Dependent Permeability Transition Research Articles

Effect of Talatisamine and its Derivate 14-O-Benzoyltalatisamine on Functional State of Rat Liver and Heart Mitochondria

Dysfunction of the mitochondria of various tissues causes the development of most pathological processes, including ischemia. In recent years, great attention has been paid to the use of plant biologically active substances in the prevention and treatment of pathological processes related to mitochondrial dysfunction. This is very relevant in relation to ischemic diseases and is of scientific and practical importance in the search for new pharmacological agents that correct the functions of damaged mitochondria for their treatment. The mitochondrial permeability transition pore (mPTP) actively participates in the regulation of mitochondrial functions, in the development of various pathological conditions and, at the same time, targets for various drugs and some biologically active substances. In vitro experiments evaluated the effects of alkaloids talatisamine and 14-O-benzoyltalatisamine on rat liver and heart Ca2+-dependent mPTP and lipid peroxidation (LPO) induced by Fe2+/ascorbate system. The investigated diterpenealkaloids inhibited the opening of the Ca2+-dependent mPTP in the membranes of rat liver and heart mitochondria. It was found that 14-O-benzoyltalatizamine inhibits the Ca2+-dependent conductance pore of rat liver and heart mitochondria more strongly than talatisamine. To compare the effects of 14-O-benzoyltalatisamine on rat liver and heart mPTP, concentrations from 1 μM to 200 μM were investigated. At these concentrations, liver mPTP was reliably inhibited by 10% to 81% and heart mPTP by 3.6% to 71.5% relative to control. The high sensitivity of diterpene alkaloids to the Ca2+-dependent permeability transition pore of liver mitochondria compared to heart mitochondria indicates their tissue specificity. The investigated alkaloids exhibited antioxidant properties by inhibiting Fe2+/ascorbate-induced mitochondrial suppression (LPO process) and MDA formation in membranes. LPO induced by Fe2+/ascorbate system in mitochondrial membranes was more actively inhibited by 14-O-benzoyltalatisamine. According to the results of the research, acylation of the hydroxyl group at the C-14 position of talatisamine by benzoyl chloride caused a rise in molecular activity of the derivative due to the introduction of the benzoyl group.

Itaconic acid impairs the mitochondrial function by the inhibition of complexes II and IV and induction of the permeability transition pore opening in rat liver mitochondria

Itaconic acid (methylene-succinic acid, ItA) is an unsaturated dicarboxylic acid that is secreted by mammalian macrophages in response to a pro-inflammatory stimulus and shows an anti-inflammatory/antibacterial effect. Being a mitochondrial metabolite, it exhibits an inhibitory activity on succinate dehydrogenase and subsequently induces mitochondrial dysfunction. The present study has shown that ItA dose-dependently inhibited ADP- and DNP-stimulated (uncoupled) respiration of rat liver mitochondria energized with succinate. This effect of ItA could be related to the suppression of the activity of complex II and the combined activity of complexes II + III of the respiratory chain. At the same time, ItA had no effect on the activity of the dicarboxylate carrier, which catalyzes the transport of succinate across the inner mitochondrial membrane. It was found that 4 mM ItA diminished the rates of ADP- and DNP-stimulated mitochondrial respiration supported by the substrates of complex I glutamate and malate. A study of the effect of ItA on the activity of complexes of the respiratory chain showed that it decreases the activity of complex IV. It was observed that 4 mM ItA inhibited the rate of H2O2 production by mitochondria. At the same time, ItA promoted the opening of the cyclosporin A-sensitive Ca2+-dependent permeability transition pore. The latter was revealed as the decrease in the calcium retention capacity of mitochondria and the stimulation of release of cytochrome c from the organelles. ItA by itself promoted the cytochrome c release from mitochondria. Possible mechanisms of the effect of ItA on mitochondrial function are discussed.

Transport of Ca2+ and Ca2+-dependent permeability transition in heart mitochondria in the early stages of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a progressive skeletal muscle disease that is associated with severe cardiac complications in the late stages. Significant mitochondrial dysfunction is reportedly responsible for the development of cardiomyopathy with age. At the same time, adaptive changes in mitochondrial metabolism in cardiomyocytes were identified in the early stages of DMD. In this work, we evaluate the functioning of calcium transport systems (MCU and NCLX), and MPT pore in the heart mitochondria of young dystrophin-deficient mice. As compared to wild-type animals, heart mitochondria of mdx mice have been found to be more efficient both in respect to Ca2+ uniport and Na+-dependent Ca2+ efflux. The data obtained indicate that the increased rate of Ca2+ uptake by heart mitochondria of mdx mice may be due to an increase in the ratio of MCU and MCUb subunits. In turn, an increase in the rate of Ca2+ efflux from organelles in DMD may be the result of a significant increase in the level of NCLX. Moreover, the heart mitochondria of mdx mice were more resistant to MPT pore opening, which may be due to an increase in the microviscosity of mitochondrial membranes of DMD mice. At the same time, the level of putative MPT pore proteins did not change. The paper discusses the effect of rearrangements of the mitochondrial proteome involved in the transport and accumulation of calcium on the adaptation of this organ to DMD.

Transport of Ca2+ and Ca2+-Dependent Permeability Transition in the Liver and Heart Mitochondria of Rats with Different Tolerance to Acute Hypoxia.

