Nitric oxide donors protect murine myocardium against infarction via modulation of mitochondrial permeability transition

Mitochondrial permeability transition (MPT) pores have recently been implicated as a potential mediator of myocardial ischemic injury. Nitric oxide (NO) donors induce a powerful late phase of cardioprotection against ischemia-reperfusion injury; however, the cellular mechanisms involved are poorly understood. The role of MPT pores as a target of cardioprotective signaling pathways activated by NO has never been explored in detail. Thus mice were administered the NO donor diethylenetriamine (DETA)/NO (4 doses of 0.1 mg/kg i.v. each) 24 h before 30 min of coronary artery occlusion followed by 24 h of reperfusion. Infarct size was significantly reduced in DETA/NO-treated mice (30 +/- 2% of risk region in treated mice vs. 50 +/- 2% in control mice; P < 0.05), which demonstrates powerful cardioprotection. To examine the role of MPT pores, mice were administered atractyloside (Atr; 25 mg/kg i.v.), which induces adenine nucleotide translocase-dependent MPT, 20 min before ischemia. Atr blocked the infarct-sparing effects of DETA/NO (infarct size, 58 +/- 1 vs. 30 +/- 2% of risk region in DETA/NO; P < 0.05), whereas Atr alone had no effect. Mitochondria isolated from DETA/NO-treated mice exhibited increased resistance to Ca(2+)-induced swelling by 20 micromol/l CaCl(2) or by the higher concentration of 200 micromol/l, which suggests that cardioprotection involves decreased propensity for MPT. Preincubation of mitochondria from control hearts with 30 nmol/l of the pore inhibitor cyclosporin A prevented swelling by 200 micromol/l CaCl(2), thereby confirming that Ca(2+) induces mitochondrial swelling via MPT. In accordance with the effects on infarct size, administration of Atr to the mice significantly abrogated DETA/NO-induced protection against Ca(2+)-induced mitochondrial swelling. These phenotypic alterations were associated with an increase in the antiapoptotic protein Bcl-2, which suggests that the underlying mechanisms may involve inhibition of cell death by Bcl-2. These data suggest that a critical process during NO donor-induced cardioprotection is to prevent MPT pore opening potentially via targeting of the adenine nucleotide translocator.     

Screening assays for the mitochondrial permeability transition using a fluorescence multiwell plate reader

Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 microM), a potential-indicating fluorophore, and Fluo-5N (1 microM), a low-affinity Ca(2+) indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl(2) (100 microM) addition led to ruthenium red-sensitive mitochondrial Ca(2+) uptake, as indicated by green Fluo-5N fluorescence. After Ca(2+) accumulation, mitochondria depolarized, released Ca(2+) into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca(2+) release were prevented by cyclosporin A (1 microM), confirming that these events represented the MPT. Measurements of Ca(2+), mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca(2+) uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca(2+) uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.     

外周苯二氮(艹卓)受体激动剂Ro5-4864抑制大鼠心肌细胞线粒体通透性转换

线粒体通透性转换（mitochondrial permeability transition,MPT）导致线粒体氧化应激性损伤。近年研究认为,位于线粒体外膜的外周苯二氮节受体（peripheral benzodiazepine receptor,PBR）参与了线粒体的重要生理功能。本研究在心肌细胞线粒体水平探讨激动PBR能否抑制Ca^2＋诱发的MPT。分离Sprague—Dawley大鼠心肌细胞线粒体,将PBR激动剂Ro5-4864（50、100、200μmol／L）和线粒体孵育,利用150μmol／L Ca^2＋诱发MPT,部分线粒体在与100μmol／L Ro5-4864孵育前5min加入MPT孔道开放剂苍术苷（atractyloside,ATR）。采用分光光度法观察线粒体膨胀情况：Westernblot检测线粒体细胞色素C（cytochrome C,CytoC）释放;利用荧光探针JC-1在激光共聚集显微镜下观察线粒体膜电位的变化。50、100、200μmol／L Ro5-4864均显著抑制Ca^2＋诱发的520nm处线粒体吸光度的下降,而且抑制Ca^2＋引起的线粒体CytoC释放和线粒体膜电位下降,但ATR可阻断R05—4864的上述作用。结果提示,PBR激动剂可抑制大鼠心肌MPT,保持线粒体CytoC含量和稳定线粒体膜电位,减轻线粒体损伤。PBR的激活可能成为减轻心肌细胞应激性损伤及心肌保护的新方法。     

