The permeability transition pore in cell death

The permeability transition pore (PT-pore) is a multi-component protein aggregate in mitochondria that comprises factors in the inner as well as in the outer mitochondrial membrane. This complex has two functions: firstly, it regulates the integration of oxidative phosphorylation into the cellular energy household and secondly, it induces cell death when converted into an unspecific channel. The latter causes a collapse of the mitochondrial membrane potential and activates a chain of events that culminate in the demise of the cell. It has been controversial for some time whether the PT-pore is causative for or only amplifies a signal of cell death but novel results confirm a central role of this protein complex for cell death induction. While a considerable body of data exist on its subunit composition, recent genetic knock-out experiments suggest that the identity of the core factors of the PT-pore is still unresolved. Moreover, accumulating evidence point to a much more complex composition of this protein complex than anticipated. Here, we review the current knowledge of its subunit composition, the evidence of a role in cell death, and we propose a model for the activation of the PT-pore for cell death.     

Participation of the mitochondrial permeability transition pore in nitric oxide-induced plant cell death

In the present study, we investigated the involvement of the mitochondrial permeability transition pore (PTP) in nitric oxide (NO)-induced plant cell death. NO donors such as sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine inhibited growth and caused death in suspension-cultured cells of Citrus sinensis. Cells treated with SNP showed chromatin condensation and fragmentation, characteristic of apoptosis. SNP caused loss of the mitochondrial membrane electrical potential, which was prevented by cyclosporin A (CsA), a specific inhibitor of PTP formation. CsA also prevented the nuclear apoptosis and subsequent Citrus cell death induced by NO. These findings indicate that mitochondrial PTP formation is involved in the signaling pathway by which NO induces apoptosis in cultured Citrus cells.     

The mitochondrial permeability transition pore and its involvement in cell death and in disease pathogenesis

Current research on the mitochondrial permeability transition pore (PTP) and its role in cell death faces a paradox. Initially considered as an in vitro artifact of little pathophysiological relevance, in recent years the PTP has received considerable attention as a potential mechanism for the execution of cell death. The recent successful use of PTP desensitizers in several disease paradigms leaves little doubt about its relevance in pathophysiology; and emerging findings that link the PTP to key cellular signalling pathways are increasing the interest on the pore as a pharmacological target. Yet, recent genetic data have challenged popular views on the molecular nature of the PTP, and called into question many early conclusions about its structure. Here we review basic concepts about PTP structure, function and regulation within the framework of intracellular death signalling, and its role in disease pathogenesis.     

BNIP3 and genetic control of necrosis-like cell death through the mitochondrial permeability transition pore

Many apoptotic signaling pathways are directed to mitochondria, where they initiate the release of apoptogenic proteins and open the proposed mitochondrial permeability transition (PT) pore that ultimately results in the activation of the caspase proteases responsible for cell disassembly. BNIP3 (formerly NIP3) is a member of the Bcl-2 family that is expressed in mitochondria and induces apoptosis without a functional BH3 domain. We report that endogenous BNIP3 is loosely associated with mitochondrial membrane in normal tissue but fully integrates into the mitochondrial outer membrane with the N terminus in the cytoplasm and the C terminus in the membrane during induction of cell death. Surprisingly, BNIP3-mediated cell death is independent of Apaf-1, caspase activation, cytochrome c release, and nuclear translocation of apoptosis-inducing factor. However, cells transfected with BNIP3 exhibit early plasma membrane permeability, mitochondrial damage, extensive cytoplasmic vacuolation, and mitochondrial autophagy, yielding a morphotype that is typical of necrosis. These changes were accompanied by rapid and profound mitochondrial dysfunction characterized by opening of the mitochondrial PT pore, proton electrochemical gradient (Deltapsim) suppression, and increased reactive oxygen species production. The PT pore inhibitors cyclosporin A and bongkrekic acid blocked mitochondrial dysregulation and cell death. We propose that BNIP3 is a gene that mediates a necrosis-like cell death through PT pore opening and mitochondrial dysfunction.     

