Nitric oxide donors, nitrosothiols and mitochondrial respiration inhibitors induce caspase activation by different mechanisms

We investigated to what extent different types of NO donors induce caspase activation by opening of the mitochondrial permeability transition pore (PTP) or inhibition of mitochondrial respiration. We found that nitrosothiols can directly open the PTP in isolated mitochondria and cause cytochrome c release, whereas NONOate donors can not. In macrophages nitrosothiols cause caspase activation that is blocked by cyclosporin A or calcium chelation, both of which prevent PTP opening, whereas caspase activation caused by NONOates is much less sensitive to these agents. Inhibitors of mitochondrial respiration did not promote PTP opening in isolated mitochondria, and although they cause caspase activation in macrophages, this activation was slower than that caused by NO donors, and was relatively insensitive to cyclosporin and calcium chelators suggesting that PTP opening was not involved.     

Antioxidant MCI-186 inhibits mitochondrial permeability transition pore and upregulates Bcl-2 expression

Reperfusion after a period of ischemia is associated with the formation of reactive oxygen species (ROS) and Ca2+ overload resulting in the opening of a nonspecific pore in the inner membrane of the mitochondria, called the mitochondrial permeability transition pore (PTP), leading to cell damage. Although endogenous antioxidants are activated because of oxidative stress following ischemia, their levels are not high enough to prevent reperfusion injury. Hence there is always a need for exogenous supplement of antioxidants, especially after acute ischemia. Here we demonstrated the effects of the antioxidant 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186) in preventing reperfusion injury of the heart by inhibition of PTP opening. Ischemia (30 min) by left coronary artery (LCA) occlusion and reperfusion (120 min) in Wistar rats after pretreatment with MCI-186 (10 mg/kg iv) infusion starting from 30 min before LCA occlusion resulted in 1) less area of myocardial infarction (19.2% vs. 61.6%), 2) well-maintained myocardial ATP content (P < 0.03 vs. control), 3) decreased mitochondrial swelling and reduced cytochrome c release, 4) increased expression of BCl-2, 5) lower prevalence of apoptotic cells (14.3% vs. 2.9%), and 6) reduced DNA fragmentation in the MCI-186-treated group. These cytoprotective effects of MCI-186 were inhibited on opening PTP before MCI-186 treatment with the PTP activators lonidamine (10 mg/kg iv) or atractyloside (5 mg/kg iv) but failed to inhibit the protective effects exerted by another antioxidant, allopurinol, suggesting that the PTP inhibiting property is specific for MCI-186. These results demonstrate that the radical scavenger MCI-186, by inhibiting the opening of the PTP, prevents necrosis and cytochrome c release and hence pathological apoptosis.     

Zn~(2+)对PTP开放和细胞色素c释放的浓度依赖性双向调节

研究Zn2+对Ca2+介导线粒体通透过渡孔道(PTP)开放和线粒体细胞色素c释放的影响,及其与线粒体膜电位(ΔΨm)和Ca2+介导的线粒体Ca2+释放(mCICR)之间的关系.提取大鼠肝线粒体,通过紫外分光光度仪检测不同浓度Zn2+作用下Ca2+介导的PTP开放状态;采用荧光分光光度仪测定不同浓度Zn2+作用下线粒体膜电位的变化;采用双波长双光束紫外分光光度仪检测不同浓度Zn2+作用下测试体系内Ca2+浓度的变化,以反映线粒体Ca2+的转运情况(即mCICR);通过免疫印迹法检测不同浓度Zn2+作用下Ca2+介导的线粒体细胞色素c的释放.高浓度Zn2+完全抑制Ca2+介导的PTP开放和细胞色素c释放.一定浓度的Zn2+部分抑制Ca2+介导的PTP开放和细胞色素c释放.适当浓度Zn2+自身介导PTP开放和细胞色素c释放.低浓度Zn2+加速Ca2+介导PTP开放和Ca2+释放;高浓度和一定浓度Zn2+分别完全或部分破坏ΔΨm;高浓度Zn2+完全抑制mCICR.当抑制mCICR时,Ca2+和Zn2+对PTP开放和细胞色素c释放的作用完全抑制.结果表明,Zn2+以浓度依赖方式双向调节PTP开放和细胞色素c释放.Zn2+的作用可能与Zn2+破坏ΔΨm和影响mCICR相关.     

