Differential effects of bcl-2 on cell death triggered under ATP-depleting conditions

The intracellular ATP concentration decides on the onset of either apoptosis or necrosis in Jurkat cells exposed to death stimuli. Bcl-2 can block apoptotic demise, which occurs preferably under conditions of high cellular ATP levels. Here, we investigated the effects of Bcl-2 on the necrotic type of cell demise that prevails under conditions of energy loss. ATP levels were modulated by using mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, in medium either lacking glucose or supplemented with glucose to stimulate glycolytic ATP generation. Under conditions of ATP depletion, staurosporine (STS) induced >90% necrosis in vector control-transfected cells, whereas bcl-2-transfected cells were protected. Thus, the antiapoptotic protein Bcl-2 can reduce the overall amount of cell death in ATP-depleted cells regardless whether it occurs by apoptosis or necrosis. Cytochrome c release, normally preceding STS-induced necrosis, was also inhibited by Bcl-2. However, Bcl-2 did not prevent an initial STS-induced drop of the mitochondrial membrane potential (DeltaPsi(m)). Therefore, the mechanisms whereby Bcl-2 prevents cell death and favors retention of cytochrome c in the mitochondria require neither the maintenance of mitochondrial DeltaPsi nor the maintenance of normal ATP levels.     

Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor alpha-induced apoptosis

The Bcl-2 family of proteins are involved in regulating the redox state of cells. However, the mode of action of Bcl-2 proteins remains unclear. This work analyzed the effects of Bcl-x(L) on the cellular redox state after treatment with tumor necrosis factor alpha (TNF-alpha) or exogenous oxidants. We show that in cells that undergo TNF-alpha-induced apoptosis, TNF-alpha induces a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) followed by high levels of reactive oxygen species (ROS). ROS scavengers delay the progression of mitochondrial depolarization and apoptotic cell death. This indicates that ROS are important mediators of mitochondrial depolarization. However, ROS scavengers fail to prevent the initial TNF-alpha-induced decrease in DeltaPsi(m). In contrast, expression of Bcl-x(L) prevents both the initial decrease in DeltaPsi(m) following TNF-alpha treatment and the subsequent induction of ROS. Bcl-x(L) itself does not act as a ROS scavenger. In addition, Bcl-x(L) does not block the initial decrease in DeltaPsi(m) following treatment with the oxidant hydrogen peroxide. However, unlike control-transfected cells, Bcl-x(L)-expressing cells can recover their mitochondrial membrane potential following the initial drop in DeltaPsi(m) induced by hydrogen peroxide. These data suggest that Bcl-x(L) plays a regulatory role in controlling the membrane potential of and ROS production by mitochondria rather than acting as a direct antioxidant.     

Macrophages require constitutive NF-kappaB activation to maintain A1 expression and mitochondrial homeostasis

NF-kappaB is a critical mediator of macrophage inflammatory responses, but its role in regulating macrophage survival has yet to be elucidated. Here, we demonstrate that constitutive NF-kappaB activation is essential for macrophage survival. Blocking the constitutive activation of NF-kappaB with pyrrolidine dithiocarbamate or expression of IkappaBalpha induced apoptosis in macrophagelike RAW 264.7 cells and primary human macrophages. This apoptosis was independent of additional death-inducing stimuli, including Fas ligation. Suppression of NF-kappaB activation induced a time-dependent loss of mitochondrial transmembrane potential (DeltaPsi(m)) and DNA fragmentation. Examination of initiator caspases revealed the cleavage of caspase 9 but not caspase 8 or the effector caspase 3. Addition of a general caspase inhibitor, z-VAD. fmk, or a specific caspase 9 inhibitor reduced DNA fragmentation but had no effect on DeltaPsi(m) collapse, indicating this event was caspase independent. To determine the pathway leading to mitochondrial dysfunction, analysis of Bcl-2 family members established that only A1 mRNA levels were reduced prior to DeltaPsi(m) loss and that ectopic expression of A1 protected against cell death following inactivation of NF-kappaB. These data suggest that inhibition of NF-kappaB in macrophages initiates caspase 3-independent apoptosis through reduced A1 expression and mitochondrial dysfunction. Thus, constitutive NF-kappaB activation preserves macrophage viability by maintaining A1 expression and mitochondrial homeostasis.     