Phosphate is essential for inhibition of the mitochondrial permeability transition pore by cyclosporin A and by cyclophilin D ablation

Energized mouse liver mitochondria displayed the same calcium retention capacity (a sensitive measure of the propensity of the permeability transition pore (PTP) to open) irrespective of whether phosphate, arsenate, or vanadate was the permeating anion. Unexpectedly, however, phosphate was specifically required for PTP desensitization by cyclosporin A (CsA) or by genetic inactivation of cyclophilin D (CyP-D). Indeed, when phosphate was replaced by arsenate, vanadate, or bicarbonate, the inhibitory effects of CsA and of CyP-D ablation on the PTP disappeared. After loading with the same amount of Ca(2+) in the presence of arsenate or vanadate but in the absence of phosphate, the sensitivity of the PTP to a variety of inducers was identical in mitochondria from wild-type mice, CyP-D-null mice, and wild-type mice treated with CsA. These findings call for a reassessment of conclusions on the role of the PTP in cell death that are based on the effects of CsA or of CyP-D ablation.     

The mitochondrial permeability transition pore: an evolving concept critical for cell life and death

In this review, we summarize current knowledge of perhaps one of the most intriguing phenomena in cell biology: the mitochondrial permeability transition pore (mPTP). This phenomenon, which was initially observed as a sudden loss of inner mitochondrial membrane impermeability caused by excessive calcium, has been studied for almost 50 years, and still no definitive answer has been provided regarding its mechanisms. From its initial consideration as an in vitro artifact to the current notion that the mPTP is a phenomenon with physiological and pathological implications, a long road has been travelled. We here summarize the role of mitochondria in cytosolic calcium control and the evolving concepts regarding the mitochondrial permeability transition (mPT) and the mPTP. We show how the evolving mPTP models and mechanisms, which involve many proposed mitochondrial protein components, have arisen from methodological advances and more complex biological models. We describe how scientific progress and methodological advances have allowed milestone discoveries on mPTP regulation and composition and its recognition as a valid target for drug development and a critical component of mitochondrial biology.     

Curcumin-induced melanoma cell death is associated with mitochondrial permeability transition pore (mPTP) opening

Here we studied the role of mitochondrial permeability transition pore (mPTP) opening in curcumin’s cytotoxicity in melanoma cells. In cultured WM-115 melanoma cells, curcumin induced mitochondrial membrane potential (MPP) decrease, cyclophilin-D (CyPD)-adenine nucleotide translocator 1 (ANT-1) (two mPTP components) mitochondrial association and cytochrome C release, indicating mPTP opening. The mPTP blocker sanglifehrin A (SfA) and ANT-1 siRNA-depletion dramatically inhibited curcumin-induced cytochrome C release and WM-115 cell death. CyPD is required for curcumin-induced melanoma cell death. The CyPD inhibitor cyclosporin A (CsA) or CyPD siRNA-depletion inhibited curcumin-induced WM-115 cell death and apoptosis, while WM-115 cells with CyPD over-expression were hyper-sensitive to curcumin. Finally, we found that C6 ceramide enhanced curcumin-induced cytotoxicity probably through facilitating mPTP opening, while CsA and SfA as well as CyPD and ANT-1 siRNAs alleviated C6 ceramide’s effect on curcumin in WM-115 cells. Together, these results suggest that curcumin-induced melanoma cell death is associated with mPTP opening.     

Inhibition of mitochondrial membrane bound-glutathione transferase by mitochondrial permeability transition inhibitors including cyclosporin A

AimsEffect of mitochondrial permeability transition (MPT) inhibitors on mitochondrial membrane-bound glutathione transferase (mtMGST1) activity in rat liver was investigated in vitro.Main methodsWhen mitochondria were incubated with MPT inhibitors, mtMGST1 activity was decreased dose dependently and their 50% inhibition concentration (IC₅₀) were 1.2 μM (cyclosporin A; CsA), 31 μM (bongkrekic acid; BKA), 1.8 mM (ADP), and 3.2 mM (ATP). The decrease of mtMGST1 activity by the MPT inhibitors was not observed in the presence of detergent Triton X-100. On the contrary, mtMGST1 inhibition by GST inhibitors such as cibacron blue (IC₅₀, 4.2 μM) and S-hexylglutathione (IC₅₀, 480 μM) was not affected in the presence of detergent. Although mtMGST1 resides in both the inner (IMM) and outer mitochondrial membranes (OMM), only mtMGST1 in the IMM was inhibited by the MPT inhibitors in the absence of detergent. GST inhibitors decreased mtMGST1 activity both in the IMM and OMM regardless of the presence or absence of detergent. Cytosolic GSTs and microsomal MGST1 were not inhibited by the MPT inhibitors.Key findingsThese results indicate that mtMGST1 is inhibited by MPT inhibitors through membrane components, not directly by the inhibitors.SignificanceSince CsA binds to cyclophilin D (Cyp-D) in the mitochondrial matrix whereas BKA or ADP binds to adenine nucleotide translocator (ANT) in the IMM, it was suggested that mtMGST1 in the IMM interacts with Cyp-D/ANT and the binding of MPT inhibitors to Cyp-D or ANT causes their conformational change followed by an alteration of mtMGST1 conformation, resulting in decreasing mtMGST1 activity.     