Nitric oxide and peroxynitrite interactions with mitochondria

Nitric oxide (*NO) and peroxynitrite (ONOO-) avidly interact with mitochondrial components, leading to a range of biological responses spanning from the modulation of mitochondrial respiration, mitochondrial dysfunction to the signaling of apoptotic cell death. Physiological levels of *NO primarily interact with cytochrome c oxidase, leading to a competitive and reversible inhibition of mitochondrial oxygen uptake. In turn, this leads to alterations in electrochemical gradients, which affect calcium uptake and may regulate processes such as mitochondrial transition pore (MTP) opening and the release of pro-apoptotic proteins. Large or persistent levels of *NO in mitochondria promote mitochondrial oxidant formation. Peroxynitrite formed either extra- or intra-mitochondrially leads to oxidative damage, most notably at complexes I and II of the electron transport chain, ATPase, aconitase and Mn-superoxide dismutase. Mitochondrial scavenging systems for peroxynitrite and peroxynitrite-derived radicals such as carbonate (CO3*-) and nitrogen dioxide radicals (*NO2) include cytochrome c oxidase, glutathione and ubiquinol and serve to partially attenuate the reactions of these oxidants with critical mitochondrial targets. Detection of nitrated mitochondrial proteins in vivo supports the concept that mitochondria constitute central loci of the toxic effects of excess reactive nitrogen species. In this review we will provide an overview of the biochemical mechanisms by which *NO and ONOO- regulate or alter mitochondrial functions.     

Genistein induces apoptosis of RPE-J cells by opening mitochondrial PTP

Although previous studies demonstrated that genistein-induced apoptosis of various cell types including RPE-J cells, the involvement of mitochondrial events in such types of apoptosis has not been demonstrated to date. In this investigation of genistein-induced apoptosis of RPE-J cells, genistein induced the reduction of the mitochondrial membrane potential and the release of cytochrome c to cytosol. A mitochondrial permeability transition pore (PTP) blocker bongkrekic acid prevented the reduction of the mitochondrial membrane potential and cytochrome c release, and consequently abolished caspase-3 activation, nuclear condensation, and DNA fragmentation. On the other hand, zVAD-fmk did not inhibit the mitochondrial event such as the reduction of the mitochondrial membrane potential and cytochrome c release although it prevented caspase-3 activation, nuclear condensation, and DNA fragmentation. Taken together, genistein induces apoptosis of RPE-J cells by opening the mitochondrial PTP, and the mitochondrial event in this type of apoptosis is caused independently of caspase.     

The peripheral-type benzodiazepine receptor is involved in control of Ca2+-induced permeability transition pore opening in rat brain mitochondria

The peripheral-type benzodiazepine receptor (PBR) is an 18 kDa mitochondrial membrane protein with still elusive function in cell death. Here, we studied whether PBR is involved in Ca2+-induced permeability transition pore (PTP) opening in isolated rat brain mitochondria (RBM). PTP opening is important in mitochondrial events leading to programmed cell death. Immunoblots revealed a single 18 kDa anti-PBR antibody-immunoreactive band in purified RBM. Adenine nucleotide transporter, a key PTP component, was found in the PBR-immunoprecipitate. In isolated intact RBM, addition of a specific anti-PBR antibody [H. Li, Z. Yao, B. Degenhardt, G. Teper, V. Papadopoulos, Cholesterol binding at the cholesterol recognition/interaction amino acid consensus (CRAC) of the peripheral-type benzodiazepine receptor and inhibition of steroidogenesis by an HIV TAT-CRAC peptide, Proc. Natl. Acad. Sci. U.S.A. 98 (2001) 1267-1272] delayed Ca2+-induced dissipation of membrane potential (psi(m)) and diminished cyclosporine A-sensitive Ca2+ efflux, which are both indicative for the suppression of PTP opening. Moreover, anti-PBR antibody caused partial retention of Ca2+ in the mitochondrial matrix in spite of psi(m) dissipation, and reduced activation of respiratory rate at Ca2+-induced PTP opening. A release of pro-apoptotic factors, AIF and cytochrome c, from RBM was shown at threshold Ca2+ load. Anti-PBR antibody blocked the release of AIF but did not affect the cytochrome c release. Addition of ATP was able to initiate PTP closing, associated with psi(m) restoration and Ca2+ re-accumulation. At the same time mitochondrial protein phosphorylation (incorporation of 32P from [gamma-32P]ATP) occurred and anti-PBR antibody was able to inhibit phosphorylation of these proteins. The endogenous PBR ligand, protoporphyrin IX, facilitated PTP opening and phosphorylation of the mitochondrial proteins, thus, inducing effects opposite to anti-PBR antibody. This study provides evidence for PBR involvement in PTP opening, controlling the Ca2+-induced Ca2+ efflux, and AIF release from mitochondria, important stages of initiation of programmed cell death.     