Mitochondrial K(ATP) channel activation reduces anoxic injury by restoring mitochondrial membrane potential

Mitochondrial membrane potential (DeltaPsi(m)) is severely compromised in the myocardium after ischemia-reperfusion and triggers apoptotic events leading to cell demise. This study tests the hypothesis that mitochondrial ATP-sensitive K(+) (mitoK(ATP)) channel activation prevents the collapse of DeltaPsi(m) in myocytes during anoxia-reoxygenation (A-R) and is responsible for cell protection via inhibition of apoptosis. After 3-h anoxia and 2-h reoxygenation, the cultured myocytes underwent extensive damage, as evidenced by decreased cell viability, compromised membrane permeability, increased apoptosis, and decreased ATP concentration. Mitochondria in A-R myocytes were swollen and fuzzy as shown after staining with Mito Tracker Orange CMTMRos and in an electron microscope and exhibited a collapsed DeltaPsi(m), as monitored by 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide (JC-1). Cytochrome c was released from mitochondria into the cytosol as demonstrated by cytochrome c immunostaining. Activation of mitoK(ATP) channel with diazoxide (100 micromol/l) resulted in a significant protection against mitochondrial damage, ATP depletion, cytochrome c loss, and stabilized DeltaPsi(m). This protection was blocked by 5-hydroxydecanoate (500 micromol/l), a mitoK(ATP) channel-selective inhibitor, but not by HMR-1098 (30 micromol/l), a putative sarcolemmal K(ATP) channel-selective inhibitor. Dissipation of DeltaPsi(m) also leads to opening of mitochondrial permeability transition pore, which was prevented by cyclosporin A. The data support the hypothesis that A-R disrupts DeltaPsi(m) and induces apoptosis, which are prevented by the activation of the mitoK(ATP) channel. This further emphasizes the therapeutic significance of mitoK(ATP) channel agonists in the prevention of ischemia-reperfusion cell injury.     

Bcl2 mitigates Ca2+ entry and mitochondrial Ca2+ overload through downregulation of L-type Ca2+ channels in PC12 cells

Altered calcium homeostasis and increased cytosolic calcium concentrations ([Ca(2+)](c)) are linked to neuronal apoptosis in epilepsy and in cerebral ischemia, respectively. Apoptotic programmed cell death is regulated by the antiapoptotic Bcl2 family of proteins. Here, we investigated the role of Bcl2 on calcium (Ca(2+)) homeostasis in PC12 cells, focusing on L-type voltage-dependent calcium channels (VDCC). Cytosolic Ca(2+) transients ([Ca(2+)](c)) and changes of mitochondrial Ca(2+) concentrations ([Ca(2+)](m)) were monitored using cytosolic and mitochondrially targeted aequorins of control PC12 cells and PC12 cells stably overexpressing Bcl2. We found that: (i) the [Ca(2+)](c) and [Ca(2+)](m) elevations elicited by K(+) pulses were markedly depressed in Bcl2 cells, with respect to control cells; (ii) such depression of [Ca(2+)](m) was not seen either in digitonin-permeabilized cells or in intact cells treated with ionomycin; (iii) the [Ca(2+)](c) transient depression seen in Bcl2 cells was reversed by shRNA transfection, as well as by the Bcl2 inhibitor HA14-1; (iv) the L-type Ca(2+) channel agonist Bay K 8644 enhanced K(+)-evoked [Ca(2+)](m) peak fourfold in Bcl2, and twofold in control cells; (v) in current-clamped cells the depolarization evoked by K(+) generated a more hyperpolarized voltage step in Bcl2, as compared to control cells. Taken together, our experiments suggest that the reduction of the [Ca(2+)](c) and [Ca(2+)](m) transients elicited by K(+), in PC12 cells overexpressing Bcl2, is related to the reduction of Ca(2+) entry through L-type Ca(2+) channels. This may be due to the fact that Bcl2 mitigates cell depolarization, thus diminishing the recruitment of L-type Ca(2+) channels, the subsequent Ca(2+) entry, and mitochondrial Ca(2+) overload.     

