Cannabinoids act as necrosis-inducing factors in Cannabis sativa

Cannabis sativa is well known to produce unique secondary metabolites called cannabinoids. We recently discovered that Cannabis leaves induce cell death by secreting tetrahydrocannabinolic acid (THCA) into leaf tissues. Examinations using isolated Cannabis mitochondria demonstrated that THCA causes mitochondrial permeability transition (MPT) though opening of MPT pores, resulting in mitochondrial dysfunction (the important feature of necrosis). Although Ca²⁺ is known to cause opening of animal MPT pores, THCA directly opened Cannabis MPT pores in the absence of Ca²⁺. Based on these results, we conclude that THCA has the ability to induce necrosis though MPT in Cannabis leaves, independently of Ca²⁺. We confirmed that other cannabinoids (cannabidiolic acid and cannabigerolic acid) also have MPT-inducing activity similar to that of THCA. Moreover, mitochondria of plants which do not produce cannabinoids were shown to induce MPT by THCA treatment, thus suggesting that many higher plants may have systems to cause THCA-dependent necrosis.Key words: cannabinoid, Cannabis sativa, cylophilin D, mitochondrial permeability transition, necrosisCannabis sativa produces unique secondary metabolites consisting of alkylresorcinol and monoterpene groups.¹ These metabolites called cannabinoids are well known to show a variety of interesting pharmacological activities including psychoactive effect and analgesic effect. Therefore, cannabinoids have attracted a great deal of attention, whereas why C. sativa produces such metabolites has long remained unclear. However, we have recently obtained evidences indicating the physiological function of THCA in Cannabis leaves.²We discovered that THCA is stored in capitate-sessile glands on Cannabis leaves and that secretion of this cannabinoid into leaf tissues causes cell death. When the properties of THCA were examined using cultured Cannabis cells, this cannabinoid induced plasmamembrane shrinkage and DNA degradation. These responses are regarded as the features of apoptotic cells, but were not suppressed by apoptosis inhibitors. In contrast, the necrosis inhibitor cyclosporine A significantly inhibited both plasmamembrane shrinkage and DNA degradation in Cannabis cells. Therefore, we assumed that THCA induces necrotic cell death in Cannabis cells and leaves.Necrosis in plants and animals is usually triggered by MPT though opening of MPT pores.^3,4 MPT is known to cause mitochondrial dysfunction by mitochondrial swelling and loss of mitochondrial membrane potential (ΔΨm),^5,6 and we also confirmed that THCA induces mitochondrial swelling and ΔΨm reduction in mitochondria isolated from Cannabis cells and that pretreatment with cyclosporine A inhibits both responses. Based on these evidences, we concluded that THCA has the activity to induce MPT-dependent necrosis.As described above, MPT pores play an important role in necrosis induction, whereas the mechanism of their opening in higher plants has not been fully understood. However, binding of cyclophilin D (a protein present in mitochondrial matrix) to MPT pores is shown to be essential for their opening in plants as well as animal.^7–9 In animal mitochondria, Ca²⁺ mediates this binding reaction, leading to opening of MPT pores. Wheat mitochondria are also shown to undergo swelling through opening of MPT pores in response to Ca²⁺,⁹ whereas MPT pores of oats,¹⁰ Arabidopsis thaliana¹¹ and C. sativa² do not open by Ca²⁺ treatment. In contrast, THCA catalyzed opening of Cannabis MPT pores in the absence of Ca²⁺, suggesting that THCA directly mediates binding of cyclophilin D to MPT pores (Fig. 1). In addition, we have now confirmed that THCA causes dysfunction though MPT in mitochondria of plants (rice, soybean, A. thaliana and Scutellaria baicalensis) lacking cannabinoid-producing ability (data not shown). Therefore, many higher plants may have the systems to induce THCA-dependent necrosis.Open in a separate windowFigure 1A model depicting the opening mechanism of MPT pores in mitochondria. CYD, cyclophilin D; CN, cannabinoid.Furthermore, we investigated whether other cannabinoids and their related compounds can mediate MPT in Cannabis mitochondria. When the MPT-inducing activity of each sample was measured by monitoring both ΔΨm reduction (Fig. 2) and mitochondrial swelling (data not shown), we confirmed that cannabinoids tested here (cannabidiolic acid and cannabigerolic acid) possess the activities similar to those of THCA. On the other hand, olivetolic acid (the akylresorcinol moiety of cannabinoid) and geraniol (the monoterpene moiety of cannabigerolic acid) showed neither ΔΨm reduction nor mitochondrial swelling (Fig. 2). These results suggested that the structures (cannabinoid skeleton) where monoterpene and olivetolic acid are coupled to each other seem essential for opening of MPT pores. Therefore, we assumed that plant cyclophilin D and MPT pores have the cannabinoid-binding site.Open in a separate windowFigure 2Change of ΔΨm by treatment with various compounds (A) and their chemical structures (B). The isolated mitochondria were stained with the ΔΨm-indicating reagent (tetramethylrhodamine methylester, TMRM) and then incubated with 200 µM of each compound for 60 min. The intensity of TMRM fluorescence was measured using a fluorescence microplate reader. A decrease of the fluorescence intensity indicates ΔΨm reduction. CBDA, cannabidiolic acid; CBGA, cannabigerolic acid; OLA, olivetolic acid.Plant cell death is shown to participate in important physiological responses such as leaf senescence, somatic embryogenesis and defense against microbial pathogens.^12,13 Based on its induction mechanism, plant cell death is largely classified into apoptosis and necrosis. Although the molecular mechanism of apoptosis has been extensively investigated, there is little precise information on plant necrosis. However, our study would provide important insight into necrosis-inducing mechanisms in higher plants.     

