BBRC

Biochemical and Biophysical Research Communications 304 (2003) 445454 www.elsevier.com/locate/ybbrc

Calcium signaling and apoptosis

Abstract Ca2 is one of the key regulators of cell survival, but Ca2 can also induce apoptosis in response to a variety of pathological conditions. The pro-apoptotic eects of Ca2 are mediated by a diverse range of Ca2 -sensitive factors that are compartmentalized in various intracellular organelles including the ER, cytoplasm, and mitochondria. The Ca2 dynamics of these organelles appear to be modulated by the apoptosis-regulating Bcl-2 family proteins. In this paper, the recent progress of research on the mechanisms mediating the apoptosis-regulating eects of Ca2 and the interactions of Bcl-2 family proteins with the Ca2 storage organelles are discussed. 2003 Elsevier Science (USA). All rights reserved.
Keywords: Apoptosis; Calcium signaling; Mitochondria; Bcl-2; Bid; VDAC; Cardiolipin; ROS

The number of studies concerned with calcium and apoptosis showed a progressive rise in the past decade and totaled approximately 1500 research papers in the past three years. By the time the research on calcium and apoptosis accelerated, ionized calcium had been established as a fundamental intracellular messenger and had also been recognized as a trigger of necrotic cell death under conditions of cellular calcium overload. Thus, a great deal of interest has been centered at the signaling mechanisms that connect alterations in calcium to the execution of apoptosis and allow for discrimination between the cell survival, apoptosis, and necrosis inducing eects of calcium. Although the work in this direction is far from being complete, numerous links that couple calcium to the extrinsic, intrinsic, and ER pathways of apoptosis have been discovered. Furthermore it has been established that selective activation of a particular cell survival- or cell death-promoting pathway by calcium utilizes the subcellular spatial and temporal pattern of the changes in [Ca2 ] and coincident detection of calcium and other signals. Studies undertaken in this direction rely heavily on the development of microscopic imaging technologies that permit monitoring cellular signals down to the single cell or subcellular level. Fur* Corresponding author. Fax: 1-S215-923-2218. E-mail address: [email protected] (G. Hajn czky). o

thermore, the critical role of changes in [Ca2 ] has been proven in a variety of tissues under various apoptosisinducing conditions. Evidence has been provided that apoptosis induced by ischemiareperfusion, UV-irradiation, reactive oxygen species, drugs (e.g., doxorubicin, cadmium, ceramide, 2-chlorodeoxyadenosine, and staurosporine), and even cytokines such as TNFa depends on calcium, at least in some cell types. Here, we shall review recent discoveries on the interaction between Bcl-2 family proteins and calcium signaling and on the mechanisms that sense and couple the changes of [Ca2 ] in ER, cytoplasm, and mitochondria to the execution of apoptosis. In 2000, we have published a review about the signaling cascades that couple IP3 /ryanodine receptor-mediated Ca2 mobilization to the control of the apoptotic machinery [1]. Thus, this paper is strictly conned to the progress achieved in the past three years. For recent reviews summarizing other aspects of the link between intracellular calcium homeostasis and apoptosis see [214]. Intracellular Ca2 + distribution: cell survival and cell death The cytoplasmic free Ca2 concentration Ca2 c is maintained at about 100 nM, a very low level relative to the extracellular uid Ca2 EC % 1:2 mM. The major

0006-291X/03/$ - see front matter 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00616-8

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mechanism to lower Ca2 c is Ca2 extrusion mediated by the plasma membrane Ca2 pumps and by the Na / Ca2 exchangers. Ca2 in the nuclear matrix Ca2 n and in the mitochondrial matrix Ca2 m is similar to Ca2 c . However, other intracellular organelles maintain a large Ca2 concentration gradient versus the cytoplasm. The most important Ca2 storage compartment is the endoplasmic reticulum (ER) (sarcoplasmic reticulum (SR) in muscle cells) in most cells. Into the lumen of SR and ER, sarco/endoplasmic reticulum Ca2 ATPases (SERCAs) pump Ca2 . The ER/SR provides a compartment from which Ca2 can be mobilized to increase Ca2 c . Ca2 distribution is depicted in the scheme shown in Fig. 1. Temporally and spatially organized increases in Ca2 c , Ca2 m , and Ca2 n represent one of the most commonly used intracellular signals. However, prolonged changes in Ca2 distribution including an elevation in Ca2 c , Ca2 m , and Ca2 n and a decrease in [Ca2 ER trigger a variety of cascades that lead to cell death. Activation of the ER/SR Ca2 release channels, IP3 receptors (IP3Rs), and ryanodine receptors (RyRs) results in a decrease of Ca2 ER and an increase of Ca2 c . Activation of plasma membrane Ca2 channels allows Ca2 to enter along the concentration and electrical gradients and also yields a Ca2 c increase. The large pores in the nuclear envelope permit Ca2 n to closely follow the changes in Ca2 c . The mitochondrial matrix is separated from the cytoplasm by two membranes and the inner mitochondrial membrane (IMM) has a very limited permeability to ions. However, conditions of elevated Ca2 c evoke activation of the Ca2 uniporter that mediates a membrane potential DWm driven Ca2 uptake to the mitochondria. Due to the

relatively low anity of the uniporter, the global Ca2 c rise %1 lM established by ER Ca2 release and Ca2 entry during physiological Ca2 c signals (yellow arrows in Fig. 1) results only a slow increase in Ca2 m . However, long-lasting global Ca2 c signals may result in accumulation of vast amounts of Ca2 in the mitochondria. Alternatively, mitochondria close to the ER or plasma membrane may sense the large and local Ca2 c increases in proximity to the activated Ca2 channels (orange arrows in Fig. 1) and exhibit a Ca2 m rise closely coupled to the rise of the Ca2 c signal. The local Ca2 c rise in the vicinity of activated Ca2 channels decays rapidly, providing for only a transient activation of mitochondrial Ca2 uptake. Control of intracellular Ca2 + transport by Bcl-2 family proteins Bcl-2 family proteins play a fundamental role in the integration of pro-apoptotic and anti-apoptotic signals. Many Bcl-2 family proteins are localized to and control the permeability properties of intracellular membranes. Bcl-xL is targeted to the mitochondria and protects the integrity of the outer mitochondrial membrane (OMM), whereas Bcl-2 distributes on several intracellular membranes and may have a major role in the control of the permeability of the ER membrane [15]. Among the proapoptotic Bcl-2 family proteins, Bak and a small fraction of Bax are associated with the mitochondria, whereas several BH3-only proteins, e.g., Bid and Bad and the major part of Bax are in the cytoplasm of surviving cells, but exhibit redistribution to the mitochondria during apoptosis (for references see [16]). Bcl-2 family proteins may interact with channels and transporters or based on results obtained with articial membranes may directly form pores to control membrane permeability. Regarding the control of Ca2 transport, most studies have been concentrated on the interaction of Bcl-2/Bcl-xL and Bax with the ER and mitochondria. Bcl-2/Bcl-xL Recent evidence suggests that overexpression of Bcl-2 results in a reduced ER Ca2 load and Ca2 ER , whereas down-regulation of Bcl-2 yields an increase in Ca2 ER [1719]. To promote a decrease in ER calcium, Bcl-2 seems to increase the passive Ca2 leak through the ER membrane without changing the activity of the SERCA pumps [17,18]. However, it is not clear whether Bcl-2 conducts Ca2 per se or activates an endogenous leak. Furthermore, Bcl-2 expression has also been reported to induce a decreased expression of the lumenal Ca2 binding chaperone, calreticulin, and SERCA2b pumps, providing additional mechanisms that may be important to lower Ca2 ER [19].

