Reactive oxygen species, but not Ca2+ overloading, trigger pH- and mitochondrial permeability transition-dependent death of adult rat myocytes after ischemia-reperfusion

We investigated the role of pH, reactive oxygen species (ROS), Ca2+, and the mitochondrial permeability transition (MPT) in pH-dependent ischemia-reperfusion injury to adult rat myocytes. Myocytes were incubated in anoxic Krebs-Ringer-HEPES buffer at pH 6.2 for 3 h to simulate ischemia. To simulate reperfusion, myocytes were reoxygenated at pH 6.2 or 7.4 for 2 h. Some myocytes were treated with MPT blockers (cyclosporin A and N-methyl-4-isoleucine cyclosporin) and antioxidants (desferal, diphenylphenylene diamine, and 2-mercaptopropionyl glycine). Mitochondrial membrane potential, inner membrane permeabilization, and ROS formation were imaged with tetramethylrhodamine methyl ester, calcein, and chloromethyldichlorofluorescein diacetate, respectively. For Ca2+ imaging, myocytes were coloaded with rhod-2 and fluo-4 to evaluate mitochondrial and cytosolic Ca2+, respectively. After 10 min of reperfusion at pH 7.4, calcein redistributed across the mitochondrial inner membrane, an event preceded by mitochondrial ROS formation and accompanied by hypercontracture, mitochondrial depolarization, and then cell death. Acidotic reperfusion, antioxidants, and MPT blockers each prevented the MPT, depolarization, hypercontraction, and cell killing. Antioxidants, but neither MPT blockers nor acidotic reperfusion, inhibited ROS formation after reperfusion. Furthermore, anoxic reperfusion at pH 7.4 prevented cell death. Both mitochondrial and cytosolic Ca2+ increased during ischemia but recovered in the first minutes of reperfusion. Mitochondrial and cytosolic Ca2+ overloading again occurred late after reperfusion. This late Ca2+ overloading was blocked by MPT inhibition. Intramitochondrial Ca2+ chelation by cold loading/warm incubation of BAPTA did not prevent cell death after reperfusion. In conclusion, mitochondrial ROS, together with normalization of pH, promote MPT onset and subsequent myocyte death after reperfusion. In contrast, Ca2+ overloading appears to be the consequence of bioenergetic failure after the MPT and is not a factor promoting MPT onset.     

Mitoferrin-2-dependent Mitochondrial Iron Uptake Sensitizes Human Head and Neck Squamous Carcinoma Cells to Photodynamic Therapy

Photodynamic therapy (PDT) is a promising approach to treat head and neck cancer cells. Here, we investigated whether mitochondrial iron uptake through mitoferrin-2 (Mfrn2) enhanced PDT-induced cell killing. Three human head and neck squamous carcinoma cell lines (UMSCC1, UMSCC14A, and UMSCC22A) were exposed to light and Pc 4, a mitochondria-targeted photosensitizer. The three cell lines responded differently: UMSCC1 and UMSCC14A cells were more resistant, whereas UMSCC22A cells were more sensitive to Pc 4-PDT-induced cell death. In non-erythroid cells, Mfrn2 is an iron transporter in the mitochondrial inner membrane. PDT-sensitive cells expressed higher Mfrn2 mRNA and protein levels compared with PDT-resistant cells. High Mfrn2-expressing cells showed higher rates of mitochondrial Fe²⁺ uptake compared with low Mfrn2-expressing cells. Bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes that causes lysosomal iron release to the cytosol, enhanced PDT-induced cell killing of both resistant and sensitive cells. Iron chelators and the inhibitor of the mitochondrial Ca²⁺ (and Fe²⁺) uniporter, Ru360, protected against PDT plus bafilomycin toxicity. Knockdown of Mfrn2 in UMSCC22A cells decreased the rate of mitochondrial Fe²⁺ uptake and delayed PDT plus bafilomycin-induced mitochondrial depolarization and cell killing. Taken together, the data suggest that lysosomal iron release and Mfrn2-dependent mitochondrial iron uptake act synergistically to induce PDT-mediated and iron-dependent mitochondrial dysfunction and subsequent cell killing. Furthermore, Mfrn2 represents a possible biomarker of sensitivity of head and neck cancers to cell killing after PDT.     