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.     

Thapsigargin directly induces the mitochondrial permeability transition.

High concentrations of thapsigargin (TG) have been used to study the process of necrotic cell death, which involves mitochondria in the cell rapidly undergoing the mitochondrial permeability transition (MPT). We therefore investigated the effects of TG on MPT in isolated liver and heart mitochondria. Using a matrix swelling assay in combination with a novel enzymatic method based on inner membrane permeability to citrate synthase substrates, TG induced MPT in a concentration-dependent manner, independent of extramitochondrial [Ca2+] and inhibitable by cyclosporin A. Evidence from alamethicin-permeabilized mitochondria suggests that TG induces MPT by causing Ca2+ release from mitochondrial matrix Ca2+-binding sites. These findings suggest that the MPT-inducing effect of TG may contribute to its pro-necrotic and pro-apoptotic effects in various cell types.     

TNF-alpha-induced cell death in ethanol-exposed cells depends on p38 MAPK signaling but is independent of Bid and caspase-8

Alcoholic liver disease is associated with an increase in the number of necrotic and apoptotic liver parenchymal cells. Part of this injury is mediated by TNF-alpha. Ethanol exposure sensitizes cells to the cytotoxic effects of TNF-alpha. This may be due, in part, to the increased propensity of the mitochondria in ethanol-exposed cells to induction of mitochondrial permeability transition (MPT) by various agents, including the proapoptotic protein Bax. This idea is supported by the observation that increased cell death induced by TNF-alpha in ethanol-exposed cells was dependent on development of the MPT. In the present study, we elucidate the pathways through which ethanol exposure enhances TNF-alpha induction of the MPT and the resulting cytotoxicity. Specifically, ethanol-exposed cells display caspase-8- and Bid-independent cell killing during TNF-alpha treatment. Moreover, the ethanol-enhanced pathway is dependent on p38 MAPK signaling, which brings about caspase-3 activation, mitochondrial depolarization, accumulation of cytochrome c in the cytosol, and the translocation of Bax to the mitochondria. Additionally, ethanol-exposed cells display a blunting of TNF-alpha-induced Akt activation and Bcl-2 antagonist of cell death phosphorylation that may account, in part, for the increased sensitivity of the mitochondria to Bax-mediated damage.     

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.     

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.     

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.     

Beta-phenylethyl isothiocyanate mediated apoptosis; contribution of Bax and the mitochondrial death pathway

The initiating events that lead to the induction of apoptosis mediated by the chemopreventative agent beta-phenyethyl isothiocyanate (PEITC) have yet to be elucidated. In the present investigation, we examined the effects of PEITC on mitochondrial function and apoptotic signaling in hepatoma HepG2 cells and isolated rat hepatocyte mitochondria. PEITC induced a conformational change in Bax leading to its translocation to mitochondria in HepG2 cells. Bax accumulation was associated with a rapid loss of mitochondrial membrane potential (Deltapsim), impaired respiratory chain enzymatic activity, release of mitochondrial cytochrome c and the activation of caspase-dependent cell death. Caspase inhibition did not prevent Bax translocation, the release of cytochrome c or the loss of Deltapsim, but blocked caspase-mediated DNA fragmentation and cell death. To determine whether PEITC dependent Bax translocation caused loss of Deltapsim by the activation of the mitochondrial permeability transition (MPT), we examined the effects of PEITC in isolated rat hepatocyte mitochondria. Interestingly, PEITC did not induce MPT in isolated rat mitochondria. Accordingly, using pharmacological inhibitors of MPT namely cyclosporine A, trifluoperazine and Bongkrekic acid we were unable to block PEITC mediated apoptosis in HepG2 cells, this suggesting that mitochondrial permeablisation is a likely consequence of Bax dependent pore formation. Taken together, our data suggest that mitochondria are a key target in PEITC induced apoptosis in HepG2 cells via the pore forming ability of pro-apoptotic Bax.     