Mitochondrial permeability transition: a common pathway to necrosis and apoptosis

Opening of high conductance permeability transition pores in mitochondria initiates onset of the mitochondrial permeability transition (MPT). The MPT is a causative event, leading to necrosis and apoptosis in hepatocytes after oxidative stress, Ca(2+) toxicity, and ischemia/reperfusion. CsA blocks opening of permeability transition pores and protects cell death after these stresses. In contrast to necrotic cell death which is a consequence of ATP depletion, ATP is required for the development of apoptosis. Reperfusion and the return of normal pH after ischemia initiate the MPT, but the balance between ATP depletion after the MPT and ATP generation by glycolysis determines whether the fate of cells will be apoptotic or necrotic death. Thus, the MPT is a common pathway leading to both necrotic and apoptotic cell death after ischemia/reperfusion.     

Measurements of mitochondrial pH in cultured cortical neurons clarify contribution of mitochondrial pore to the mechanism of glutamate-induced delayed Ca2+ deregulation

To clarify the role of the mitochondrial permeability transition pore (MPT) in the mechanism of the glutamate-induced delayed calcium deregulation (DCD) and mitochondrial depolarization (MD), we studied changes in cytosolic (pH(c)) and mitochondrial pH (pH(m)) induced by glutamate in cultured cortical neurons expressing pH-sensitive fluorescent proteins. We found that DCD and MD were associated with a prominent pH(m) decrease which presumably resulted from MPT opening. This pH(m) decrease occurred with some delay after the onset of DCD and MD. This argued against the hypothesis that MPT opening plays a dominant role in triggering of DCD. This conclusion was also supported by experiments in which Ca(2+) was replaced with antagonist of MPT opening Sr(2+). We found that in Sr(2+)-containing medium glutamate-induced delayed strontium deregulation (DSD), similar to DCD, which was accompanied by a profound MD. Analysis of the changes in pH(c) and pH(m) associated with DSD led us to conclude that MD in Sr(2+)-containing medium occurred without involvement of the pore. In contrast, in Ca(2+)-containing medium such "non-pore mechanism" was responsible only for MD initiation while in the final stages of MD development the MPT played a major role.     

Mitochondrial swelling and cytochrome c release: sensitivity to cyclosporin A and calcium

The opening of mitochondrial membrane permeability transition (MPT) pores, which results in a cyclosporin A (CsA)-sensitive and Ca(2+)-dependent dissipation of the membrane potential (delta psi) and swelling (classical MPT), has been postulated to play an important role in the release of cytochrome c (Cyt.c) and also in apoptotic cell death. Recently, it has been reported that CsA-insensitive or Ca(2+)-independent MPT can be classified as non-classic MPT. Therefore, we studied the effects of apoptosis-inducing agents on mitochondrial functions with respect to their CsA-sensitivity and Ca(2+)-dependency. CsA-sensitive mitochondrial swelling, depolarization, and the release of Ca2+ and Cyt.c were induced by low concentrations of arachidonic acid, triiodothyronine (T3), or 6-hydroxdopamine but not by valinomycin and high concentrations of the fatty acid or T3. Fe2+/ADP and 2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) induced swelling of mitochondria and the release of Ca2+ and Cyt.c were not coupled with depolarization or CsA-sensitivity while dibucaine-induced swelling occurred without depolarization, Cyt.c-release or by a CsA-sensitive mechanism. A protonophoric FCCP and SF-6847 induced depolarization and Ca(2+)-release occurred in a CsA-insensitive manner and failed to stimulate the release of Cyt.c. These results indicate that ambient conditions of mitochondria can greatly influence the state of membrane stability and that Cyt.c release may occur not only via a CsA-sensitive MPT but also by way of a CsA-insensitive membrane deterioration.     