Cell death during ischemia: relationship to mitochondrial depolarization and ROS generation

Ischemia-reperfusion injury induces cell death, but the responsible mechanisms are not understood. This study examined mitochondrial depolarization and cell death during ischemia and reperfusion. Contracting cardiomyocytes were subjected to 60-min ischemia followed by 3-h reperfusion. Mitochondrial membrane potential (DeltaPsi(m)) was assessed with tetramethylrhodamine methyl ester. During ischemia, DeltaPsi(m) decreased to 24 +/- 5.5% of baseline, but no recovery was evident during reperfusion. Cell death assessed by Sytox Green was minimal during ischemia but averaged 66 +/- 7% after 3-h reperfusion. Cyclosporin A, an inhibitor of mitochondrial permeability transition, was not protective. However, pharmacological antioxidants attenuated the fall in DeltaPsi(m) during ischemia and cell death after reperfusion and decreased lipid peroxidation as assessed with C11-BODIPY. Cell death was also attenuated when residual O(2) was scavenged from the perfusate, creating anoxic ischemia. These results suggested that reactive oxygen species (ROS) were important for the decrease in DeltaPsi(m) during ischemia. Finally, 143B-rho(0) osteosarcoma cells lacking a mitochondrial electron transport chain failed to demonstrate a depletion of DeltaPsi(m) during ischemia and were significantly protected against cell death during reperfusion. Collectively, these studies identify a central role for mitochondrial ROS generation during ischemia in the mitochondrial depolarization and subsequent cell death induced by ischemia and reperfusion in this model.     

Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.

Treatment of elicited peritoneal macrophages or the macrophage cell line RAW 264.7 with high concentrations of nitric oxide donors is followed by apoptotic cell death. Analysis of the changes in the mitochondrial transmembrane potential (DeltaPsi(m)) with specific fluorescent probes showed a rapid and persistent increase of DeltaPsi(m), a potential that usually decreases in cells undergoing apoptosis through mitochondrial-dependent mechanisms. Using confocal microscopy, the release of cytochrome c from the mitochondria to the cytosol was characterized as an early event preceding the rise of DeltaPsi(m). The cytochrome c from cells treated with nitric oxide donors was modified chemically, probably through the formation of nitrotyrosine residues, suggesting the synthesis of peroxynitrite in the mitochondria. These results indicate that nitric oxide-dependent apoptosis in macrophages occurs in the presence of a sustained increase of DeltaPsi(m), and that the chemical modification and release of cytochrome c from the mitochondria precede the changes of DeltaPsi(m).-Hortelano, S., Alvarez, A. M., Boscá, L. Nitric oxide induces tyrosine nitration and release of cytochrome c preceding an increase of mitochondrial transmembrane potential in macrophages.     

MEK/ERK pathway protects ionizing radiation-induced loss of mitochondrial membrane potential and cell death in lymphocytic leukemia cells

MEK/ERK-mediated signals have recently been found to inhibit Fas-mediated cell death through inhibition of caspase-8 activity. It remains unknown whether MEK/ERK-mediated signals affect ionizing radiation (IR)-induced cell death. Here we demonstrate that MEK/ERK-mediated signals selectively inhibit IR-induced loss of mitochondrial membrane potential (DeltaPsi(m)) and subsequent cell death. In Jurkat cells, TPA strongly activated ERK and inhibited the IR-induced caspase-8/Bid cleavage and the loss of DeltaPsi(m). The inhibitory effect of TPA was mostly abrogated by pretreatment of a specific MEK inhibitor PD98059, indicating that the effect depends upon MEK/ERK-mediated signals. Moreover, BAF-B03 transfectants expressing IL-2 receptor (IL-2R) beta(c) chain lacking the acidic region, which is responsible for MEK/ERK-mediated signals, revealed higher sensitivity to IR than the transfectants expressing wild-type IL-2R. Interestingly, the signals could neither protect the DeltaPsi(m) loss nor cell death in UV-irradiated cells. These data imply that the anti-apoptotic effect of MEK/ERK-mediated signals appears to selectively inhibit the IR-induced cell death through protection of the DeltaPsi(m) loss. Our data enlighten an anti-apoptotic function of MEK/ERK pathway against IR-induced apoptosis, thereby implying its contribution to radioresistance.     

Mitochondrial alterations and tendency to apoptosis in peripheral blood cells from children with Down syndrome

Different types of cells from subjects with Down syndrome (DS) have an increased susceptibility to cell death. We have studied apoptosis and mitochondrial (mt) membrane potential (DeltaPsi(m)) in peripheral blood mononuclear cells (PBMC) from DS children and age-matched healthy donors after in vitro treatment with apoptogenic molecules, along with mtDNA content. We found that PBMC from DS and healthy controls had a similar tendency to undergo apoptosis and a similar amount of mtDNA. However, in cells from DS subjects, mitochondria showed a higher loss of DeltaPsi(m), underlying the presence of an increasing susceptibility of these organelles to damaging agents.     