Effects of cannabinoids Δ(9)-tetrahydrocannabinol, Δ(9)-tetrahydrocannabinolic acid and cannabidiol in MPP+ affected murine mesencephalic cultures

Cannabinoids derived from Cannabis sativa demonstrate neuroprotective properties in various cellular and animal models. Mitochondrial impairment and consecutive oxidative stress appear to be major molecular mechanisms of neurodegeneration. Therefore we studied some major cannabinoids, i.e. delta-9-tetrahydrocannabinolic acid (THCA), delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in mice mesencephalic cultures for their protective capacities against 1-methyl-4-phenyl pyridinium (MPP(+)) toxicity. MPP(+) is an established model compound in the research of parkinsonism that acts as a complex I inhibitor of the mitochondrial respiratory chain, resulting in excessive radical formation and cell degeneration. MPP(+) (10 μM) was administered for 48 h at the 9th DIV with or without concomitant cannabinoid treatment at concentrations ranging from 0.01 to 10 μM. All cannabinoids exhibited in vitro antioxidative action ranging from 669 ± 11.1 (THC), 16 ± 3.2 (THCA) to 356 ± 29.5 (CBD) μg Trolox (a vitamin E derivative)/mg substance in the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) assay. Cannabinoids were without effect on the morphology of dopaminergic cells stained by tyrosine hydroxylase (TH) immunoreaction. THC caused a dose-dependent increase of cell count up to 17.3% at 10 μM, whereas CBD only had an effect at highest concentrations (decrease of cell count by 10.1-20% at concentrations of 0.01-10 μM). It influenced the viability of the TH immunoreactive neurons significantly, whereas THCA exerts no influence on dopaminergic cell count. Exposure of cultures to 10 μM of MPP(+) for 48 h significantly decreased the number of TH immunoreactive neurons by 44.7%, and shrunken cell bodies and reduced neurite lengths could be observed. Concomitant treatment of cultures with cannabinoids rescued dopaminergic cells. Compared to MPP(+) treated cultures, THC counteracted toxic effects in a dose-dependent manner. THCA and CBD treatment at a concentration of 10 μM lead to significantly increased cell counts to 123% and 117%, respectively. Even though no significant preservation or recovery of neurite outgrowth to control values could be observed, our data show that cannabinoids THC and THCA protect dopaminergic neurons against MPP(+) induced cell death.     

Specific cleavage of ribosomal RNA and mRNA during victorin-induced apoptotic cell death in oat

Here we report that rRNA and mRNA are specifically degraded in oat (Avena sativa L.) cells during apoptotic cell death induced by victorin, a host-selective toxin produced by Cochliobolus victoriae. Northern analysis indicated that rRNA species from the cytosol, mitochondria and chloroplasts were all degraded via specific degradation intermediates during victorin-induced apoptotic cell death but, in contrast, they were randomly digested in necrotic cell death induced by 30 mM CuSO(4) and heat shock. This indicates that specific rRNA cleavage could be controlled by an intrinsic program. We also observed specific cleavage of mRNA of housekeeping genes such as actin and ubiquitin during victorin-induced cell death. Interestingly, no victorin-induced mRNA degradation was detected with stress-responding genes such as PR-1, PR-10 and GPx throughout the experimental period. The RNA degradation mostly, but not always, occurred in parallel with DNA laddering, but pharmacological studies indicated that these processes are regulated by different signaling pathways with some overlapping upstream signals.     

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.     

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.     

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.     