Sphingosine impairs mitochondrial function by opening permeability transition pore

Growing evidence suggest that, in the heart, sphingosine participates to contractile dysfunction by altering calcium transients and mitochondria function. However, mechanisms underlying sphingosine-induced cardiac mitochondria dysfunction are poorly understood. Here, we studied the effects of sphingosine on isolated cardiac mitochondria of either wild-type or Bcl-2 overexpressing transgenic mice. Sphingosine induced reductions in ADP-coupled respiration, membrane potential, mitochondrial cytochrome c content and ATP production, which were partially prevented by cyclosporine A and mitochondrial Bcl-2 overexpression. These data suggest that sphingosine promotes mitochondrial permeability transition pore opening, which may result in uncoupled respiration and participate in cardiac contractile dysfunction.     

Salt stress-induced programmed cell death in tobacco protoplasts is mediated by reactive oxygen species and mitochondrial permeability transition pore status

The status of mitochondrial permeability transition pore (PTP) and levels of reactive oxygen species (ROS) play key roles in regulating apoptosis in animal cells. To investigate if the PTP and cellular oxidation-reduction state are also involved in salt stress-induced programmed cell death (PCD) in tobacco (Nicotiana tabacum, cultivar BY-2) protoplasts, flow cytometry was used to simultaneously monitor ROS levels, PTP status and PCD. Increased ROS and decreased mitochondrial membrane potential (delta psi(m)) were observed before the appearance of PCD. Pre-treatment with an inhibitor of the PTP opening, cyclosporin A (CsA), effectively retarded the onset of PCD, the delta psi(m) decrease and the ROS content increase. Addition of ascorbic acid (AsA) during the salt stress significantly decreased the percentage of protoplasts undergoing PCD and ROS levels but increased delta psi(m). Hydrogen peroxide effectively induced the appearance of PCD and caused an increase in ROS and a decrease in delta psi(m). Pre-treatment of protoplasts with CsA weakened the effects of H2O2. All these results suggest that the open state of PTP and ROS are necessary elements for salt stress-induced PCD in tobacco protoplasts. The open states of PTP and ROS could promote each other suggesting that ROS could lead to a self-amplifying process. This positive feedback loop may act as an all-or-nothing switch, which is in good accordance with the hypothesis that PTP is an important coordinator and executioner of PCD in both animals and plants.     

The role of the mitochondrial permeability transition in cell death

The mitochondrial permeability transition (MPT) is a non-selective inner membrane permeabilization that occurs in response to increased calcium load and redox stress. Currently, two models of the MPT exist including the, largely hypothetical, native proteinaceous pore model and the oxidized inner membrane protein model which may reflect the extremes in a continuum of changes that occur to the inner membrane prior to its permeabilization. Here I discuss evidence that the MPT per se leads to necrosis, but not cytochrome c release and apoptosis. However, data also suggest that signaling crosstalk between the MPT and Bcl-2 family proteins occurs indicating an important role for the MPT in apoptosis.     