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.     

Mitochondrial calcium uniporter blocker effectively prevents brain mitochondrial dysfunction caused by iron overload

AimsAlthough iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction.Main methodsIsolated brain mitochondria from male Wistar rats were used. Iron (Fe^2 + and Fe^3 +) at 0–286 μM were applied onto mitochondria at various incubation times (5–30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied.Key findingsBoth Fe^2 + and Fe^3 + entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe^2 + caused more severe mitochondrial dysfunction than Fe^3 +. Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization.SignificanceOur findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.     

Nitric oxide: a signaling molecule against mitochondrial permeability transition- and pH-dependent cell death after reperfusion

Reperfusion of ischemic tissue can precipitate cell death. Much of this cell killing is related to the return of physiological pH after the tissue acidosis of ischemia. The mitochondrial permeability transition (MPT) is a key mechanism contributing to this pH-dependent reperfusion injury in hepatocytes, myocytes, and other cell types. When ATP depletion occurs after the MPT, necrotic cell death ensues. If ATP levels are maintained, at least in part, the MPT initiates apoptosis caused by mitochondrial swelling and release of cytochrome c and other proapoptotic factors. Cyclosporin A and acidotic pH inhibit opening of permeability transition pores and protect cells against oxidative stress and ischemia/reperfusion injury, whereas Ca²⁺, mitochondrial reactive oxygen species, and pH above 7 promote mitochondrial inner membrane permeabilization. Reperfusion with nitric oxide (NO) donors also blocks the MPT via a guanylyl cyclase and protein kinase G-dependent signaling pathway, which in turn prevents reperfusion-induced cell killing. In isolated mitochondria, a combination of cGMP, cytosolic extract, and ATP blocks the Ca²⁺-induced MPT, an effect that is reversed by protein kinase G inhibition. Thus, NO prevents pH-dependent cell killing after ischemia/reperfusion by a guanylyl cyclase/cGMP/protein kinase G signaling cascade that blocks the MPT.     

Mitochondrial Dysfunction in the Pathogenesis of Necrotic and Apoptotic Cell Death

Mitochondria are frequently the target of injury after stresses leading to necrotic and apoptoticcell death. Inhibition of oxidative phosphorylation progresses to uncoupling when opening ofa high conductance permeability transition (PT) pore in the mitochondrial inner membraneabruptly increases the permeability of the mitochondrial inner membrane to solutes of molecularmass up to 1500 Da. Cyclosporin A (CsA) blocks this mitochondrial permeability transition(MPT) and prevents necrotic cell death from oxidative stress, Ca²⁺ ionophore toxicity,Reye-related drug toxicity, pH-dependent ischemia/reperfusion injury, and other models of cell injury.Confocal fluorescence microscopy directly visualizes onset of the MPT from the movementof green-fluorescing calcein into mitochondria and the simultaneous release from mitochondriaof red-fluorescing tetramethylrhodamine methylester, a membrane potential-indicatingfluorophore. In oxidative stress to hepatocytes induced by tert-butylhydroperoxide, NAD(P)Hoxidation, increased mitochondrial Ca²⁺, and mitochondrial generation of reactive oxygen speciesprecede and contribute to onset of the MPT. Confocal microscopy also shows directly thatthe MPT is a critical event in apoptosis of hepatocytes induced by tumor necrosis factor-.Progression to necrotic and apoptotic cell killing depends, at least in part, on the effect theMPT has on cellular ATP levels. If ATP levels fall profoundly, necrotic killing ensues. If ATPlevels are at least partially maintained, apoptosis follows the MPT. Cellular features of bothapoptosis and necrosis frequently occur together after death signals and toxic stresses. A newterm, necrapoptosis, describes such death processes that begin with a common stress or deathsignal, progress by shared pathways, but culminate in either cell lysis (necrosis) or programmedcellular resorption (apoptosis) depending on modifying factors such as ATP.     