The mitochondrial permeability transition regulates cytochrome c release for apoptosis during endoplasmic reticulum stress by remodeling the cristae junction

The role of the mitochondrial permeability transition (MPT) in apoptosis and necrosis is controversial. Here we show that the MPT regulates the release of cytochrome c for apoptosis during endoplasmic reticulum (ER) stress by remodeling the cristae junction (CJ). CEM cells, HCT116 colon cancer cells, and murine embryo fibroblast cells were treated with the ER stressor thapsigargin (THG), which led to cyclophilin D-dependent mitochondrial release of the profusion GTPase optic atrophy 1 (OPA1), which controls CJ integrity, and cytochrome c, leading to apoptosis. Interference RNA knockdown of Bax blocked OPA1 and cytochrome c release after THG treatment but did not prevent the MPT, showing that Bax was essential for the release of cytochrome c by MPT. In isolated mitochondria, MPT led to OPA1 and cytochrome c release independently of voltage-dependent anion channel and the outer membrane, indicating that the MPT is an inner membrane phenomenon. Last, the MPT was regulated by the electron transport chain but not mitochondrial reactive oxygen species, since THG-induced cell death was not blocked by antioxidants and did not occur in cells lacking mitochondrial DNA. Our results show that the MPT regulates CJ remodeling for cytochrome c-dependent apoptosis induced by ER stress and that mitochondrial electron transport is indispensable for this process.     

The Coenzyme Q10 analog decylubiquinone inhibits the redox-activated mitochondrial permeability transition: role of mitcohondrial [correction mitochondrial] complex III

The mitochondrial permeability transition (MPT) is a key event in apoptotic and necrotic cell death and is controlled by the cellular redox state. To further investigate the mechanism(s) involved in regulation of the MPT, we used diethylmaleate to deplete GSH in HL60 cells and increase mitochondrial reactive oxygen species (ROS) production. The site of mitochondrial ROS production was determined to be mitochondrial respiratory complex III (cytochrome bc1), because 1). stigmatellin, a Qo site inhibitor, blocked ROS production and prevented the MPT and cell death and 2). cytochrome bc1 activity was abolished in cells protected from the redox-dependent MPT by stigmatellin. We next investigated the effect of pretreating cells with coenzyme Q10 analogs decylubiquinone (dUb) and ubiquinone 0 (Ub0) on the redox-dependent MPT. Pretreatment of HL60 cells with dUb blocked ROS production induced by GSH depletion and prevented activation of the MPT and cell death, whereas Ub0 did not. Since we also found that dUb did not inhibit cytochrome bc1 activity, the mechanism of protection against redox-dependent MPT by dUb may depend on its ability to scavenge ROS generated by cytochrome bc1. These results indicate that dUb, like the clinically used ubiquinone analog idebenone, may serve as a candidate antioxidant compound for the development of pharmacological agents to treat diseases where there is an oxidative stress component.     

Heat shock suppresses the permeability transition in rat liver mitochondria

Heat shock proteins inhibit apoptotic and necrotic cell death in various cell types. However, the specific mechanism underlying protection by heat shock proteins remains unclear. To test the hypothesis that heat shock proteins inhibit cell death by blocking opening of mitochondrial permeability transition (MPT) pores, mitochondria from heat-preconditioned rat livers were isolated by differential centrifugation. Heat shock inhibited MPT pore opening induced by 50 microm CaCl(2) plus 5 microm HgCl(2) or 1 microm mastoparan and by 200 microm CaCl(2) alone. Half-maximal swelling was delayed 15 min or more after heat shock compared with control. Heat shock also increased the threshold of unregulated (Ca(2+)-independent and cyclosporin A-insensitive) MPT pore opening induced by higher doses of HgCl(2) and mastoparan. Heat shock treatment decreased mitochondrial reactive oxygen species formation by 27% but did not change mitochondrial respiration, membrane potential, Ca(2+) uptake, or total glutathione in mitochondrial and cytosolic extracts of liver. Western blot analysis showed that mitochondrial Hsp25 increased, whereas Hsp10, Hsp60, Hsp70, Hsp75, cyclophilin D, and voltage-dependent anion channel did not change after heat shock. These results indicate that heat shock causes resistance to opening of MPT pores, which may contribute to heat shock protection against cellular injury.     

Mechanism of 3-nitropropionic acid-induced membrane permeability transition of isolated mitochondria and its suppression by L-carnitine

3-Nitropropionic acid (3NP) functions as an irreversible inhibitor of succinic acid dehydrogenase (complex II) and induces neuronal disorders in rats similar to those in patients with Huntington's disease. It is well known that L-carnitine (LC), a carrier of long chain fatty acid into the mitochondrial matrix, attenuates the neuronal degeneration in 3NP-treated rats. From these findings it has been suggested that 3NP induces certain neuronal cell death through mitochondrial dysfunction and that LC preserves the neurons against the dysfunction of mitochondria caused by 3NP. However, the detailed mechanism of cell death by 3NP and the protective actions of LC against the mitochondrial dysfunction have not been fully elucidated yet. Thus, we studied the molecular mechanism of the effects of 3NP and LC on isolated rat liver mitochondria. 3NP inhibited succinate respiration and the decreased respiratory control ratio of isolated mitochondria without affecting oxidative phosphorylation. 3NP induced a membrane permeability transition (MPT), which plays an important role in the mechanism of apoptotic cell death. 3NP stimulated Ca2+ release from mitochondria, decreased membrane potential, induced mitochondrial swelling, and stimulated cytochrome c release from mitochondria. 3NP-induced swelling was suppressed by bovine serum albumin, inhibitors of phospholipase A(2) and by an inhibitor of classic MPT, cyclosporin A. Furthermore, LC suppressed the changes brought about by 3NP in mitochondrial functions in the presence of ATP. These results suggest that MPT underlies the mechanism of 3NP-induced cell death, and that LC attenuates mitochondrial MPT by decreasing long chain fatty acids generated by phospholipase A(2).     