Mitochondrial membrane permeability transition and cell death

Mitochondria are important organelles for energy production, Ca2+ homeostasis, and cell death. In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received much attention. In apoptotic and necrotic death, an increase of mitochondrial membrane permeability is considered to be one of the key events, although the detailed mechanism remains to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca2+-dependent increase in the permeability of the mitochondrial membrane that leads to loss of Deltapsi, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel, which is termed the permeability transition pore (PTP) and putatively consists of the voltage-dependent anion channel (VDAC), the adenine nucleotide translocator (ANT), cyclophilin D (Cyp D: a mitochondrial peptidyl prolyl-cis, trans-isomerase), and other molecule(s). Our studies of mice lacking Cyp D have revealed that it is essential for occurrence of the MPT and that the Cyp D-dependent MPT regulates some forms of necrotic cell death, but not apoptotic death. We have also shown that two anti-apoptotic proteins, Bcl-2 and Bcl-x(L), block the MPT by directly inhibition of VDAC activity. Here we summarize a role of the MPT in cell death.     

Limited mitochondrial permeabilization is an early manifestation of palmitate-induced lipotoxicity in pancreatic beta-cells

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.     

Voltage-dependent anion channels are dispensable for mitochondrial-dependent cell death

Mitochondria are critically involved in necrotic cell death induced by Ca(2+) overload, hypoxia and oxidative damage. The mitochondrial permeability transition (MPT) pore - a protein complex that spans both the outer and inner mitochondrial membranes - is considered the mediator of this event and has been hypothesized to minimally consist of the voltage-dependent anion channel (Vdac) in the outer membrane, the adenine-nucleotide translocase (Ant) in the inner membrane and cyclophilin-D in the matrix. Here, we report the effects of deletion of the three mammalian Vdac genes on mitochondrial-dependent cell death. Mitochondria from Vdac1-, Vdac3-, and Vdac1-Vdac3-null mice exhibited a Ca(2+)- and oxidative stress-induced MPT that was indistinguishable from wild-type mitochondria. Similarly, Ca(2+)- and oxidative-stress-induced MPT and cell death was unaltered, or even exacerbated, in fibroblasts lacking Vdac1, Vdac2, Vdac3, Vdac1-Vdac3 and Vdac1-Vdac2-Vdac3. Wild-type and Vdac-deficient mitochondria and cells also exhibited equivalent cytochrome c release, caspase cleavage and cell death in response to the pro-death Bcl-2 family members Bax and Bid. These results indicate that Vdacs are dispensable for both MPT and Bcl-2 family member-driven cell death.     

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.     

Mitochondrial permeability transition in rat hepatocytes after anoxia/reoxygenation: role of Ca2+-dependent mitochondrial formation of reactive oxygen species