Catalytic consumption of nitric oxide by prostaglandin H synthase-1 regulates platelet function

Nitric oxide (( small middle dot)NO) plays a central role in vascular homeostasis via regulation of smooth muscle relaxation and platelet aggregation. Although mechanisms for ( small middle dot)NO formation are well known, removal pathways are less well characterized, particularly in cells that respond to ( small middle dot)NO through activation of soluble guanylate cyclase. Herein, we report that ( small middle dot)NO is catalytically consumed by prostaglandin H synthase-1 (PGHS-1) through acting as a reducing peroxidase substrate. With purified ovine PGHS-1, ( small middle dot)NO consumption requires peroxide (LOOH or H(2)O(2)), with a K(m)( (app)) for 15(S)hydroperoxyeicosatetraenoic acid (HPETE) of 3. 27 +/- 0.35 microm. During this, 2 mol ( small middle dot)NO are consumed per mol HPETE, and loss of HPETE hydroperoxy group occurs with retention of the conjugated diene spectrum. Hydroperoxide-stimulated ( small middle dot)NO consumption requires heme incorporation, is not inhibited by indomethacin, and is further stimulated by the reducing peroxidase substrate, phenol. PGHS-1-dependent ( small middle dot)NO consumption also occurs during arachidonate, thrombin, or activation of platelets (1-2 microm.min(-1) for typical plasma platelet concentrations) and prevents ( small middle dot)NO stimulation of platelet soluble guanylate cyclase. Platelet sensitivity to ( small middle dot)NO as an inhibitor of aggregation is greater using a platelet-activating stimulus () that does not cause ( small middle dot)NO consumption, indicating that this mechanism overcomes the anti-aggregatory effects of ( small middle dot)NO. Catalytic consumption of ( small middle dot)NO during eicosanoid synthesis thus represents both a novel proaggregatory function for PGHS-1 and a regulated mechanism for vascular ( small middle dot)NO removal.     

Nitric oxide inhibition of mitochondrial respiration and its role in cell death

Nitric oxide (NO) or its derivatives (reactive nitrogen species, RNS) inhibit mitochondrial respiration in two different ways: (i) an acute, potent, and reversible inhibition of cytochrome oxidase by NO in competition with oxygen; and, (ii) irreversible inhibition of multiple sites by RNS. NO inhibition of respiration may impinge on cell death in several ways. Inhibition of respiration can cause necrosis and inhibit apoptosis due to ATP depletion, if glycolysis is also inhibited or is insufficient to compensate. Inhibition of neuronal respiration can result in excitotoxic death of neurons due to induced release of glutamate and activation of NMDA-type glutamate receptors. Inhibition of respiration may cause apoptosis in some cells, while inhibiting apoptosis in other cells, by mechanisms that are not clear. However, NO can induce (and inhibit) cell death by a variety of mechanisms unrelated to respiratory inhibition.     