Role of the mitochondrial membrane permeability transition in cell death

In recent years, the role of the mitochondria in both apoptotic and necrotic cell death has received considerable attention. An increase of mitochondrial membrane permeability is one of the key events in apoptotic or necrotic death, although the details of the mechanism involved remain to be elucidated. The mitochondrial membrane permeability transition (MPT) is a Ca²⁺-dependent increase of mitochondrial membrane permeability that leads to loss of Δψ, mitochondrial swelling, and rupture of the outer mitochondrial membrane. The MPT is thought to occur after the opening of a channel that is known as the permeability transition pore (PTP), which 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). Recently, significant progress has been made by studies performed with mice lacking Cyp D at several laboratories, which have convincingly demonstrated that Cyp D is essential for the MPT to occur and that the Cyp D-dependent MPT regulates some forms of necrotic, but not apoptotic, cell death. Cyp D-deficient mice have also been used to show that the Cyp D-dependent MPT plays a crucial role in ischemia/reperfusion injury. The anti-apoptotic proteins Bcl-2 and Bcl-x_L have the ability to block the MPT, and can therefore block MPT-dependent necrosis in addition to their well-established ability to inhibit apoptosis.     

Physiological roles of the mitochondrial permeability transition pore

Neurons experience high metabolic demand during such processes as synaptic vesicle recycling, membrane potential maintenance and Ca²⁺ exchange/extrusion. The energy needs of these events are met in large part by mitochondrial production of ATP through the process of oxidative phosphorylation. The job of ATP production by the mitochondria is performed by the F₁F_O ATP synthase, a multi-protein enzyme that contains a membrane-inserted portion, an extra-membranous enzymatic portion and an extensive regulatory complex. Although required for ATP production by mitochondria, recent findings have confirmed that the membrane-confined portion of the c-subunit of the ATP synthase also houses a large conductance uncoupling channel, the mitochondrial permeability transition pore (mPTP), the persistent opening of which produces osmotic dysregulation of the inner mitochondrial membrane, uncoupling of oxidative phosphorylation and cell death. Recent advances in understanding the molecular components of mPTP and its regulatory mechanisms have determined that decreased uncoupling occurs in states of enhanced mitochondrial efficiency; relative closure of mPTP therefore contributes to cellular functions as diverse as cardiac development and synaptic efficacy.     

Lead and calcium produce rod photoreceptor cell apoptosis by opening the mitochondrial permeability transition pore

Calcium overload is suggested to play a fundamental role in the process of rod apoptosis in chemical-induced and inherited retinal degenerations. However, this hypothesis has not been tested directly. We developed an in vitro model utilizing isolated rat retinas to determine the mechanisms underlying Ca(2+)- and/or Pb(2+)-induced retinal degeneration. Confocal microscopy, histological, and biochemical studies established that the elevated [Ca(2+)] and/or [Pb(2+)] were localized to photoreceptors and produced rod-selective apoptosis. Ca(2+) and/or Pb(2+) induced mitochondrial depolarization, swelling, and cytochrome c release. Subsequently caspase-9 and caspase-3 were sequentially activated. Caspase-7 and caspase-8 were not activated. The effects of Ca(2+) and Pb(2+) were additive and blocked completely by the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A, whereas the calcineurin inhibitor FK506 had no effect. The caspase inhibitors carbobenzoxy-Leu-Glu-His-Asp-CH(2)F and carbobenzoxy-Asp-Glu-Val-Asp-CH(2)F, but not carbobenzoxy-Ile-Glu-Thr-Asp-CH(2)F, differentially blocked post-mitochondrial events. The levels of reduced and oxidized glutathione and pyridine nucleotides in rods were unchanged. Our results demonstrate that rod mitochondria are the target site for Ca(2+) and Pb(2+). Moreover, they suggest that Ca(2+) and Pb(2+) bind to the internal metal (Me(2+)) binding site of the PTP and subsequently open the PTP, which initiates the cytochrome c-caspase cascade of apoptosis in rods.     

The mitochondrial megachannel is the permeability transition pore

Single-channel electrophysiological recordings from rat liver mitoplast membranes showed that the 1.3-nS mitochondrial megachannel was activated by Ca⁺⁺ and inhibited by Mg⁺⁺, Cyclosporin A, and ADP, probably acting at matrix-side sites. These agents are known to modulate the so-called mitochondrial permeability transition pore (Gunter, T. E., and Pfeiffer, D. R. (1990)Am. J. Physiol. 258, C755–C786) in the same manner. Furthermore, the megachannel is unselective, and the minimum pore size calculated from its conductance is in agreement with independent estimates of the minimum size of the permeabilization pore. The results support the tentative identification of the megachannel with the pore believed to be involved in the permeabilization process.Abbreviations used: PT: permeability transition; PTP: permeability transition pore; MMC: mitochondrial megachannel; IMAC: inner membrane anion channel. P_A: permeability of ion A. CSP: Cyclosporin A.     