Onset of the mitochondrial permeability transition (MPT) is the penultimate event leading to lethal cellular ischemia-reperfusion injury, but the mechanisms precipitating the MPT after reperfusion remain unclear. Here, we investigated the role of mitochondrial free Ca(2+) and reactive oxygen species (ROS) in pH- and MPT-dependent reperfusion injury to hepatocytes. Cultured rat hepatocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 4 h and then reoxygenated at pH 7.4 to simulate ischemia-reperfusion. Some cells were loaded with the Ca(2+) chelators, BAPTA/AM and 2-[(2-bis-[carboxymethyl]aono-5-methoxyphenyl)-methyl-6-methoxy-8-bis[carboxymethyl]aminoquinoline, either by a cold loading protocol for intramitochondrial loading or by warm incubation for cytosolic loading. Cell death was assessed by propidium iodide fluorometry and immunoblotting. Mitochondrial Ca(2+), inner membrane permeability, membrane potential, and ROS formation were monitored with Rhod-2, calcein, tetramethylrhodamine methylester, and dihydrodichlorofluorescein, respectively. Necrotic cell death increased after reoxygenation. Necrosis was blocked by 1 μM cyclosporin A, an MPT inhibitor, and by reoxygenation at pH 6.2. Confocal imaging of Rhod-2, calcein, and dichlorofluorescein revealed that an increase of mitochondrial Ca(2+) and ROS preceded onset of the MPT after reoxygenation. Intramitochondrial Ca(2+) chelation, but not cytosolic Ca(2+) chelation, prevented ROS formation and subsequent necrotic and apoptotic cell death. Reoxygenation with the antioxidants, desferal or diphenylphenylenediamine, also suppressed MPT-mediated cell death. However, inhibition of cytosolic ROS by apocynin or diphenyleneiodonium chloride failed to prevent reoxygenation-induced cell death. In conclusion, Ca(2+)-dependent mitochondrial ROS formation is the molecular signal culminating in onset of the MPT after reoxygenation of anoxic hepatocytes, leading to cell death.     

Prooxidants open both the mitochondrial permeability transition pore and a low-conductance channel in the inner mitochondrial membrane

Mitochondria can be induced by a variety of agents/conditions to undergo a permeability transition (MPT), which nonselectively increases the permeability of the inner membrane (i.m.) to small (<1500 Da) solutes. Prooxidants are generally considered to trigger the MPT, but some investigators suggest instead that prooxidants open a Ca(2+)-selective channel in the inner mitochondrial membrane and that the opening of this channel, when coupled with Ca(2+) cycling mediated by the Ca(2+) uniporter, leads ultimately to the observed increase in mitochondrial permeability [see, e.g., Schlegel et al. (1992) Biochem. J. 285, 65]. S. A. Novgorodov and T. I. Gudz [J. Bioenerg. Biomembr. (1996) 28, 139] propose that the i.m. contains a pore that, upon exposure to prooxidants, can open to two states, one of which conducts only H(+) and one of which is the classic MPT pore. Given the current interest in increased mitochondrial permeability as a factor in apoptotic cell death, it is important to determine whether i.m. permeability is regulated in one or multiple ways and, in the latter event, to characterize each regulatory mechanism in detail. This study examined the effects of the prooxidants diamide and t-butylhydroperoxide (t-BuOOH) on the permeability of isolated rat liver mitochondria. Under the experimental conditions used, t-BuOOH induced mitochondrial swelling only in the presence of exogenous Ca(2+) (>2 microM), whereas diamide was effective in its absence. In the absence of exogenous inorganic phosphate (P(i)), (1) both prooxidants caused a collapse of the membrane potential (DeltaPsi) that preceded the onset of mitochondrial swelling; (2) cyclosporin A eliminated the swelling induced by diamide and dramatically slowed that elicited by t-BuOOH, without altering prooxidant-induced depolarization; (3) collapse of DeltaPsi was associated with Ca(2+) efflux but not with efflux of glutathione; (4) neither Ca(2+) efflux nor DeltaPsi collapse was sensitive to ruthenium red; (5) collapse of DeltaPsi was accompanied by an increase in matrix pH; no stimulation of respiration was observed; (6) Sr(2+) was able to substitute for Ca(2+) in supporting t-BuOOH-induced i.m. depolarization, but not swelling; (7) in addition to being insensitive to CsA, the collapse of DeltaPsi was also resistant to trifluoperazine, spermine, and Mg(2+), all of which block the MPT; and (8) DeltaPsi was restored (and its collapse was inhibited) upon addition of dithiothreitol, ADP, ATP or EGTA. We suggest that these results indicate that prooxidants open two channels in the i.m.: the classic MPT and a low-conductance channel with clearly distinct properties. Opening of the low-conductance channel requires sulfhydryl group oxidation and the presence of a divalent cation; both Ca(2+) and Sr(2+) are effective. The channel permits the passage of cations, including Ca(2+), but not of protons. It is insensitive to inhibitors of the classic MPT.     