Up-regulation of Bak and Bim via JNK downstream pathway in the response to nitric oxide in human glioblastoma cells

Nitric oxide (NO) is a chemical messenger implicated in neuronal damage associated with ischemia neurodegenerative disease and excitotoxicity. In the present study, we examined the biological effects of NO and its mechanisms in human malignant glioblastoma cells. Addition of a NO donor, S-nitroso-N-acetyl-penicillamine (SNAP), induced apoptosis in U87MG human glioblastoma cells, accompanied by opening mitochondrial permeability transition pores, release of cytochrome c and AIF, and subsequently by caspase activation. NO-induced apoptosis occurred concurrently with significantly increased levels of the Bak and Bim. Treatment with SNAP resulted in sustained activation of JNK and its downstream pathway, c-Jun/AP-1. The expression of dominant-negative (DN)-JNK1 and DN-c-Jun suppressed the activation of AP-1, the induction of Bak and Bim, and the SNAP-induced apoptosis. In addition, de novo protein synthesis was required for the initiation of apoptosis in that the protein synthesis inhibitor, cycloheximide (CHX), inhibited NO-induced apoptotic cell death as well as up-regulation of Bak and Bim. These results suggest that NO activates an apoptotic cascade, involving sustained JNK activation, AP-1 DNA binding activity, and subsequent Bak and Bim induction, followed by cytochrome c and AIF releases and caspases cascade activation, resulting in human malignant brain tumor cell death.     

Biphasic regulation of leukocyte superoxide generation by nitric oxide and peroxynitrite

Activation of the NADPH oxidase-derived oxidant burst of polymorphonuclear leukocytes (PMNs) is of critical importance in inflammatory disease. PMN-derived superoxide (O(2)) can be scavenged by nitric oxide (NO( small middle dot)) with the formation of peroxynitrite (ONOO(-)); however, questions remain regarding the effects and mechanisms by which NO( small middle dot) and ONOO(-) modulate the PMN oxidative burst. Therefore, we directly measured the dose-dependent effects of NO( small middle dot) and ONOO(-) on O(2) generation from human PMNs stimulated with phorbol 12-myristate 13-acetate using EPR spin trapping. Pretreatment with low physiological (microm) concentrations of NO( small middle dot) from NO( small middle dot) gas had no effect on PMN O(2) generation, whereas high levels (> or =50 microm) exerted inhibition. With ONOO(-) pretreatment, however, a biphasic modulation of O(2) generation was seen with stimulation by microm levels, but inhibition at higher levels. With the NO( small middle dot) donor NOR-1, which provides more sustained release of NO( small middle dot) persisting at the time of O(2) generation, a similar biphasic modulation of O(2) generation was seen, and this was inhibited by ONOO(-) scavengers. The enhancement of O(2) generation by low concentrations of ONOO(-) or NOR-1 was associated with activation of the ERK MAPKs and was blocked by their inhibition. Thus, low physiological levels of NO( small middle dot) present following PMN activation are converted to ONOO(-), which enhances O(2) generation through activation of the ERK MAPK pathway, whereas higher levels of NO( small middle dot) or ONOO(-) feed back and inhibit O(2) generation. This biphasic concentration-dependent regulation of the PMN oxidant burst by NO( small middle dot)-derived ONOO(-) may be of critical importance in regulating the process of inflammation.     

Hypoxia sensitizes cells to nitric oxide-induced apoptosis

Nitric oxide (NO) can induce apoptosis in a variety of cell types. A non-toxic concentration of nitric oxide under normal oxygen conditions triggered cell death under hypoxic conditions (1.5% O(2)) in fibroblasts. Nitric oxide administered during hypoxia induced the release of cytochrome c, caspase-9 activation, and the loss of mitochondrial membrane potential followed by DNA fragmentation and lactate dehydrogenase release (markers of cell death). Bcl-X(L) protected cells from nitric oxide-induced apoptosis during hypoxia by preventing the release of cytochrome c, caspase-9 activation, and by maintaining a mitochondrial membrane potential. Murine embryonic fibroblasts from bax(-/-) bak(-/-) mice exposed to nitric oxide during hypoxia did not die, indicating that pro-apoptotic Bcl-2 family members are required for NO-induced apoptosis during hypoxia. The nitric oxide-induced cell death during hypoxia was independent of cGMP and peroxynitrite. Cells devoid of mitochondrial DNA (rho secondary-cells) lack a functional electron transport chain and were resistant to nitric oxide-induced cell death during hypoxia, suggesting that a functional electron transport chain is required for nitric oxide-induced apoptosis during hypoxia.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2 transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2 transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mClCR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mClCR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mClCR and PTP opening. mClCR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2 transport and PTP opening are largely dependent on electron transport and energy coupling.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca2+ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca2+ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca2+ transport and PTP opening are largely dependent on electron transport and energy coupling.     