Fig. 1. Ca2 distribution in the cell. High and low Ca2 domains are marked with orange and blue, respectively. Global and local pathways of Ca2 signal propagation from Ca2 entry and ER Ca2 release to the mitochondria are marked with yellow and orange arrows, respectively.

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In Bcl-2 overexpressing cells, mobilization of ER Ca2 evoked by IP3 -linked agonists or thapsigargin, an inhibitor of the SERCA pumps, results in a relatively small Ca2 c signal that may be a consequence of a reduction in ER Ca2 loading. Furthermore, overexpression of Bcl-xL has been reported to evoke a decrease in IP3R protein (types 1 and 3) and this mechanism may also account for a decrease in the IP3 -dependent Ca2 c signal [20]. In addition, down-regulation of capacitative Ca2 entry may also be important for attenuation of the Ca2 c signal in Bcl-2-overexpressing cells [17,19]. However this point remains controversial, since upregulation of capacitative and swelling-induced Ca2 entry has also been observed in Bcl-2-overexpressing cells [18,21,22]. Overexpression of Bcl-2 results in an enhancement of the mitochondrial Ca2 uptake. Bcl-2 has been reported to promote the reduced redox state of pyridine nucleotides, providing a mechanism that protects against opening of the permeability transition pore (PTP) under conditions of Ca2 uptake and oxidative stress [23,24]. The Bcl-2-induced increase in mitochondrial volume may also be important for the control of mitochondrial Ca2 uptake [25]. Furthermore, Bcl-xL has been reported to control the opening of VDAC [26] and evidence is emerging that VDAC is part of the mitochondrial Ca2 uptake pathway [27,28]. Despite the increased mitochondrial Ca2 uptake capacity, the Ca2 m signal evoked by IP3R-mediated Ca2 mobilization from the ER is relatively small in Bcl-2 overexpressing cells [17]. This may be a consequence of the attenuated Ca2 mobilization from the ER and/or the increased mitochondrial volume in Bcl-2 overexpressing cells. Bax/Bak Similar to overexpression of Bcl-2, enforced expression of Bax or Bak has been reported to induce depletion of the ER Ca2 store ([29,30], but no eect in [31]). In Bax or Bak overexpressing cells, an early increase in mitochondrial calcium has also been observed and under conditions of inhibited mitochondrial Ca2 uptake the Bax/Bak-induced apoptosis was also inhibited [30,32]. These results indicate a Bax/Bak-induced, early redistribution of Ca2 from ER to the mitochondria. However the mechanisms underlying the ER Ca2 depletion and the activation of mitochondrial Ca2 uptake sites remain elusive. Bid In response to engagement of the death receptors, Bid, a BH3-only Bcl-2 family protein, is cleaved by caspase-8, and subsequently, the truncated C-terminus Bid (tBid) is translocated from cytoplasm to the mito-

chondria to induce release of apoptotic factors. During tBid-induced release of apoptotic factors from the mitochondria, the inner mitochondrial membrane (IMM) barrier function is maintained in several experimental models, e.g., [3335], although remodeling of the IMM [36] and permeabilization of both the outer mitochondrial membrane (OMM) have also been reported [37]. In permeabilized RBL-2H3 cells, we found that tBid evoked rapid permeabilization of the OMM, but the IMM barrier was preserved [38]. We used this model to evaluate the eect of the tBid-induced selective OMM-permeabilization on ER and mitochondrial Ca2 handling. tBid did not aect the ER Ca2 store and IP3 induced Ca2 release. By contrast, a marked increase in the IP3 -induced Ca2 m signal was observed, suggesting that tBid promoted propagation of the IP3 -induced Ca2 c signal to the mitochondria. However, tBid failed to aect the Ca2 m rise during sustained and global Ca2 c elevations evoked by addition of Ca2 or thapsigargin [38]. Thus, the OMM seems to attenuate exposure of the mitochondrial Ca2 uptake sites to the transient, high Ca2 c microdomains generated by the neighboring IP3Rs, but the OMM Ca2 permeability is sucient to ensure optimal activation of the Ca2 uniporter during sustained Ca2 c signals. tBid may optimize local Ca2 delivery from the IP3Rs to the IMM via selective permeabilization of the OMM.

Eectors of Ca2 + in the ER, cytoplasm, and mitochondria ER Physiological stimuli trigger transient and partial discharge of the ER Ca2 store and Bcl-2 seems to utilize a partial decrease in ER Ca2 to promote cell survival [39]. However, massive depletion of the ER Ca2 store is an ER stress condition that initiates execution of apoptosis. Release of ER Ca2 provides for a Ca2 c elevation that may activate Ca2 -sensitive cytoplasmic enzymes to initiate apoptosis (see below). However, in a variety of paradigms, the Ca2 c elevation is not essential for execution of the ER stress-induced apoptosis. Thus, depletion of ER calcium per se promotes activation of an apoptotic cascade. Potential eectors of Ca2 are the lumenal Ca2 binding chaperones such as calreticulin. Calreticulin is believed to play a critical role in quality control processes during protein synthesis and folding. Calreticulin interacts in a Ca2 dependent fashion with other lumenal chaperones, e.g., protein disulde isomerase and ERp57, with unfolded glycoproteins and with Ca2 transporters at the ER membrane (for references see [40]). Based on a report by Nakamura et al. [40], calreticulin decient cells are resistant to apoptosis, whereas calreticulin overexpressing