Partial contribution of mitochondrial permeability transition to t-butyl hydroperoxide-induced cell death

Mitochondrial permeability transition (MPT) is thought to determine cell death under oxidative stress. However, MPT inhibitors only partially suppress oxidative stress-induced cell death. Here, we demonstrate that cells in which MPT is inhibited undergo cell death under oxidative stress. When C6 cells were exposed to 250 μM t-butyl hydroperoxide (t-BuOOH), the loss of a membrane potential-sensitive dye (tetramethylrhodamine ethyl ester, TMRE) from mitochondria was observed, indicating mitochondrial depolarization leading to cell death. The fluorescence of calcein entrapped in mitochondria prior to addition of t-BuOOH was significantly decreased to 70% after mitochondrial depolarization. Cyclosporin A suppressed the decrease in mitochondrial calcein fluorescence, but not mitochondrial depolarization. These results show that t-BuOOH induced cell death even when it did not induce MPT. Prior to MPT, lactate production and respiration were hampered. Taken together, these data indicate that the decreased turnover rate of glycolysis and mitochondrial respiration may be as vital as MPT for cell death induced under moderate oxidative stress.     

Role of protein kinase C and mitochondrial permeability transition pore in the neuroprotective effect of ceramide in ischemia-induced cell death

In this study, we investigated the role of protein kinase C (PKC) and mitochondrial permeability transition pore (mPTP) on the effect of ceramide in an in vitro model of ischemia in SH-SY5Y neuroblastoma cells. In ischemic cell viability studies, a dual effect of ceramide was observed, depending on ceramide concentration. PKC isoforms are involved in the protective effect of low concentrations of ceramide. During ischemia, ceramide treatment leads to an increase in the formation of reactive oxygen species (ROS), which induces a controlled opening of mPTP. This fact prevents mitochondrial Ca²⁺ overload, which is clearly protective.     

The role of mitochondrial permeability transition pore in physiology and pathology of the cell

Mitochondrial megachannel, a multiprotein complex, is localized in close contacts of the outer and the inner mitochondrial membranes. It plays important role in many aspects of cell physiology and its opening can have different consequences. This review summarizes the present knowledge about structure and function of the megachannel in the cell.     

Regulation of cyclosporin A sensitive mitochondrial permeability transition by the redox state of pyridine nucleotides

The mechanisms involved in the induction of cyclosporine A sensitive mitochondrial swelling by oxidative stress were investigated in isolated guinea pig liver mitochondria. The aim of our study was to investigate, if swelling is inevitably associated with the oxidation of pyridine nucleotides, and if the oxidized pyridine nucleotides have to be hydrolysed for the induction of mitochondrial swelling. Quantitative measurement of oxidized pyridine nucleotides was performed with HPLC. Mitochondrial swelling was recorded by monitoring the decrease in light scattering of the mitochondrial suspension. Reduction and oxidation of pyridine nucleotides were followed by monitoring the changes of the autofluorescence signal of reduced pyridine nucleotides. Qualitative measurement of mitochondrial membrane potential was performed with the fluorescence indicator rhodamine 123. Neither t-butyl hydroperoxide nor the dissipation of the mitochondrial inner membrane potential with FCCP (carbonyl cyanide-p-trifluoromethoxyphenyl hydrazone) induced the opening of the membrane permeability transition pore, unless an extensive oxidation of mitochondrial pyridine nucleotides took place. Mitochondrial swelling induced by our experimental conditions was always sensitive to cyclosporine A and accompanied by a cyclosporine A sensitive release of inner mitochondrial pyridine nucleotides without pyridine nucleotide hydrolysis. Not the cycling of calcium across the mitochondrial inner membrane but the accumulation of calcium inside the mitochondria was a prerequisite for mitochondrial swelling. The mitochondrial membrane permeability transition is neither caused nor accompanied by the hydrolysis of mitochondrial pyridine nucleotides.