Poly(ADP-ribose) polymerase-1-mediated cell death in astrocytes requires NAD+ depletion and mitochondrial permeability transition

Extensive activation of poly(ADP-ribose) polymerase-1 (PARP-1) by DNA damage is a major cause of caspase-independent cell death in ischemia and inflammation. Here we show that NAD(+) depletion and mitochondrial permeability transition (MPT) are sequential and necessary steps in PARP-1-mediated cell death. Cultured mouse astrocytes were treated with the cytotoxic concentrations of N-methyl-N'-nitro-N-nitrosoguanidine or 3-morpholinosydnonimine to induce DNA damage and PARP-1 activation. The resulting cell death was preceded by NAD(+) depletion, mitochondrial membrane depolarization, and MPT. Sub-micromolar concentrations of cyclosporin A blocked MPT and cell death, suggesting that MPT is a necessary step linking PARP-1 activation to cell death. In astrocytes, extracellular NAD(+) can raise intracellular NAD(+) concentrations. To determine whether NAD(+) depletion is necessary for PARP-1-induced MPT, NAD(+) was restored to near-normal levels after PARP-1 activation. Restoration of NAD(+) enabled the recovery of mitochondrial membrane potential and blocked both MPT and cell death. Furthermore, both cyclosporin A and NAD(+) blocked translocation of the apoptosis-inducing factor from mitochondria to nuclei, a step previously shown necessary for PARP-1-induced cell death. These results suggest that NAD(+) depletion and MPT are necessary intermediary steps linking PARP-1 activation to AIF translocation and cell death.     

The victorin-induced mitochondrial permeability transition precedes cell shrinkage and biochemical markers of cell death, and shrinkage occurs without loss of membrane integrity

Summary In this study, we determined the timing of events associated with cell death induced by the host-selective toxin, victorin. We show that the victorin-induced collapse in mitochondrial transmembrane potential (Deltapsi(m)), indicative of a mitochondrial permeability transition (MPT), on a per cell basis, did not occur simultaneously in the entire mitochondrial population. The loss of Deltapsi(m) in a predominant population of mitochondria preceded cell shrinkage by 20-35 min. Rubisco cleavage, DNA laddering, and victorin binding to the P protein occurred concomitantly with cell shrinkage. During and following cell shrinkage, tonoplast rupture did not occur, and membranes, including the plasma membrane and tonoplast, retained integrity. Ethylene signaling was implicated upstream of a victorin-induced loss in mitochondrial motility and the collapse in Deltapsi(m). Results suggest that the victorin-induced collapse in Deltapsi(m) is a consequence of an MPT and that the timing of the victorin-induced MPT is poised to influence the cell death response. The retention of plasma membrane and tonoplast integrity during cell shrinkage supports the interpretation that victorin induces an apoptotic-like cell death response.     

The course of etoposide-induced apoptosis in Jurkat cells lacking p53 and Bax

Jurkat T-lymphocytes lack p53 and Bax but contain p73 and Bid and are killed by etoposide (ETO). With ETO c-abl is phosphorylated and phosphorylated p73 increased. Translocation of full-length Bid to mitochondria follows, with induction of the mitochondrial permeability transition (MPT) and release of cytochrome c into the cytosol. Pronounced swelling of mitochondria was evident ultrastructurally, and the MPT inhibitor cyclosporin A prevented the release of cytochrome c. Overexpression of Bcl-2 prevented the translocation of Bid, the release of cytochrome c, and cell death. The pan-caspase inhibitor ZVAD-FMK prevented the cell killing, but not the initial release of cytochrome c. An accumulation of tBid occurred at later times in association with Bid degradation. A sequence is proposed that couples DNA damage to Bid translocation via activation of c-abl and p73. Bid translocation induces the MPT, the event that causes release of cytochrome c, activation of caspases, and cell death.