Hypothermia causes a marked injury to rat proximal tubular cells that is aggravated by all currently used preservation solutions

Cold preservation results in cell death via iron-dependent formation of reactive oxygen species, leading to apoptosis during rewarming. We aimed to study cold-induced damage (i.e., injury as a consequence of hypothermia itself and not cold ischemia) in proximal tubular cells (PTC) in various preservation solutions presently applied and to clarify the role of mitochondria in this injury. Primary cultures of rat PTC were incubated at 4 degrees C for 24 h in culture medium, UW, Euro-Collins or HTK solution with and without the iron chelator desferal and rewarmed at 37 degrees C in culture medium. Cell damage, morphology, and apoptosis were studied and mitochondrial membrane potential was assessed by fluorescence microscopy. Cold incubation of PTC in culture medium followed by rewarming caused marked cell damage compared to warm incubation alone (LDH release 39+/-10% vs. 1.6+/-0.3%). Cold-induced damage was aggravated in all preservation solutions (LDH release 85+/-2% for UW; similar in Euro-Collins and HTK). After rewarming, cells showed features suggestive for apoptosis. Desferal prevented cell injury in all solutions (e.g., 8+/-2% for UW). Mitochondrial membrane potential was lost during rewarming and this loss could also be inhibited by desferal. Trifluoperazine, which is known to inhibit mitochondrial permeability transition (MPT), was able to prevent cold-induced injury (LDH 85+/-5% vs. 12+/-2%). We conclude that cold-induced injury occurs in PTC and is aggravated by UW, Euro-Collins, and HTK solution. Iron-dependent MPT is suggested to play a role in this damage. Strategies to prevent cold-induced injury should aim at reducing the availability of "free" iron.     

Mitochondrial ATP-Sensitive K<Superscript>+</Superscript> Channels Prevent Oxidative Stress,Permeability Transition and Cell Death

Ischemia followed by reperfusion results in impairment of cellular and mitochondrial functionality due to opening of mitochondrial permeability transition pores. On the other hand, activation of mitochondrial ATP-sensitive K⁺ channels (mitoK_ATP) protects the heart against ischemic damage. This study examined the effects of mitoK_ATP and mitochondrial permeability transition on isolated rat heart mitochondria and cardiac cells submitted to simulated ischemia and reperfusion (cyanide/aglycemia). Both mitoK_ATP opening, using diazoxide, and the prevention of mitochondrial permeability transition, using cyclosporin A, protected against cellular damage, without additive effects. MitoK_ATP opening in isolated rat heart mitochondria slightly decreased Ca²⁺ uptake and prevented mitochondrial reactive oxygen species production, most notably in the presence of added Ca²⁺. In ischemic cells, diazoxide decreased ROS generation during cyanide/aglycemia while cyclosporin A prevented oxidative stress only during simulated reperfusion. Collectively, these studies indicate that opening mitoK_ATP prevents cellular death under conditions of ischemia/reperfusion by decreasing mitochondrial reactive oxygen species release secondary to Ca²⁺ uptake, inhibiting mitochondrial permeability transition.     

Effect of taurine on ischemia–reperfusion injury

Taurine is an abundant β-amino acid that regulates several events that dramatically influence the development of ischemia–reperfusion injury. One of these events is the extrusion of taurine and Na⁺ from the cell via the taurine/Na⁺ symport. The loss of Na⁺ during the ischemia–reperfusion insult limits the amount of available Na⁺ for Na⁺/Ca²⁺ exchange, an important process in the development of Ca²⁺ overload and the activation of the mitochondrial permeability transition, a key process in ischemia–reperfusion mediated cell death. Taurine also prevents excessive generation of reactive oxygen species by the respiratory chain, an event that also limits the activation of the MPT. Because taurine is an osmoregulator, changes in taurine concentration trigger “osmotic preconditioning,” a process that activates an Akt-dependent cytoprotective signaling pathway that inhibits MPT pore formation. These effects of taurine have clinical implications, as experimental evidence reveals potential promise of taurine therapy in preventing cardiac damage during bypass surgery, heart transplantation and myocardial infarction. Moreover, severe loss of taurine from the heart during an ischemia–reperfusion insult may increase the risk of ventricular remodeling and development of heart failure.     