Glutamate Interacts with VDAC and Modulates Opening of the Mitochondrial Permeability Transition Pore

The amino acid glutamate, synthesized in the mitochondria, serves multiple functions, including acting as a neurotransmitter and participating in degradative and synthetic pathways. We have previously shown that glutamate modulates the channel activity of bilayer-reconstituted voltage-dependent anion channel (VDAC). In this study, we demonstrate that glutamate also modulates the opening of the mitochondrial permeability transition pore (PTP), of which VDAC is an essential component. Glutamate inhibited PTP opening, as monitored by transient Ca2+ accumulation, mitochondrial swelling and accompanying release of cytochrome c. Exposure to L-glutamate delayed the onset of PTP opening up to 3-times longer, with an IC50 of 0.5 mM. Inhibition of PTP opening by L-glutamate is highly specific, not being mimicked by D-glutamate, L-glutamine, L-aspartate, or L-asparagine. The interaction of L-glutamate with VDAC and its inhibition of VDAC's channel activity and PTP opening suggest that glutamate may also act as an intracellular messenger in the mitochondria-mediated apoptotic pathway.     

The mitochondrial permeability transition, release of cytochrome c and cell death. Correlation with the duration of pore openings in situ

We investigated the relationship between opening of the permeability transition pore (PTP), mitochondrial depolarization, cytochrome c release, and occurrence of cell death in rat hepatoma MH1C1 cells. Treatment with arachidonic acid or induces PTP opening in situ with similar kinetics, as assessed by the calcein loading-Co(2+) quenching technique (Petronilli, V., Miotto, G., Canton, M., Colonna, R., Bernardi, P., and Di Lisa, F. (1999) Biophys. J. 76, 725-734). Yet depolarization, as assessed from the changes of mitochondrial tetramethylrhodamine methyl ester (TMRM) fluorescence, is rapid and extensive with arachidonic acid and slow and partial with. Cyclosporin A-inhibitable release of cytochrome c and cell death correlate with the changes of TMRM fluorescence but not with those of calcein fluorescence. Since pore opening must be accompanied by depolarization, we conclude that short PTP openings are detected only by trapped calcein and may have little impact on cell viability, while changes of TMRM distribution require longer PTP openings, which cause release of cytochrome c and may result in cell death. Modulation of the open time appears to be the key element in determining the outcome of stimuli that converge on the PTP.     

Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation.

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca(2+) dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291-296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038-11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca(2+) by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca(2+)-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca(2+) levels induce apoptosis.     

Bupivacaine myotoxicity is mediated by mitochondria.

We have investigated the effects of the myotoxic local anesthetic bupivacaine on rat skeletal muscle mitochondria and isolated myofibers from flexor digitorum brevis, extensor digitorum longus, soleus, and from the proximal, striated portion of the esophagus. In isolated mitochondria, bupivacaine caused a concentration-dependent mitochondrial depolarization and pyridine nucleotide oxidation, which were matched by an increased oxygen consumption at bupivacaine concentrations of 1.5 mm or less at pH 7.4, whereas respiration was inhibited at higher concentrations. As a consequence of depolarization, bupivacaine caused the opening of the permeability transition pore (PTP), a cyclosporin A-sensitive inner membrane channel that plays a key role in many forms of cell death. In intact flexor digitorum brevis fibers bupivacaine caused mitochondrial depolarization and pyridine nucleotides oxidation that were matched by increased concentrations of cytosolic free Ca(2+), release of cytochrome c, and eventually, hypercontracture. Both mitochondrial depolarization and cytochrome c release were inhibited by cyclosporin A, indicating that PTP opening rather than bupivacaine as such was responsible for these events. Similar responses to bupivacaine were observed in the soleus, which is highly oxidative. In contrast, fibers from the esophagus (which we show to be more fatigable than flexor digitorum brevis fibers) and from the highly glycolytic extensor digitorum longus didn't undergo pyridine nucleotide oxidation upon the addition of bupivacaine and were resistant to bupivacaine toxicity. These results suggest that active oxidative metabolism is a key determinant in bupivacaine toxicity, that bupivacaine myotoxicity is a relevant model of mitochondrial dysfunction involving the PTP and Ca(2+) dysregulation, and that it represents a promising system to test new PTP inhibitors that may prove relevant in spontaneous myopathies where mitochondria have long been suspected to play a role.     