Mechanism of A23187-induced apoptosis in HL-60 cells: dependency on mitochondrial permeability transition but not on NADPH oxidase

Calcium ions (Ca(2+)) are involved in a number of physiological cellular functions including apoptosis. An elevation in intracellular levels of Ca(2+) in A23187-treated HL-60 cells was associated with the generation of both intracellular and extracellular reactive oxygen species (ROS) and induction of apoptotic cell death. A23187-induced apoptosis was prevented by cyclosporin A, a potent inhibitor of mitochondrial permeability transition (MPT). The generation of extracellular ROS was suppressed by the NADPH oxidase inhibitor diphenylene iodonium, and by superoxide dismutase, but these agents had no effect on A23187-induced apoptosis. In contrast, the blocking of intracellular ROS by a cell-permeant antioxidant diminished completely the induction of MPT and apoptosis. In isolated mitochondria, the addition of Ca(2+) induced a typical MPT concomitant with the generation of ROS, which leads to augmentation of intracellular ROS levels. These results indicate that intracellular not extracellular ROS generated by A23187 is associated with the opening of MPT pores that leads to apoptotic cell death.     

Mitochondria and Ca(2+)in cell physiology and pathophysiology

There is now a consensus that mitochondria take up and accumulate Ca(2+)during physiological [Ca(2+)](c)signalling. This contribution will consider some of the functional consequences of mitochondrial Ca(2+)uptake for cell physiology and pathophysiology. The ability to remove Ca(2+)from local cytosol enables mitochondria to regulate the [Ca(2+)] in microdomains close to IP3-sensitive Ca(2+)-release channels. The [Ca(2+)] sensitivity of these channels means that, by regulating local [Ca(2+)](c), mitochondrial Ca(2+)uptake modulates the rate and extent of propagation of [Ca(2+)](c)waves in a variety of cell types. The coincidence of mitochondrial Ca(2+)uptake with oxidative stress may open the mitochondrial permeability transition pore (mPTP). This is a catastrophic event for the cell that will initiate pathways to cell death either by necrotic or apoptotic pathways. A model is presented in which illumination of an intramitochondrial fluorophore is used to generate oxygen radical species within mitochondria. This causes mitochondrial Ca(2+)loading from SR and triggers mPTP opening. In cardiomyocytes, mPTP opening leads to ATP consumption by the mitochondrial ATPase and so results in ATP depletion, rigor and necrotic cell death. In central mammalian neurons exposed to glutamate, a cellular Ca(2+)overload coincident with NO production also causes loss of mitochondrial potential and cell death, but mPTP involvement has proven more difficult to demonstrate unequivocally.     

Effect of Ca2+ on induction of the mitochondrial pore opening in the rat myocardium

The mitochondrial role opening (MPT) induced by Ca2+ has been studied in isolated rat heart mitochondria. MPT was characterized as cyclosporine A-inhibited swelling accompanied by the loss of membrane potential (deltapsim) and Ca2+ efflux after the Ca2+ -loading which was followed spectrophotometrically after the Ca2+ -arsenaso-III complex formation. It has been shown that in suspension of isolated mitochondria MPT was activated by low (with maximum at about 20 microM Ca2+) and high concentrations of Ca2+ (the concentration curve shows a saturation at about 1.0-1.5 mM). In all the cases an access of Ca2+ ions to the matrix space of the mitochondria was necessary for MPT induction. MPT activated by low concentrations of Ca2+ was accompanied by slow decrease of deltapsim and slow release of Ca2+, enhanced by ruthenium red (RR), and was independent of the substrate used (glutamate or succinate). It had not been observed if the respiratory chain was inhibited, even if the Ca2+ access to the inner mitochondrial membrane was provided by Ca2+ -ionophore A23187. At high Ca2+ concentrations rapid Ca2+ -uptake and release via Ca2+ -uniporter (inhibited by ruthenium red) followed by extensive swelling (pore formation) have been observed. It had been supposed that rapid MPT at high concentrations of Ca2+ was the result of Ca2+ entrance to the mitochondrial matrix and depolarisation of the mitochondrial membrane. The data obtained show two different mechanisms of Ca2+ -induced MPT. The one is sensitive to the redox-state of the electron transport chain and is abolished if the respiration is inhibited. The other is independent of mitochondrial respiration and needs only Ca2+ access to the inner mitochondrial membrane and Ca2+ binding to some specific sites leading to MPT opening.     