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cells exhibit enhanced sensitivity to apoptosis. Interestingly, calreticulin seems to control both thapsigarginand staurosporine-induced apoptosis [41], indicating that it has a role both in the ER Ca2 depletion-induced and in the mitochondrial apoptotic pathways. Of note, calreticulin also serves as a Ca2 buer species in the lumen of the ER and so changes in calreticulin expression are associated with substantial changes in Ca2 handling by the ER and in calcium signal propagation from ER to mitochondria [40]. Interestingly, in Caenorhabditis elegans, mutation of calreticulin or knockdown of calnexin, another Ca2 binding chaperone, suppressed the MEC-4(d) ion channel induced cell death, but thapsigargin-induced ER Ca2 release could restore the cell death when calreticulin was absent [42]. Based on this result, calreticulin is not essential for ER Ca2 depletion-induced apoptosis in the worm. In addition to the chaperones, ER membrane proteins that display a Ca2 binding site on the lumenal side such as the IP3R and RyR may also sense Ca2 depletion. In RyR overexpressing CHO cells, stimulation of RyR-mediated Ca2 release triggered apoptosis, but IP3R-mediated store depletion failed to promote cell death [43]. This result suggests that RyRs could mediate ER Ca2 release but did not serve to sense the Ca2 depletion mediated by the IP3Rs. Thus, further studies will be necessary to identify the protein(s) that sense the change in ER Ca2 content and trigger activation of an apoptotic cascade. Cytoplasm Elevation of Ca2 c may result in activation of a wide variety of Ca2 -sensitive enzymes. These enzymes may generate signaling molecules for recruitment of mitochondria to the apoptotic cascade or for activation of the caspase enzymes. Calpain, a ubiquitous cysteine protease, and calcineurin, a serine/threonine phosphatase, are among the cytoplasmic targets of Ca2 , which are directly connected to the executioners of apoptosis. The Ca2 -sensitive NO synthase is discussed together with other sources of the reactive oxygen species (see below). Calpain has been implicated in the cleavage and targeting of Bax to the mitochondria. Recently, calpain has also been reported to cleave Bid, a BH3-only Bcl-2 family protein, resulting in a cleavage product that induces cytochrome c (cyto c) release from mitochondria [44,45]. Calpain mediated Bid-cleavage has been shown to be important in cisplatin-induced apoptosis [44] and in ischemia/reperfusion injury [46]. Importantly, the calpain activation seems to be downstream of a rise in Ca2 c in both cisplatin- and ischemia/reperfusion-induced apoptosis. Calpain has also been shown to cleave both Bid and Bcl-2 in calcium-ionophore-treated cells [47]. Truncation enhances the Bids capacity to induce mitochondrial membrane permeabilization (see above),

whereas the cleavage may decrease the Bcl-2s capacity to protect the OMM barrier. Another emerging target of calpain is cain/cabin1, an endogenous inhibitor of calcineurin. During calcium-ionophore induced cell death, calpain has been reported to cleave the calcineurinbinding domain of cain/cabin1 to activate calcineurin [48]. Calcineurin-mediated dephosphorylation of Bad promotes the translocation of Bad to the mitochondria and in turn, the association of Bad with Bcl-xL . Calcium-induced, calpain mediated cleavage of cabin1 also results in activation of myocyte enhancer factor 2 (MEF2) and consequent transcription of Nur77, a nuclear receptor. This sequence has been found to be important in TCR-mediated thymocyte apoptosis [49]. Interestingly, immortalization of B cells evoked by EpsteinBarr virus infection also seems to depend on an interaction with Nur77, which prevents Nur77 from targeting mitochondria in response to apoptotic stimuli [50]. Finally, calpain has also been reported to cleave and in turn, activate a caspase (caspase-12) that is localized on the cytoplasmic side of ER [51]. This pathway might allow execution of apoptosis to proceed without recruitment of the mitochondria. Mitochondria Calcium signal propagation to the mitochondria results in stimulation of ATP production through activation of the Ca2 -sensitive dehydrogenases and yields important feed back eects on cytoplasmic calcium signaling. By contrast, excessive Ca2 load to the mitochondria may induce apoptosis by stimulating the release of apoptosis promoting factors from the mitochondrial intermembrane space to the cytoplasm (cyto c, AIF, Smac/DIABLO, OMI/HtrA2, pro-caspases, etc.; for a recent review see [52]) and by impairing mitochondrial function, e.g., reactive oxygen species (ROS) generation by the damaged respiratory chain. Under certain predisposing conditions, small amounts of Ca2 , e.g., physiological Ca2 m spikes also trigger mitochondrial membrane permeabilization and apoptosis instead of a metabolic response. For example, in C2-ceramide pretreated permeabilized HepG2 cells, a relatively small Ca2 loading evoked complete depolarization followed by partial release of cyto c (Fig. 2). Subsequently, addition of tBid, a pro-apoptotic Bcl-2 family protein, caused completion of the cyto c release (Fig. 2). Both the Ca2 -induced depolarization and cyto c release were prevented by cyclosporin A (CsA), whereas the tBid-induced cyto c release was insensitive to CsA in this model [53,59]. Thus, the Ca2 -induced depolarization was due to opening of the permeability transition pore (PTP) and the PTP opening was also critical for the subsequent cyto c release. Importantly, the Ca2 -induced depolarization appeared as a wave, indicating coordinated recruitment of mitochondria to

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Fig. 2. Temporal and spatial organization of mitochondrial depolarization and cyto c release induced by C2-ceramide+Ca2 and tBid. Fluorescence imaging of DWm was carried out simultaneously with cyto c distribution in cyto c-GFP-expressing and TMRE-loaded HepG2 cells. Sequential uorescence images are shown as green (Fcyto c-GFP ), red (FTMRE ) overlays. Depolarization appears as a loss of the red uorescence, whereas cyto c release appears as a loss of the green uorescence. Cyto c-GFP was expressed only in three cells that are shown in the upper right quadrant. Permeabilized cells were pretreated with C2-ceramide (40 lM for 5 min) before addition of Ca2 (50 lM CaCl2 ) and, subsequently, tBid (25 nM). In the absence of C2-ceramide, a relatively small Ca2 -induced depolarization and no cyto c-GFP release were observed (not shown). The Ca2 -induced mitochondrial depolarization initiated in discrete subcellular regions and propagated as waves through cells (direction of the wave is marked by the white arrow in the upper left image). Time courses of the uorescence changes are plotted in the graph. For experimental details see [53,59,87].