Mitochondrial Ca2+ transport and permeability transition pore opening and mitochondrial energetic status

The relationship between mitochondrial Ca²⁺ transport and permeability transition pore (PTP) opening as well as the effects of mitochondrial energetic status on mitochondrial Ca²⁺ transport and PTP opening were studied. The results showed that the calcium-induced calcium release from mitochondria (mCICR) induced PTP opening. Inhibitors for electron transport of respiratory chain inhibited mCICR and PTP opening. Partial recovery of electron transport in respiratory chain resulted in partial recovery of mCICR and PTP opening. mCICR and PTP opening were also inhibited by CCCP which eliminated transmembrane proton gradient. The results indicated that mitochondrial Ca²⁺ transport and PTP opening are largely dependent on electron transport and energy coupling.     

Nitric oxide protects against mitochondrial permeabilization induced by glutathione depletion: role of S-nitrosylation?

Nitric oxide (NO) is known to mediate a multitude of biological effects including inhibition of respiration at cytochrome c oxidase (COX), formation of peroxynitrite (ONOO-) by reaction with mitochondrial superoxide (O2*-), and S-nitrosylation of proteins. In this study, we investigated pathways of NO metabolism in lymphoblastic leukemic CEM cells in response to glutathione (GSH) depletion. We found that NO blocked mitochondrial protein thiol oxidation, membrane permeabilization, and cell death. The effects of NO were: (1) independent of respiratory chain inhibition since protection was also observed in CEM cells lacking mitochondrial DNA (rho0) which do not possess a functional respiratory chain and (2) independent of ONOO- formation since nitrotyrosine (a marker for ONOO- formation) was not detected in extracts from cells treated with NO after GSH depletion. However, NO increased the level of mitochondrial protein S-nitrosylation (SNO) determined by the Biotin Switch assay and by the release of NO from mitochondrial fractions treated with mercuric chloride (which cleaves SNO bonds to release NO). In conclusion, these results indicate that NO blocks cell death after GSH depletion by preserving the redox status of mitochondrial protein thiols probably by a mechanism that involves S-nitrosylation of mitochondrial protein thiols.     

Functional consequences of the sustained or transient activation by Bax of the mitochondrial permeability transition pore.

The overexpression of Bax kills cells by a mechanism that depends on induction of the mitochondrial permeability transition (MPT) (Pastorino, J. G., Chen, S.-T., Tafani, M., Snyder, J. W., and Farber, J. L. (1998) J. Biol. Chem. 273, 7770-7775). In the present study, purified, recombinant Bax opened the mitochondrial permeability transition pore (PTP). Depending on its concentration, Bax had two distinct effects. At a concentration of 125 nM, Bax caused the release of the intermembranous proteins cytochrome c and adenylate kinase and the release from the matrix of sequestered calcein, effects prevented by the inhibitor of the PTP cyclosporin A (CSA). At this concentration of Bax, there was no detectable mitochondrial swelling or depolarization. These effects of low Bax concentrations are interpreted as the consequence of transient, non-synchronous activation of the PTP followed by a prompt recovery of mitochondrial integrity. By contrast, Bax concentrations between 250 nM and 1 microM caused a sustained opening of the PTP with consequent persistent mitochondrial swelling and deenergization (the MPT). CSA prevented the MPT induced by Bax. Increasing concentrations of calcium caused a greater proportion of the mitochondria to undergo the MPT in the presence of Bax. Importantly, two known mediators of apoptosis, ceramide and GD3 ganglioside, potentiated the induction by Bax of the MPT. The data imply that Bax mediates the opening of the mitochondrial PTP with the resultant release of cytochrome c from the intermembranous space.