Cold-induced apoptosis of hepatocytes: mitochondrial permeability transition triggered by nonmitochondrial chelatable iron

We previously described that the cold-induced apoptosis of cultured hepatocytes is mediated by an increase in the cellular chelatable iron pool. We here set out to assess whether a mitochondrial permeability transition (MPT) is involved in cold-induced apoptosis. When cultured hepatocytes were rewarmed after 18 h of cold (4°C) incubation in cell culture medium or University of Wisconsin solution, the vast majority of cells rapidly lost mitochondrial membrane potential. This loss was due to MPT as assessed by confocal laser scanning microscopy and as evidenced by the inhibitory effect of the MPT inhibitors trifluoperazine plus fructose. The occurrence of the MPT was iron-dependent: it was strongly inhibited by the iron chelators 2,2′-dipyridyl and deferoxamine. Addition of trifluoperazine plus fructose also strongly inhibited cold-induced apoptosis, suggesting that the MPT constitutes a decisive intermediate event in the pathway leading to cold-induced apoptosis. Further experiments employing the non-site-specific iron indicator Phen Green SK and specifically mitochondrial iron indicators and chelators (rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester, RPA, and rhodamine B-[(2,2′-bipyridin-4-yl)aminocarbonyl]benzyl ester, RDA) suggest that it is the cold-induced increase in cytosolic chelatable iron that triggers the MPT and that mitochondrial chelatable iron is not involved in this process.     

Interplay between Ca2+ cycling and mitochondrial permeability transition pores promotes reperfusion-induced injury of cardiac myocytes

Uncontrolled release of Ca(2+) from the sarcoplasmic reticulum (SR) contributes to the reperfusion-induced cardiomyocyte injury, e.g. hypercontracture and necrosis. To find out the underlying cellular mechanisms of this phenomenon, we investigated whether the opening of mitochondrial permeability transition pores (MPTP), resulting in ATP depletion and reactive oxygen species (ROS) formation, may be involved. For this purpose, isolated cardiac myocytes from adult rats were subjected to simulated ischemia and reperfusion. MPTP opening was detected by calcein release and by monitoring the ΔΨ(m). Fura-2 was used to monitor cytosolic [Ca(2+)](i) or mitochondrial calcium [Ca(2+)](m), after quenching the cytosolic compartment with MnCl(2). Mitochondrial ROS [ROS](m) production was detected with MitoSOX Red and mag-fura-2 was used to monitor Mg(2+) concentration, which reflects changes in cellular ATP. Necrosis was determined by propidium iodide staining. Reperfusion led to a calcein release from mitochondria, ΔΨ(m) collapse and disturbance of ATP recovery. Simultaneously, Ca(2+) oscillations occurred, [Ca(2+)](m) and [ROS](m) increased, cells developed hypercontracture and underwent necrosis. Inhibition of the SR-driven Ca(2+) cycling with thapsigargine or ryanodine prevented mitochondrial dysfunction, ROS formation and MPTP opening. Suppression of the mitochondrial Ca(2+) uptake (Ru360) or MPTP (cyclosporine A) significantly attenuated Ca(2+) cycling, hypercontracture and necrosis. ROS scavengers (2-mercaptopropionyl glycine or N-acetylcysteine) had no effect on these parameters, but reduced [ROS](m). In conclusion, MPTP opening occurs early during reperfusion and is due to the Ca(2+) oscillations originating primarily from the SR and supported by MPTP. The interplay between Ca(2+) cycling and MPTP promotes the reperfusion-induced cardiomyocyte hypercontracture and necrosis. Mitochondrial ROS formation is a result rather than a cause of MPTP opening.     

Activation of the oxygen-sensing signal cascade prevents mitochondrial injury after mouse liver ischemia-reperfusion