Mitochondrial Ca2+ overload underlies Abeta oligomers neurotoxicity providing an unexpected mechanism of neuroprotection by NSAIDs

Dysregulation of intracellular Ca(2+) homeostasis may underlie amyloid beta peptide (Abeta) toxicity in Alzheimer's Disease (AD) but the mechanism is unknown. In search for this mechanism we found that Abeta(1-42) oligomers, the assembly state correlating best with cognitive decline in AD, but not Abeta fibrils, induce a massive entry of Ca(2+) in neurons and promote mitochondrial Ca(2+) overload as shown by bioluminescence imaging of targeted aequorin in individual neurons. Abeta oligomers induce also mitochondrial permeability transition, cytochrome c release, apoptosis and cell death. Mitochondrial depolarization prevents mitochondrial Ca(2+) overload, cytochrome c release and cell death. In addition, we found that a series of non-steroidal anti-inflammatory drugs (NSAIDs) including salicylate, sulindac sulfide, indomethacin, ibuprofen and R-flurbiprofen depolarize mitochondria and inhibit mitochondrial Ca(2+) overload, cytochrome c release and cell death induced by Abeta oligomers. Our results indicate that i) mitochondrial Ca(2+) overload underlies the neurotoxicity induced by Abeta oligomers and ii) inhibition of mitochondrial Ca(2+) overload provides a novel mechanism of neuroprotection by NSAIDs against Abeta oligomers and AD.     

Visualization of cyclosporin A and Ca2+-sensitive cyclical mitochondrial depolarizations in cell culture

Mitochondria not only facilitate chemiosmotic energy transduction, but also are excitable organelles that are important participants in intracellular Ca2+ signaling and are obligate participants in the active cell death cascade known as apoptosis. Underlying these functions is the cyclosporin A (CSA)-sensitive mitochondrial permeability transition pore (MTP), which can open transiently in a low conductance mode (MTPL) to relieve excess Ca2+, and irreversibly during the initiation of apoptosis. Here we image for the first time CSA- and Ca2+-sensitive cyclical mitochondrial depolarizations in cultures of the SH-SY5Y human neuroblastoma cell. In addition, we show that mitochondrial transmembrane potential (DeltaPsi) increases in response to CSA, indicating a baseline channel activity. Moreover, networks of mitochondria are shown to behave as an excitable system that may use Ca2+ as a diffusible messenger to recruit neighboring mitochondria to depolarize. We propose that these depolarizations represent MTPL activity. Our data further reinforce the notion that mitochondria are excitable organelles and suggest coordinated activation of MTPL.     

Extracellular superoxide released from mitochondria mediates mast cell death by advanced glycation end products

Advanced glycation end products (AGEs) accumulate during aging and to higher extents under pathological conditions such as diabetes. Since we previously showed that mast cells expressed the AGE-binding protein, receptor for AGEs (RAGE) on their cell surface, we examined whether AGE affected mast cell survival. Herein, we demonstrate that mast cells undergo apoptosis in response to AGE. Glycated albumin (GA), an AGE, but not stimulation with the high-affinity IgE receptor (FcepsilonRI), can induce mast cell death, as measured by annexin V/propidium iodide double-staining. GA (> or =0.1 mg/ml) exhibited this pro-apoptotic activity in a concentration-dependent manner. GA and FcepsilonRI stimulation increased the cytosolic Ca(2+) levels to a similar extent, whereas GA, but not FcepsilonRI stimulation, caused mitochondrial Ca(2+) overload and membrane potential collapse, resulting in mitochondrial integrity disruption, cytochrome c release and caspase-3/7 activation. In addition, GA, but not FcepsilonRI stimulation, induced extracellular release of superoxide from mitochondria, and this release played a key role in the disruption of Ca(2+) homeostasis. Knockdown of RAGE expression using small interfering RNA abolished GA-induced apoptosis, mitochondrial Ca(2+) overload, and superoxide release, demonstrating that RAGE mediates the GA-induced mitochondrial death pathway. AGE-induced mast cell apoptosis may contribute to the immunocompromised and inflammatory conditions.