Activation of the herpes simplex virus type-1 origin-binding protein (UL9) by heat shock proteins.

Heat shock proteins participate in the initiation of DNA replication of different organisms by facilitating the assembly of initiation complexes. We have examined the effects of human heat shock proteins (Hsp40 and Hsp70) on the interaction of the herpes simplex virus type-1 initiator protein (UL9) with oriS, one of the viral origins of replication. Hsp40 and Hsp70 act substoichiometrically to increase the affinity of UL9 for oriS. The major contributor to this effect is Hsp40. Heat shock proteins also stimulate the ATPase activity of UL9 with oriS and increase opening of the origin. In contrast, heat shock proteins have no effect on the origin-independent activities of UL9 suggesting that their role is not merely in refolding denatured protein. These observations are consistent with a role for heat shock proteins in activating UL9 to efficiently initiate viral origin-dependent DNA replication. The action of heat shock proteins in this capacity is analogous to their role in activating the initiator proteins of other organisms.     

The characterization of mitochondrial permeability transition in clonal pancreatic beta-cells. Multiple modes and regulation

Mitochondrial permeability transition (MPT), which contributes substantially to the regulation of normal mitochondrial metabolism, also plays a crucial role in the initiation of cell death. It is known that MPT is regulated in a tissue-specific manner. The importance of MPT in the pancreatic beta-cell is heightened by the fact that mitochondrial bioenergetics serve as the main glucose-sensing regulator and energy source for insulin secretion. In the present study, using MIN6 and INS-1 beta-cells, we revealed that both Ca(2+)-phosphate- and oxidant-induced MPT is remarkably different from other tissues. Ca(2+)-phosphate-induced transition is accompanied by a decline in mitochondrial reactive oxygen species production related to a significant potential dependence of reactive oxygen species formation in beta-cell mitochondria. Hydroperoxides, which are indirect MPT co-inducers active in liver and heart mitochondria, are inefficient in beta-cell mitochondria, due to the low mitochondrial ability to metabolize them. Direct cross-linking of mitochondrial thiols in pancreatic beta-cells induces the opening of a low conductance ion permeability of the mitochondrial membrane instead of the full scale MPT opening typical for liver mitochondria. Low conductance MPT is independent of both endogenous and exogenous Ca(2+), suggesting a novel type of nonclassical MPT in beta-cells. It results in the conversion of electrical transmembrane potential into DeltapH instead of a decrease in total protonmotive force, thus mitochondrial respiration remains in a controlled state. Both Ca(2+)- and oxidant-induced MPTs are phosphate-dependent and, through the "phosphate flush" (associated with stimulation of insulin secretion), are expected to participate in the regulation in beta-cell glucose-sensing and secretory activity.     

Hypercholesterolemia increases mitochondrial oxidative stress and enhances the MPT response in the porcine myocardium: beneficial effects of chronic exercise