The mitochondrial permeability transition (MPT) plays an important role in hepatocyte death caused by ischemia-reperfusion (IR). This study investigated whether activation of the cellular oxygen-sensing signal cascade by prolyl hydroxylase inhibitors (PHI) protects against the MPT after hepatic IR. Ethyl 3,4-dihyroxybenzoate (EDHB, 100 mg/kg ip), a PHI, increased mouse hepatic hypoxia-inducible factor-1alpha and heme oxygenase-1 (HO-1). EDHB-treated and untreated mice were subjected to 1 h of warm ischemia to approximately 70% of the liver followed by reperfusion. Mitochondrial polarization, cell death, and the MPT were assessed by intravital confocal/multiphoton microscopy of rhodamine 123, propidium iodide, and calcein. EDHB largely blunted alanine aminotransferase (ALT) release and necrosis after reperfusion. In vehicle-treated mice at 2 h after reperfusion, viable cells with depolarized mitochondria were 72%, and dead cells were 2%, indicating that depolarization preceded necrosis. Mitochondrial voids excluding calcein disappeared, indicating MPT onset in vivo. NIM811, a specific inhibitor of the MPT, blocked mitochondrial depolarization after IR, further confirming that mitochondrial depolarization was due to MPT onset. EDHB decreased mitochondrial depolarization to 16% and prevented the MPT. Tin protoporphyrin (10 micromol/kg sc), an HO-1 inhibitor, partially abrogated protection by EDHB against ALT release, necrosis, and mitochondrial depolarization. In conclusion, IR causes the MPT and mitochondrial dysfunction, leading to hepatocellular death. PHI prevents MPT onset and liver damage through an effect mediated partially by HO-1.     

Does the calcein-AM method assay the total cellular 'labile iron pool' or only a fraction of it?

The calcein-AM (calcein-acetoxymethyl ester) method is a widely used technique that is supposed to assay the intracellular 'labile iron pool' (LIP). When cells in culture are exposed to this ester, it passes the plasma membrane and reacts with cytosolic unspecific esterases. One of the reaction products, calcein, is a fluorochrome and a hydrophilic alcohol to which membranes are non-permeable and which, consequently, is retained within the cytosol of cells. Calcein fluorescence is quenched following chelation of low-mass labile iron, and the degree of quenching gives an estimate of the amounts of chelatable iron. However, a requirement for the assay to be able to demonstrate cellular LIP in total is that such iron be localized in the cytosol and not in a membrane-limited compartment. For some time it has been known that a major part of cellular, redox-active, labile, low-mass iron is temporarily localized in the lysosomal compartment as a result of the autophagic degradation of ferruginous materials, such as mitochondrial complexes and ferritin. Even if some calcein-AM may escape cytosolic esterases and enter lysosomes to be cleaved by lysosomal acidic esterases, the resulting calcein does not significantly chelate iron at 相似文献   

Involvement of cyclophilin D in mitochondrial permeability transition induction in intact cells

The mitochondrial permeability transition (MPT) is involved in both Ca²⁺ signaling and cell death. The present study aimed to clarify the involvement of cyclophilin D, a peptidyl prolyl cis-trans isomerase (PPIase), in MPT induction in intact cells. To achieve this, we used C6 cells overexpressing wild-type or PPIase-deficient cyclophilin D, and measured the inner mitochondrial membrane permeability to calcein, a 623-Da hydrophilic fluorescent molecule, to evaluate MPT induction. In vector control cells, the percentage of MPT induction by ionomycin increased as the Ca²⁺ concentration in the extracellular medium increased. This result indicates that the present method is valid for numerical evaluation of MPT induction. In C6 cells expressing the PPIase-deficient mutant, the percentage of MPT induction was significantly decreased compared with wild-type CypD-overexpressing cells or vector control cells. These results suggest that cyclophilin D is involved in MPT induction by Ca²⁺ in intact cells.     

Synaptic and nonsynaptic mitochondria demonstrate a different degree of calcium-induced mitochondrial dysfunction