the PTP opening (Fig. 2, direction of wave propagation is marked by a white arrow in the rst image). In addition to the PTP, ROS, cardiolipin, and Ca2 -activated K channels (mitoKCa ) have been emerging as key players in the control of the mitochondrial phase of the Ca2 -dependent apoptosis. Mitochondria are the major source of ROS in the cell. Superoxide O is generated by the operation of 2 complexes I and III in the matrix and is converted to hydrogen peroxide H2 O2 by Mn-superoxide dismutase. O can also rapidly react with another free radical, 2 namely, nitric oxide (NO), yielding more toxic ROS such as peroxynitrite. Among other factors, Ca2 accumulation has been reported to stimulate mitochondrial ROS production. Notably, if mitochondrial Ca2 uptake triggers signicant depolarization, this may cause a decrease in the DWm -dependent complex III-mediated ROS formation. Mitochondrial Ca2 has been reported to trigger the mitochondrial NO synthase activity and

cytoplasmic Ca2 may also control the availability of NO through activation of the Ca2 -sensitive cytoplasmic NO synthase for references see [5458]. Oxidants can directly interact with and damage a range of mitochondrial proteins and membrane lipids. For example, O (generated by xanthine + xanthine 2 oxidase) can induce permeabilization of the OMM independent of Ca2 and recruitment of Bax to the mitochondria [59]. Furthermore, ROS also targets proteins (e.g., PTP) and lipids (e.g., cardiolipin) that are controlled by Ca2 m , providing a means to increase or decrease their sensitivity to Ca2 m and in turn, to change the specicity of mitochondrial calcium signaling. In addition, ROS may induce ROS release from mitochondria to the cytoplasm [60] and exposure to ROS changes the activity of many Ca2 transporters. Recently, mitochondrial ROS production has been reported to induce Ca2 release from ER, which in turn, stimulated mitochondrial Ca2 loading. The amplication

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of oxidative stress and Ca2 loading culminated in mitochondrial membrane permeabilization and cell death [61]. Cardiolipin and other anionic phospholipids bind Ca2 with high anity. Cardiolipin is conned to the IMM and to the contact sites and is associated with a variety of mitochondrial proteins such as cyto c, NADH:ubiquinone oxidoreductase, cyto c oxidase, F1 F0 ATPase, and adenine nucleotide translocase. The structure and concentration of cardiolipin is central to the function of these proteins (for references see [62,63]). Cardiolipin at the contact sites has been claimed to be the target of tBid that translocates to the mitochondria and induces OMM permeabilization during apoptosis [64]. Based on in vitro studies, binding of Ca2 to cardiolipin results in a change in the membrane topography favoring cardiolipin peroxidation [65]. In addition, Ca2 loading to the mitochondrial matrix facilitates ROS production (see above). In several apoptotic paradigms, peroxidation of cardiolipin is an early event that is envisioned to allow cyto c release, e.g., [66,67]. Interaction of Ca2 with cardiolipin containing membranes also promotes Bax-induced membrane leakage [68] and this eect of Ca2 may account for the mitochondrial Ca2 uptake-dependent potentiation of Bax-induced cyto c release in rat liver mitochondria [69]. Thus, during elevation of Ca2 m , binding of Ca2 to cardiolipin may provide mechanisms that lead to a damage to the membrane barrier. However, further studies will be necessary to establish conditions of mitochondrial membrane permeabilization, which depend on the interaction of accumulated Ca2 with cardiolipin. The mitochondrial PTP represents a non-specic pore that may exhibit cut-o in permeability at 1.5 kDa or at <300 Da and opens under conditions of elevated Ca2 m . Most commonly, Ca2 loading to the mitochondria triggers PTP opening that appears as IMM permeabilization, uncoupling, and often as massive mitochondrial swelling. The PTP seems to be formed by a multi-protein complex that is assembled at the contact sites between IMM and OMM but the exact molecular composition of the PTP has not been elucidated. A widely considered model suggests that the primary components of the PTP include the voltage-dependent anion channel (VDAC) in the OMM, adenine nucleotide translocase (ANT) in the inner membrane, and cyclophilin D (CypD) within the mitochondrial matrix (for recent reviews see [7072]). PTPs have been established to play an important role in many paradigms of both apoptotic and necrotic cell death. In addition to VDAC, ANT, and CypD, proteins that are associated with PTP including hexokinase, peripheral benzodiazepine receptor, mitochondrial creatine kinase and Bcl-2 family proteins or lipids concentrated at the contact sites (e.g., cardiolipin, see above) have also been implicated in apoptosis (recently reviewed in [7378]).

It is clear that activation of the PTP by Ca2 requires Ca2 accumulation to the mitochondrial matrix but the respective role of CypD, ANT, and VDAC in sensing Ca2 m has not been established. The signicance of CypD has been underscored by the observation that cyclosporin A-induced dissociation of CypD from the mitochondrial membrane is accompanied by protection against Ca2 -induced PTP opening in many models. However, multiple Ca2 regulatory sites may be important in opening of the PTP. For example, a recent study demonstrated that micromolar concentrations of Ca2 regulate the protein-protein interaction between VDAC and the mitochondrial creatine kinase using surface plasmon resonance spectroscopy [79]. Importantly, opening of the PTP by Ca2 is also aected by other modulators of the PTP including ROS, pH, DWm , and adenine nucleotides (reviewed in [80,81]). Opening of the PTP by mitochondrial Ca2 is also potentiated by arachidonic acid generated by phospholipase A2 activation in TNFa-treated cells [82]. The interaction between ROS and Ca2 , which results in activation of the PTP, is illustrated in Fig. 3. Xanthine + xanthine oxidase (X + XO) and menadione (MEN) were added to permeabilized HepG2 cells to enhance O generation in the 2 cytoplasm and in the mitochondrial matrix, respectively. In the presence of both X + XO and MEN, mitochondrial depolarization evoked by Ca2 pulses was augmented and prolonged (Figs. 3A and C). Simultaneously, both in the X + XO and MEN pretreated cells, mitochondria lost their capacity to keep sequestered the exogenously added Ca2 (Figs. 3B and D). Enhanced depolarization and impaired Ca2 uptake exhibited by both X + XO and menadione treated cells reected opening of PTP as it was inhibited by CsA (Figs. 3AD). Thus, Ca2 -induced PTP opening is largely facilitated by O generated either in the cytosol or 2 in the mitochondria. To determine whether conversion of O to H2 O2 may also be important for sensitization 2 of the PTP, H2 O2 was also added to the permeabilized cells. H2 O2 that permeates through the mitochondrial membranes also potentiated the Ca2 -induced mitochondrial depolarization and decreased Ca2 retention in mitochondria, suggesting that H2 O2 also promotes the interaction of Ca2 with the PTP (Figs. 3E and F). In contrast to the eect of ROS, overexpression of Bcl-2 has been reported to suppress Ca2 activation of a mitochondrial megachannel that is considered to be a manifestation of the PTP [83]. This result indicates that Bcl-2 and other proteins that are associated with the PTP may also modulate the activation by Ca2 m . An important implication of the modulation of Ca2 activation by other regulators of the PTP is that pro-apoptotic signals may promote mitochondrial membrane permeabilization by increasing the Ca2 sensitivity of the PTP, whereas anti-apoptotic signals may strengthen the mitochondrial membrane barrier by suppressing the