Hypercholesterolemia has been suggested to have direct negative effects on myocardial function due to increased reactive oxygen species (ROS) generation and increased myocyte death. Mitochondrial permeability transition (MPT) is a significant mediator of cell death, which is enhanced by ROS generation and attenuated by exercise training. The purpose of this study was to investigate the effect of hypercholesterolemia on the MPT response of cardiac mitochondria. We tested the hypothesis that familial hypercholesterolemic (FH) pigs would have an enhanced MPT response and that exercise training could reverse this phenotype. MPT was assessed by mitochondrial swelling in response to 10-100 μM Ca(2+). FH pigs did show an increased MPT response to Ca(2+) that was associated with decreases in the expression of the putative MPT pore components mitochondrial phosphate carrier (PiC) and cyclophilin-D (CypD). FH also caused increased oxidative stress, depicted by increased protein nitrotyrosylation, as well as decreased levels of reduced GSH in cardiac mitochondria. Expression of the mitochondrial antioxidant enzymes manganese superoxide dismutase (MnSOD), thioredoxin-2 (Trx2), and peroxiredoxin-3 (Prx3) was greatly reduced in the FH pigs. In contrast, cytosolic catalase expression and activity were increased. However, chronic exercise training was able to normalize the MPT response in FH pigs, reduce mitochondrial oxidative stress, and return MnSOD, Trx2, Prx3, and catalase expression/activities to normal. We conclude that FH reduces mitochondrial antioxidants, increases mitochondrial oxidative stress, and enhances the MPT response in the porcine myocardium, and that exercise training can reverse these detrimental alterations.     

Apoptosis driven by IP(3)-linked mitochondrial calcium signals

Increases of mitochondrial matrix [Ca(2+)] ([Ca(2+)](m)) evoked by calcium mobilizing agonists play a fundamental role in the physiological control of cellular energy metabolism. Here, we report that apoptotic stimuli induce a switch in mitochondrial calcium signalling at the beginning of the apoptotic process by facilitating Ca(2+)-induced opening of the mitochondrial permeability transition pore (PTP). Thus [Ca(2+)](m) signals evoked by addition of large Ca(2+) pulses or, unexpectedly, by IP(3)-mediated cytosolic [Ca(2+)] spikes trigger mitochondrial permeability transition and, in turn, cytochrome c release. IP(3)-induced opening of PTP is dependent on a privileged Ca(2+) signal transmission from IP(3) receptors to mitochondria. After the decay of Ca(2+) spikes, resealing of PTP occurs allowing mitochondrial metabolism to recover, whereas activation of caspases is triggered by cytochrome c released to the cytosol. This organization provides an efficient mechanism to establish caspase activation while mitochondrial metabolism is maintained to meet ATP requirements of apoptotic cell death.     

Alteration in mitochondrial thiol enhances calcium ion dependent membrane permeability transition and dysfunction in vitro: a cross-talk between mtThiol, Ca2+, and ROS

Mitochondrial permeability transition (MPT) and dysfunctions play a pivotal role in many patho-physiological and toxicological conditions. The interplay of mitochondrial thiol (mtThiol), MPT, Ca(2+) homeostasis, and resulting dysfunctions still remains controversial despite studies by several research groups. Present study was undertaken to ascertain the correlation between Ca(2+) homeostasis, mtThiol alteration and reactive oxygen species (ROS) in causing MPT leading to mitochondrial dysfunction. mtThiol depletion significantly enhanced Ca(2+) dependent MPT (swelling) and depolarization of mitochondria resulting in release of pro-apoptotic proteins like Cyt c, AIF, and EndoG. mtThiol alteration and Ca(2+) overload caused reduced mitochondrial electron flow, oxidation of pyridine nucleotides (NAD(P)H) and significantly enhanced ROS generation (DHE and DCFH-DA fluorescence). Studies with MPT inhibitor (Cyclosporin A), Ca(2+) uniport blocker (ruthenium red) and Ca(2+) chelator (BAPTA) indicated that mitochondrial dysfunction was more pronounced under dual stress of altered mtThiol and Ca(2+) overload in comparison with single stress of excessive Ca(2+). Transmission electron microscopy confirmed the changes in mitochondrial integrity under stress. Our findings suggest that the Ca(2+) overload itself is not solely responsible for structural and functional impairment of mitochondria. A multi-factorial cross-talk between mtThiol, Ca(2+) and ROS is responsible for mitochondrial dysfunction. Furthermore, minor depletion of mtThiol was found to be an important factor along with Ca(2+) overload in triggering MPT in isolated mitochondria, tilting the balance towards disturbed functionality.