AimsSince variety in response to Ca²⁺-induced mitochondrial dysfunction in different neuronal mitochondrial populations is associated with the pathogenesis of several neurological diseases, we investigated the effects of Ca²⁺ overload on synaptic (SM) and nonsynaptic mitochondrial (NM) dysfunction and probed the effects of cyclosporin A (CsA), 4′-chlorodiazepam (CDP) and Ru360 on relieving mitochondrial damage.Main methodsSM and NM mitochondria were isolated from rats' brains (n = 5/group) and treated with various concentrations (5, 10, 100, and 200 μM) of Ca²⁺, with and without CsA (mPTP blocker), CDP (PBR/TSPO blocker) and Ru360 (MCU blocker) pretreatments. Mitochondrial function was determined by mitochondrial swelling, ROS production and mitochondrial membrane potential changes (ΔΨm).Key findingsAt 200-μM Ca²⁺, SM presented mitochondrial swelling to a greater extent than NM. At 100 and 200-μM Ca²⁺, the ROS production of SM was higher than that of NM and ΔΨm dissipation of SM was also larger. CsA, CDP and Ru360 could reduce ROS production of SM and NM with exposure to 200-μM Ca²⁺. However, only Ru360 could completely inhibit ROS generation in both SM and NM, whereas CsA and CDP could only partially reduce the ROS level in SM. Moreover, CsA and CDP pretreatments were not able to restore ΔΨm. However, Ru360 pretreatment could protect ΔΨm dissipation in both SM and NM, with complete protection observed only in NM.SignificanceOur findings suggested that mitochondrial calcium uniporter is a possible major pathway for calcium uptake in both mitochondrial populations. However, SM might have additional pathways involved in the calcium uptake.     

Mechanisms of Rapid Reactive Oxygen Species Generation in Response to Cytosolic Ca2+ or Zn2+ Loads in Cortical Neurons

Excessive “excitotoxic” accumulation of Ca²⁺ and Zn²⁺ within neurons contributes to neurodegeneration in pathological conditions including ischemia. Putative early targets of these ions, both of which are linked to increased reactive oxygen species (ROS) generation, are mitochondria and the cytosolic enzyme, NADPH oxidase (NOX). The present study uses primary cortical neuronal cultures to examine respective contributions of mitochondria and NOX to ROS generation in response to Ca²⁺ or Zn²⁺ loading. Induction of rapid cytosolic accumulation of either Ca²⁺ (via NMDA exposure) or Zn²⁺ (via Zn²⁺/Pyrithione exposure in 0 Ca²⁺) caused sharp cytosolic rises in these ions, as well as a strong and rapid increase in ROS generation. Inhibition of NOX activation significantly reduced the Ca²⁺-induced ROS production with little effect on the Zn²⁺- triggered ROS generation. Conversely, dissipation of the mitochondrial electrochemical gradient increased the cytosolic Ca²⁺ or Zn²⁺ rises caused by these exposures, consistent with inhibition of mitochondrial uptake of these ions. However, such disruption of mitochondrial function markedly suppressed the Zn²⁺-triggered ROS, while partially attenuating the Ca²⁺-triggered ROS. Furthermore, block of the mitochondrial Ca²⁺ uniporter (MCU), through which Zn²⁺ as well as Ca²⁺ can enter the mitochondrial matrix, substantially diminished Zn²⁺ triggered ROS production, suggesting that the ROS generation occurs specifically in response to Zn²⁺ entry into mitochondria. Finally, in the presence of the sulfhydryl-oxidizing agent 2,2''-dithiodipyridine, which impairs Zn²⁺ binding to cytosolic metalloproteins, far lower Zn²⁺ exposures were able to induce mitochondrial Zn²⁺ uptake and consequent ROS generation. Thus, whereas rapid acute accumulation of Zn²⁺ and Ca²⁺ each can trigger injurious ROS generation, Zn²⁺ entry into mitochondria via the MCU may do so with particular potency. This may be of particular relevance to conditions like ischemia in which cytosolic Zn²⁺ buffering is impaired due to acidosis and oxidative stress.     

Mitochondrial ROS-induced ROS release: an update and review

Unstable mitochondrial membrane potential and redox transitions can occur following insults including ischemia/reperfusion injury and toxin exposure, with negative consequences for mitochondrial integrity and cellular survival. These transitions can involve mechanisms such as the recently described process, "Reactive Oxygen Species (ROS)-induced ROS-release" (RIRR), and be generated by circuits where the mitochondrial permeability transition (MPT) pore and the inner membrane anion channel (IMAC) are involved. The exposure to excessive oxidative stress results in an increase in ROS reaching a threshold level that triggers the opening of one of the requisite mitochondrial channels. In turn, this leads to the simultaneous collapse of the mitochondrial membrane potential and a transient increased ROS generation by the electron transfer chain. Generated ROS can be released into cytosol and trigger RIRR in neighboring mitochondria. This mitochondrion-to-mitochondrion ROS-signaling constitutes a positive feedback mechanism for enhanced ROS production leading to potentially significant mitochondrial and cellular injury. This review and update considers a variety of RIRR mechanisms (involving MPT, IMAC and episodes of mitochondrial transient hyperpolarization). RIRR could be a general cell biology phenomenon relevant to the processes of programmed mitochondrial destruction and cell death, and may contribute to other mechanisms of post-ischemic pathologies, including arrhythmias.     