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Fig. 3. Eect of ROS on Ca2 -induced PTP opening. For the simultaneous measurements of DWm and mitochondrial Ca2 uptake, the permeabilized cells were supplemented with JC1 (800 nM) and fura2FF/FA (0.5 lM) and ratiometric uorescence measurement was carried out in a multiwavelength-excitation dual wavelength-emission uorimeter. (A,B) O generating system (xanthine, 0.1 mM + xanthine oxidase, 20 mU/ml), (C,D) 2 menadione (10 lM), and (E,F) H2 O2 (90 mM) augmented Ca2 -induced depolarization (three pulses, 30 lM CaCl2 each) and decreased mito2 chondrial Ca uptake. These eects were inhibited by CsA (1 lM). Addition of X + XO, MEN, H2 O2 , and Ca2 is marked by arrows. For experimental details see [35].

sensitivity to Ca2 . In many cell types, physiological Ca2 m spikes result in activation of the Ca2 -sensitive matrix dehydrogenases and stimulate ATP production, but fail to induce PTP opening. However, in the presence of C2-ceramide or staurosporine, the Ca2 m spikes evoke PTP opening that is followed by cyto c release, caspase activation, and execution of apoptosis [53]. Thus coincidence detection by the PTP may allow a switch from a Ca2 m spiking-induced survival promoting program to a Ca2 m spiking-induced apoptotic cascade. Recent studies have revealed a similar principle of induction of apoptosis in other systems [39,84]. Another remarkable feature of the activation of PTP by Ca2 is that the opening of the PTP permits release of the accumulated Ca2 that is subsequently taken up by adjacent mitochondria. This mechanism may result in a regenerative response that spreads throughout the entire mitochondrial population of the cell (for recent reviews see [85,86]). Notably, PTP-dependent intermitochondrial calcium signaling has been reported to recruit mitochondria to the apoptotic process [87]. As a high density of mitochondria facilitates communication between neighboring organelles, the local signaling machinery can be utilized eectively in the large myotubes

of heart and skeletal muscle that are among the cells most abundant in mitochondria. Emerging evidence supports the fact that ATP-sensitive (mitoKATP ) and Ca2 -activated K channels (mitoKCa ) are present on the IMM in a variety of cell types (for references see [88,89]). The mitoKCa appears to be a BK-type Ca2 -activated K channel [90]. The IMM K channels are believed to be important determinants of resistance to ischemic damage and apoptosis (for references see [89]). Openers of the mitoKATP have been shown to induce a large decrease in DWm [91,92], however, this eect was not necessarily due to opening of mitoKATP because of the potentially confounding nonspecic eects of the drugs at high concentrations. Based on studies of the pharmacological and toxic effects of K channel openers, the increased K inux associated with opening of mitoKATP has been reported to cause only 12 mV depolarization but induced a signicant increase in matrix volume [93]. Thus mitoKATP may be primarily involved in the volume regulation. In myocytes, mitoKCa -mediated current was detected at resting Ca2 c (approx. 200 nM) and was largely increased by elevation of Ca2 c to 40 lM. Mitochondrial Ca2 uptake stimulated by the rise in Ca2 c may provide

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G. Hajnczky et al. / Biochemical and Biophysical Research Communications 304 (2003) 445454 o [9] C.W. Distelhorst, Recent insights into the mechanism of glucocorticosteroid-induced apoptosis, Cell Death Dier. 9 (2002) 619. [10] G. Ermak, K.J. Davies, Calcium and oxidative stress: from cell signaling to cell death, Mol. Immunol. 38 (2002) 713721. [11] C. Gill, R. Mestril, A. Samali, Losing heart: the role of apoptosis in heart diseasea novel therapeutic target?, FASEB J. 16 (2002) 135146. [12] J.J. Lemasters, T. Qian, L. He, J.S. Kim, S.P. Elmore, W.E. Cascio, D.A. Brenner, Role of mitochondrial inner membrane permeabilization in necrotic cell death, apoptosis, and autophagy, Antioxid. Redox Signal. 4 (2002) 769781. [13] P. Pinton, D. Ferrari, E. Rapizzi, F. Di Virgilio, T. Pozzan, R. Rizzuto, A role for calcium in Bcl-2 action?, Biochimie 84 (2002) 195201. [14] S.S. Smaili, Y.T. Hsu, R.J. Youle, J.T. Russell, Mitochondria in Ca2 signaling and apoptosis, J. Bioenerg. Biomembr. 32 (2000) 3546. [15] T. Kaufmann, S. Schlipf, J. Sanz, K. Neubert, R. Stein, C. Borner, Characterization of the signal that directs Bcl-xL, but not Bcl-2, to the mitochondrial outer membrane, J. Cell Biol. 160 (2003) 5364. [16] M. Karbowski, Y.J. Lee, B. Gaume, S.Y. Jeong, S. Frank, A. Nechushtan, A. Santel, M. Fuller, C.L. Smith, R.J. Youle, Spatial and temporal association of Bax with mitochondrial ssion sites, Drp1, and Mfn2 during apoptosis, J. Cell Biol. 159 (2002) 931 938. [17] P. Pinton, D. Ferrari, P. Magalhaes, K. Schulze-Ostho, F. Di Virgilio, T. Pozzan, R. Rizzuto, Reduced loading of intracellular Ca(2+) stores and downregulation of capacitative Ca(2+) inux in Bcl-2-overexpressing cells, J. Cell Biol. 148 (2000) 857862. [18] R. Foyouzi-Yousse, S. Arnaudeau, C. Borner, W.L. Kelley, J. Tschopp, D.P. Lew, N. Demaurex, K.H. Krause, Bcl-2 decreases the free Ca2 concentration within the endoplasmic reticulum, Proc. Natl. Acad. Sci. USA 97 (2000) 57235728. [19] F. Vanden Abeele, R. Skryma, Y. Shuba, F. Van Coppenolle, C. Slomianny, M. Roudbaraki, B. Mauroy, F. Wuytack, N. Prevarskaya, Bcl-2-dependent modulation of Ca(2+) homeostasis and store-operated channels in prostate cancer cells, Cancer Cell 1 (2002) 169179. [20] C. Li, C.J. Fox, S.R. Master, V.P. Bindokas, L.A. Chodosh, C.B. Thompson, Bcl-X(L) aects Ca(2+) homeostasis by altering expression of inositol 1,4,5-trisphosphate receptors, Proc. Natl. Acad. Sci. USA 99 (2002) 98309835. [21] S.S. Williams, J.N. French, M. Gilbert, A.A. Rangaswami, J. Walleczek, S.J. Knox, Bcl-2 overexpression results in enhanced capacitative calcium entry and resistance to SKF-96365-induced apoptosis, Cancer Res. 60 (2000) 43584361. [22] M.R. Shen, T.P. Yang, M.J. Tang, A novel function of BCL-2 overexpression in regulatory volume decrease. Enhancing swelling-activated Ca(2+) entry and Cl()) channel activity, J. Biol. Chem. 277 (2002) 1559215599. [23] E. Gottlieb, M.G. Vander Heiden, C.B. Thompson, Bcl-x(L) prevents the initial decrease in mitochondrial membrane potential and subsequent reactive oxygen species production during tumor necrosis factor a-induced apoptosis, Mol. Cell Biol. 20 (2000) 56805689. [24] A.J. Kowaltowski, A.E. Vercesi, G. Fiskum, Bcl-2 prevents mitochondrial permeability transition and cytochrome c release via maintenance of reduced pyridine nucleotides, Cell Death Dier. 7 (2000) 903910. [25] A.J. Kowaltowski, R.G. Cosso, C.B. Campos, G. Fiskum, Eect of Bcl-2 overexpression on mitochondrial structure and function, J. Biol. Chem. 277 (2002) 4280242807. [26] M.G. Vander Heiden, X.X. Li, E. Gottleib, R.B. Hill, C.B. Thompson, M. Colombini, Bcl-xL promotes the open conguration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane, J. Biol. Chem. 276 (2001) 1941419419.

Ca2 for the regulatory site that is likely to face the matrix [89]. Activation of mitoKCa has been proposed to be important in protection against ischemic injury [89]. Along this line, activation of mitoKCa may be envisioned as a Ca2 m -dependent antiapoptotic mechanism, however, the exact role of mitoKCa in cell death remains an important subject for future investigations.

Conclusions Bcl-2 family proteins emerge as major regulators of cellular calcium handling. In addition, apoptotic factors released from the mitochondria and activated caspase enzymes may also control Ca2 transport proteins (e.g., control of IP3 receptors and SERCA by ROS or caspase-3). These eects on calcium handling often provide for amplication loops for the apoptotic cascade or enhance the security of antiapoptotic mechanisms. Usually, depletion of ER Ca2 and elevation of Ca2 c or Ca2 m are involved in the execution of apoptosis, but Ca2 c and Ca2 m signals may also be important for cell survival mechanisms. In each compartment, several Ca2 eectors are present and many of these eectors may serve as a link to the control of the apoptotic cascade. However, it remains a goal for future studies to delineate the Ca2 dependent mechanisms that are required for activation or inhibition of an apoptotic cascade. Overall, such studies may also help to understand the pathogenesis of a number of disorders that involve impaired control of Ca2 transport or Ca2 eectors. References
[1] G. Hajn czky, G. Csords, M. Madesh, P. Pacher, Control of o a apoptosis by IP3 and ryanodine receptor driven calcium signals, Cell Calcium 28 (2000) 349363. [2] M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signalling, Nat. Rev. Mol. Cell Biol. 1 (2000) 1121. [3] L.P. Zhu, X.D. Yu, S. Ling, R.A. Brown, T.H. Kuo, Mitochondrial Ca(2+) homeostasis in the regulation of apoptotic and necrotic cell deaths, Cell Calcium 28 (2000) 107117. [4] J. Jacobson, M.R. Duchen, What nourishes me, destroys me: towards a new mitochondrial biology, Cell Death Dier. 8 (2001) 963966. [5] M.P. Mattson, D.S. Gary, S.L. Chan, W. Duan, Perturbed endoplasmic reticulum function, synaptic apoptosis and the pathogenesis of Alzheimers disease, Biochem. Soc. Symp. 67 (2001) 151162. [6] P. Pacher, G. Csords, G. Hajnczky, Mitochondrial Ca2 a o signaling and cardiac apoptosis, Biol. Signal. Recept. 10 (2001) 200223. [7] M.S. Suleiman, A.P. Halestrap, E.J. Griths, Mitochondria: a target for myocardial protection, Pharmacol. Ther. 89 (2001) 29 46. [8] S.P. Yu, L.M. Canzoniero, D.W. Choi, Ion homeostasis and apoptosis, Curr. Opin. Cell Biol. 13 (2001) 405411.

G. Hajnczky et al. / Biochemical and Biophysical Research Communications 304 (2003) 445454 o [27] D. Gincel, H. Zaid, V. Shoshan-Barmatz, Calcium binding and translocation by the voltage-dependent anion channel: a possible regulatory mechanism in mitochondrial function, Biochem. J. 358 (2001) 147155. [28] E. Rapizzi, P. Pinton, G. Szabadkai, M.R. Wieckowski, G. Vandecasteele, G. Baird, R.A. Tuft, K.E. Fogarty, R. Rizzuto, Recombinant expression of the voltage-dependent anion channel enhances the transfer of Ca2 microdomains to mitochondria, J. Cell Biol. 159 (2002) 613624. [29] Z. Pan, M.B. Bhat, A.L. Nieminen, J. Ma, Synergistic movements of Ca(2+) and Bax in cells undergoing apoptosis, J. Biol. Chem. 276 (2001) 3225732263. [30] L.K. Nutt, A. Pataer, J. Pahler, B. Fang, J. Roth, D.J. McConkey, S.G. Swisher, Bax and Bak promote apoptosis by modulating endoplasmic reticular and mitochondrial Ca2 stores, J. Biol. Chem. 277 (2002) 92199225. [31] S. Aiba-Masago, Xb.XB. Liu, R. Masago, N. Vela-Roch, F. Jimenez, C.M. Lau, V.C. Frohlich, N. Talal, H. Dang, Bax gene expression alters Ca(2+) signal transduction without aecting apoptosis in an epithelial cell line, Oncogene 21 (2002) 27622767. [32] L.K. Nutt, J. Chandra, A. Pataer, B. Fang, J.A. Roth, S.G. Swisher, R.G. ONeil, D.J. McConkey, Bax-mediated Ca2 mobilization promotes cytochrome c release during apoptosis, J. Biol. Chem. 277 (2002) 2030120308. [33] O. von Ahsen, C. Renken, G. Perkins, R.M. Kluck, E. BossyWetzel, D.D. Newmeyer, Preservation of mitochondrial structure and function after Bid- or Bax-mediated cytochrome c release, J. Cell Biol. 150 (2000) 10271036. [34] V.K. Mootha, M.C. Wei, K.F. Buttle, L. Scorrano, V. Panoutsakopoulou, C.A. Mannella, S.J. Korsmeyer, A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c, EMBO J. 20 (2001) 661 671. [35] M. Madesh, G. Hajnczky, VDAC-dependent permeabilization of o the outer mitochondrial membrane by superoxide induces rapid and massive cytochrome c release, J. Cell Biol. 155 (2001) 1003 1015. [36] L. Scorrano, M. Ashiya, K. Buttle, S. Weiler, S.A. Oakes, C.A. Mannella, S.J. Korsmeyer, A distinct pathway remodels mitochondrial cristae and mobilizes cytochrome c during apoptosis, Dev. Cell 2 (2002) 5567. [37] N. Zamzami, C. El Hamel, C. Maisse, C. Brenner, C. MunozPinedo, A.S. Belzacq, P. Costantini, H. Vieira, M. Loeer, G. Molle, G. Kroemer, Bid acts on the permeability transition pore complex to induce apoptosis, Oncogene 19 (2000) 6342 6350. [38] G. Csords, M. Madesh, B. Antonsson, G. Hajnczky, tcBid a o promotes Ca2 signal propagation to the mitochondria: control of Ca2 permeation through the outer mitochondrial membrane, EMBO J. 21 (2002) 21982206. [39] P. Pinton, D. Ferrari, E. Rapizzi, F.D. Di Virgilio, T. Pozzan, R. Rizzuto, The Ca2 concentration of the endoplasmic reticulum is a key determinant of ceramide-induced apoptosis: signicance for the molecular mechanism of Bcl-2 action, EMBO J. 20 (2001) 26902701. [40] S. Arnaudeau, M. Frieden, K. Nakamura, C. Castelbou, M. Michalak, N. Demaurex, Calreticulin dierentially modulates calcium uptake and release in the endoplasmic reticulum and mitochondria, J. Biol. Chem. 277 (2002) 4669646705. [41] A. Asumendi, M.C. Morales, A. Alvarez, J. Arechaga, G. PerezYarza, Implication of mitochondria-derived ROS and cardiolipin peroxidation in N-(4-hydroxyphenyl)retinamide-induced apoptosis, Br. J. Cancer 86 (2002) 19511956. [42] K. Xu, N. Tavernarakis, M. Driscoll, Necrotic cell death in Caenorhabditis elegans requires the function of calreticulin and regulators of Ca(2+) release from the endoplasmic reticulum, Neuron 31 (2001) 957971.