Mitochondrial permeability transition in pH-dependent reperfusion injury to rat hepatocytes

To simulateischemia and reperfusion, cultured rat hepatocytes were incubated inanoxic buffer at pH 6.2 for 4 h and reoxygenated at pH 7.4. Duringanoxia, intracellular pH (pH_i)decreased to 6.3, mitochondria depolarized, and ATP decreased to <1%of basal values, but the mitochondrial permeability transition (MPT)did not occur as assessed by confocal microscopy from theredistribution of cytosolic calcein into mitochondria. Moreover, cellviability remained >90%. After reperfusion at pH 7.4, pH_i returned to pH 7.2, the MPToccurred, and most hepatocytes lost viability. In contrast, afterreperfusion at pH 6.2 or withNa⁺-free buffer at pH 7.4, pH_i did not rise and cellviability remained >80%. After acidotic reperfusion, the MPT did notoccur. When hepatocytes were reperfused with cyclosporin A (0.5-1µM) at pH 7.4, the MPT was prevented and cell viability remained>80%, although pH_i increased to7.2. Reperfusion with glycine (5 mM) also prevented cell killing butdid not block recovery of pH_i orthe MPT. Retention of cell viability was associated with recovery of30-40% of ATP. In conclusion, preventing the rise ofpH_i after reperfusion blocked theMPT, improved ATP recovery, and prevented cell death. Cyclosporin Aalso prevented cell killing by blocking the MPT without blocking recovery of pH_i. Glycine preventedcell killing but did not inhibit recovery ofpH_i or the MPT.

     

High fluence low‐power laser irradiation induces mitochondrial permeability transition mediated by reactive oxygen species

High fluence low‐power laser irradiation (HF‐LPLI) can induce cell apoptosis via the mitochondria/caspase‐3 pathway. Here, we further investigated the mechanism involved in the apoptotic process in human lung adenocarcinoma cells (ASTC‐a‐1) at a laser irradiation fluence of 120 J/cm² (633 nm). Cytochrome c release was ascribed to mitochondrial permeability transition (MPT) because the release was prevented by cyclosporine (CsA), a specific inhibitor of MPT. Furthermore, mitochondrial permeability for calcein (～620 Da) was another evidence for the MPT induction under HF‐LPLI treatment. A high‐level intracellular reactive oxygen species (ROS) generation was observed after irradiation. The photodynamically produced ROS caused onset of MPT, as the ROS scavenger docosahexaenoic acid (DHA) prevented the MPT. However, CsA failed to prevented cell death induced by HF‐LPLI, indicating the existence of other signaling pathways. Following laser irradiation, Bax activation occurred after mitochondrial depolarization and cytochrome c release, indicating Bax activation was a downstream event. In the presence of CsA, Bax was still activated at the end‐stage of apoptotic process caused by HF‐LPLI, suggesting that Bax was involved in an alternative‐signaling pathway, which was independent of MPT. Under HF‐LPLI treatment, cell viabilities due to pre‐treatment with DHA, CsA, or Bax small interfering RNA (siRNA) demonstrated that the MPT signaling pathway was dominant, while Bax signaling pathway was secondary, and more importantly ROS mediated both pathways. Taken together, these results showed that HF‐LPLI induced cell apoptosis via the CsA‐sensitive MPT, which was ROS‐dependent. Furthermore, there existed a secondary signaling pathway through Bax activation. The observed link between MPT and triggering ROS could be a fundamental phenomenon in HF‐LPLI‐induced cell apoptosis. J. Cell. Physiol. 218: 603–611, 2009. © 2008 Wiley‐Liss, Inc.     

Oxidative Stress Underlies the Mechanism for Ca2+-induced Permeability Transition of Mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca²⁺ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca²⁺ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca²⁺ increased the generation of H²O² by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca²⁺ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca²⁺-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca²⁺ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.