453

[43] Z. Pan, D. Damron, A.L. Nieminen, M.B. Bhat, J. Ma, Depletion of intracellular Ca2 by caeine and ryanodine induces apoptosis of chinese hamster ovary cells transfected with ryanodine receptor, J. Biol. Chem. 275 (2000) 1997819984. [44] A. Mandic, K. Viktorsson, L. Strandberg, T. Heiden, J. Hansson, S. Linder, M.C. Shoshan, Calpain-mediated Bid cleavage and calpain-independent Bak modulation: two separate pathways in cisplatin-induced apoptosis, Mol. Cell. Biol. 22 (2002) 3003 3013. [45] M. Chen, H. He, S. Zhan, S. Krajewski, J.C. Reed, R.A. Gottlieb, Calpain and mitochondria in ischemia/reperfusion injury, J. Biol. Chem. 277 (2002) 2918129186. [46] M. Chen, D.J. Won, S. Krajewski, R.A. Gottlieb, Calpain and mitochondria in ischemia/reperfusion injury, J. Biol. Chem. 277 (2002) 2918129186. [47] S. Gil-Parrado, A. Fernandez-Montalvan, I. Assfalg-Machleidt, O. Popp, F. Bestvater, A. Holloschi, T.A. Knoch, E.A. Auerswald, K. Welsh, J.C. Reed, H. Fritz, P. Fuentes-Prior, E. Spiess, G.S. Salvesen, W. Machleidt, Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members, J. Biol. Chem. 277 (2002) 2721727226. [48] M.J. Kim, D.G. Jo, G.S. Hong, B.J. Kim, M. Lai, D.H. Cho, K.W. Kim, A. Bandyopadhyay, Y.M. Hong, H. Kim do, C. Cho, J.O. Liu, S.H. Snyder, Y.K. Jung, Calpain-dependent cleavage of cain/cabin1 activates calcineurin to mediate calcium-triggered cell death, Proc. Natl. Acad. Sci. USA 99 (2002) 98709875. [49] H.D. Youn, J.O. Liu, Cabin1 represses MEF2-dependent Nur77 expression and T cell apoptosis by controlling association of histone deacetylases and acetylases with MEF2, Immunity 13 (2000) 8594. [50] J.M. Lee, K.H. Lee, M. Weidner, B.A. Osborne, S.D. Hayward, Epstein-Barr virus EBNA2 blocks Nur77-mediated apoptosis, Proc. Natl. Acad. Sci. USA 99 (2002) 1187811883. [51] T. Nakagawa, J. Yuan, Cross-talk between two cysteine protease families. Activation of caspase-12 by calpain in apoptosis, J. Cell Biol. 150 (2000) 887894. [52] X. Wang, The expanding role of mitochondria in apoptosis, Genes Dev. 15 (2001) 29222933. [53] G. Szalai, R. Krishnamurthy, G. Hajnczky, Apoptosis driven by o IP(3)-linked mitochondrial calcium signals, EMBO J. 18 (1999) 63496361. [54] P. Ghafourifar, U. Schenk, S.D. Klein, C. Richter, Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation, J. Biol. Chem. 274 (1999) 3118531188. [55] D.G. Nicholls, S.L. Budd, Mitochondria and neuronal survival, Physiol. Rev. 80 (2000) 315360 (Review). [56] A.J. Kowaltowski, R.F. Castilho, A.E. Vercesi, Mitochondrial permeability transition and oxidative stress, FEBS Lett. 495 (2001) 1215. [57] M. Szibor, C. Richter, P. Ghafourifar, Redox control of mitochondrial functions, Antioxid. Redox Signal. 3 (2001) 515 523. [58] A.A. Starkov, B.M. Polster, G. Fiskum, Regulation of hydrogen peroxide production by brain mitochondria by calcium and Bax, J. Neurochem. 83 (2002) 220228. [59] M. Madesh, B. Antonsson, Srinivasula, E. Alnemri, G. Hajnczky, Rapid kinetics of tBid-induced cytochrome c and o Smac/DIABLO release and mitochondrial depolarization, J. Biol. Chem. 277 (2002) 56515659. [60] D.B. Zorov, C.R. Filburn, L.O. Klotz, J.L. Zweier, S.J. Sollott, Reactive oxygen species (ROS)-induced ROS release: a new phenomenon accompanying induction of the mitochondrial permeability transition in cardiac myocytes, J. Exp. Med. 192 (2000) 10011014. [61] J. Jacobson, M.R. Duchen, Mitochondrial oxidative stress and cell death in astrocytes-requirement for stored Ca2 and sustained

454

G. Hajnczky et al. / Biochemical and Biophysical Research Communications 304 (2003) 445454 o opening of the permeability transition pore, J. Cell Sci. 115 (2002) 11751188. M. Garcia Fernandez, L. Troiano, L. Moretti, M. Nasi, M. Pinti, S. Salvioli, J. Dobrucki, A. Cossarizza, Early changes in intramitochondrial cardiolipin distribution during apoptosis, Cell Growth Dier. 13 (2002) 449455. J. Jacobson, M.R. Duchen, S.J. Heales, Intracellular distribution of the uorescent dye nonyl acridine orange responds to the mitochondrial membrane potential: implications for assays of cardiolipin and mitochondrial mass, J. Neurochem. 82 (2002) 224233. M. Lutter, G.A. Perkins, X. Wang, The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites, BMC Cell Biol. 2 (2001) 22. M.T. Grijalba, A.E. Vercesi, S. Schreier, Ca2 -induced increased lipid packing and domain formation in submitochondrial particles. A possible early step in the mechanism of Ca2 -stimulated generation of reactive oxygen species by the respiratory chain, Biochemistry 38 (1999) 1327913287. G. Petrosillo, F.M. Ruggiero, M. Pistolese, G. Paradies, Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef-heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis, FEBS Lett. 509 (2001) 435438. A. Asumendi, M.C. Morales, A. Alvarez, J. Arechaga, G. PerezYarza, Implication of mitochondria-derived ROS and cardiolipin peroxidation in N-(4-hydroxyphenyl)retinamide-induced apoptosis, Br. J. Cancer 86 (2002) 19511956. R.F. Epand, J.C. Martinou, S. Montessuit, R.M. Epand, Membrane perturbations induced by the apoptotic Bax protein, Biochem. J. 367 (2002) 849855. V. Gogvadze, J.D. Robertson, B. Zhivotovsky, S. Orrenius, Cytochrome c release occurs via Ca2 -dependent and Ca2 independent mechanisms that are regulated by Bax, J. Biol. Chem. 276 (2001) 1906619071. A.P. Halestrap, G.P. McStay, S.J. Clarke, The permeability transition pore complex: another view, Biochimie 84 (2002) 153 166. L. He, J.J. Lemasters, Regulated and unregulated mitochondrial permeability transition pores: a new paradigm of pore structure and function?, FEBS Lett. 512 (2002) 17. N. Zamzami, G. Kroemer, The mitochondrion in apoptosis: how Pandoras box opens, Nat. Rev. Mol. Cell Biol. 2 (2001) 6771. S. Desagher, J.C. Martinou, Mitochondria as the central control point of apoptosis, Trends Cell Biol. 10 (2000) 369377. H.L. Vieira, D. Haouzi, C. El Hamel, E. Jacotot, A.S. Belzacq, C. Brenner, G. Kroemer, Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator, Cell Death Dier. 7 (2000) 11461154 (Review). A.S. Belzacq, H.L. Vieira, G. Kroemer, C. Brenner, The adenine nucleotide translocator in apoptosis, Biochimie 84 (2002) 167176. P. Bernardi, V. Petronilli, F. Di Lisa, M. Forte, A mitochondrial perspective on cell death, Trends Biochem. Sci. 26 (2001) 112117. M. Castedo, J.L. Perfettini, G. Kroemer, Mitochondrial apoptosis and the peripheral benzodiazepine receptor: a novel target for viral and pharmacological manipulation, J. Exp. Med. 196 (2002) 11211125. Y. Tsujimoto, S. Shimizu, The voltage-dependent anion channel: an essential player in apoptosis, Biochimie 84 (2002) 187193. U. Schlattner, M. Dolder, T. Wallimann, M. Tokarska-Schlattner, Mitochondrial creatine kinase and mitochondrial outer membrane porin show a direct interaction that is modulated by calcium, J. Biol. Chem. 276 (2001) 4802748030. P. Bernardi, Mitochondrial transport of cations: channels, exchangers, and permeability transition, Physiol. Rev. 79 (1999) 11271155. M. Crompton, The mitochondrial permeability transition pore and its role in cell death, Biochem. J. 341 (1999) 233249. L. Scorrano, D. Penzo, V. Petronilli, F. Pagano, P. Bernardi, Arachidonic acid causes cell death through the mitochondrial permeability transition. Implications for tumor necrosis factor-a aopototic signaling, J. Biol. Chem. 276 (2001) 1203512040. R.C. Murphy, E. Schneider, K.W. Kinnally, Overexpression of Bcl-2 suppresses the calcium activation of a mitochondrial megachannel, FEBS Lett. 497 (2001) 7376. J.V. Gerasimenko, O.V. Gerasimenko, A. Palejwala, A.V. Tepikin, O.H. Petersen, A.J. Watson, Menadione-induced apoptosis: roles of cytosolic Ca(2+) elevations and the mitochondrial permeability transition pore, J. Cell Sci. 115 (2002) 485497. F. De Giorgi, L. Lartigue, F. Ichas, Electrical coupling and plasticity of the mitochondrial network, Cell Calcium 28 (2000) 365370. G. Hajn czky, P. Pacher, X. Lin, Spatio-temporal organization of o the mitochondrial phase of apoptosis, IUBMB Life 52 (2001) 237 245. P. Pacher, G. Hajnczky, Propagation of the apoptotic signal by o mitochondrial waves, EMBO J. 20 (2001) 41074121. R. Bajgar, S. Seetharaman, A.J. Kowaltowski, K.D. Garlid, P. Paucek, Identication and properties of a novel intracellular (mitochondrial) ATP-sensitive potassium channel in brain, J. Biol. Chem. 276 (2001) 3336933374. W. Xu, Y. Liu, S. Wang, T. McDonald, J.E. Van Eyk, A. Sidor, B. ORourke, Cytoprotective role of Ca2 - activated K channels in the cardiac inner mitochondrial membrane, Science 298 (2002) 10291033. D. Siemen, C. Loupatatzis, J. Borecky, E. Gulbins, F. Lang, Ca2 activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line, Biochem. Biophys. Res. Commun. 257 (1999) 549554. E.L. Holmuhamedov, L. Wang, A. Terzic, ATP-sensitive K channel openers prevent Ca2 overload in rat cardiac mitochondria, J. Physiol. 519 (1999) 347360. Y. Liu, T. Sato, J. Seharaseyon, A. Szewczyk, B. ORourke, E. Marban, Mitochondrial ATP-dependent potassium channels. Viable candidate eectors of ischemic preconditioning, Ann. N. Y. Acad. Sci. 874 (1999) 2737 (Review). A.J. Kowaltowski, S. Seetharaman, P. Paucek, K.D. Garlid, Bioenergetic consequences of opening the ATP-sensitive K(+) channel of heart mitochondria, Am. J. Physiol. Heart Circ. Physiol. 280 (2001) H649H657.

[62]

[78] [79]

[63]

[80]

[64]

[81] [82]

[65]

[83]

[66]

[84]

[67]

[85]

[68]

[86]

[69]

[87] [88]

[70]

[89]

[71]

[72] [73] [74]

[90]

[91]

[92]

[75] [76] [77]

[93]