The work examines the kinetic parameters of Ca2+ uptake via the mitochondrial calcium uniporter complex (MCUC) and the opening of the Ca2+-dependent permeability transition pore (MPT pore) in the liver and heart mitochondria of rats with high resistance (HR) and low resistance (LR) to acute hypoxia. We found that the rate of Ca2+ uptake by mitochondria of the liver and heart in HR rats is higher than that in LR rats, which is associated with a higher level of the channel-forming subunit MCU in liver mitochondria of HR rats and a lower content of the dominant-negative channel subunit MCUb in heart mitochondria of HR rats. It was shown that the liver mitochondria of HR rats are more resistant to the induction of the MPT pore than those of LR rats (the calcium retention capacity of liver mitochondria of HR rats was found to be 1.3 times greater than that of LR rats). These data correlate with the fact that the level of F0F1-ATP synthase, a possible structural element of the MPT pore, in the liver mitochondria of HR rats is lower than in LR rats. In heart mitochondria of rats of the two phenotypes, no statistically significant difference in the formation of the MPT pore was revealed. The paper discusses how changes in the expression of the MCUC subunits and the putative components of the MPT pore can affect Ca2+ homeostasis of mitochondria in animals with originally different tolerance to hypoxia and in hypoxia-induced tissue injury.

Transport of Ca2+ and Ca2+-Dependent Permeability Transition in Rat Liver Mitochondria under the Streptozotocin-Induced Type I Diabetes

Although diabetes mellitus is known to be a disease associated with mitochondrial dysfunction, not everything is clear about mitochondrial Ca2+ transport and Ca2+-induced permeability transition in diabetic cells. The objective of this work was to study the operation of MCU and Ca2+-dependent mitochondrial permeabilization in the liver cells of Sprague-Dawley rats under the streptozotocin-induced type I diabetes. It was shown that two weeks after the induction of diabetes, the rate of Ca2+ uptake by the mitochondria of diabetic animals increased ~1.4-fold. The expression of MCU and MICU1 subunits did not change, yet the quantity of dominant-negative MCUb channel subunits was almost twice as lower. The organelles also became more resistant to the induction of CsA-sensitive MPT pore and less resistant to the induction of CsA-insensitive palmitate/Ca2+-induced pore. The mitochondria of diabetic liver cells also showed changes in the lipid matrix of their membranes. The content of fatty acids in the membranes grew, and microviscosity of the lipid bilayer (assessed with laurdan) increased. At the same time, lipid peroxidation (assessed by the production of malonic dialdehyde) was stimulated. The paper discusses the consequences of the diabetes-related changes in mitochondria in the context of cell physiology.

Induction of the Ca2+-Dependent Permeability Transition in Liver Mitochondria by α,ω-Hexadecanedioic Acid is Blocked by Inorganic Phosphate in the Presence of Cyclosporin A

The effect of α,ω-hexadecanedioic acid (HDA) as an inducer of Ca2+-dependent permeability of the inner membrane (pore opening) of isolated rat liver mitochondria was studied in the absence and presence of pore blocker cyclosporin A (CsA) and one of its effective modulator – inorganic phosphate (Pi). It was shown that the addition of HDA at a concentration of 30 µM to Ca2+-loaded mitochondria induces swelling of the organelles, rapid Ca2+ release from the matrix, and almost total drop in Δψ, which indicates the induction of the Ca2+-dependent permeability of the inner membrane. It was found that 1 µM CsA or 1 mM Pi added separately do not affect these effects of HDA. At the same time, in the presence of both CsA and Pi, HDA added to Ca2+-loaded mitochondria does not induce their swelling, Ca2+ release from the matrix, and a drop in Δψ. It is found that unlike HDA, the induction of Ca2+-dependent lipid pore by palmitic acid is not blocked by the combined action of CsA and Pi. On the basis of the obtained data Pi is considered as a blocker of the HDA-induced Ca2+-dependent pore in the presence of CsA. In this case, Pi can not be replaced by a similar permeable anion vanadate. It was established that this effect of Pi was eliminated if mitochondria were incubated with SH-reagents mersalyl (10 nmol/mg protein) and n-ethylmaleimide (200 nmol/mg protein), which are known as Pi-carrier inhibitors. We conclude that the mechanisms of the effects of HDA and palmitic acid as inducers of the Ca2+-dependent permeability of liver mitochondria differ significantly. The Ca2+-dependent effect of HDA can be considered as the formation of a pore sensitive to the combined action of CsA and Pi, while the Ca2+-dependent effect of palmitic acid is the formation of a lipid pore. Possible causes of the blocking action of Pi on the HDA-induced Ca2+-dependent mitochondrial pore are discussed.

Modulation of mitochondrial ion transport by inorganic polyphosphate - essential role in mitochondrial permeability transition pore

Inorganic polyphosphate (polyP) is a biopolymer of phosphoanhydride-linked orthophosphate residues. PolyP is involved in multiple cellular processes including mitochondrial metabolism and cell death. We used artificial membranes and isolated mitochondria to investigate the role of the polyP in mitochondrial ion transport and in activation of PTP. Here, we found that polyP can modify ion permeability of de-energised mitochondrial membranes but not artificial membranes. This permeability was selective for Ba2+ and Ca2+ but not for other monovalent and bivalent cations and can be blocked by inhibitors of the permeability transition pore – cyclosporine A or ADP. Lower concentrations of polyP modulate calcium dependent permeability transition pore opening. Increase in polyP concentrations and elongation chain length of the polymer causes calcium independent swelling in energized conditions. Physiologically relevant concentrations of inorganic polyP can regulate calcium dependent as well calcium independent mitochondrial permeability transition pore opening. This raises the possibility that cytoplasmic polyP can be an important contributor towards regulation of the cell death.

B4G2 Induces Mitochondrial Apoptosis by the ROS-Mediated Opening of Ca2+-Dependent Permeability Transition Pores

Background/Aims: Hepatocellular carcinoma (HCC) is the most common type of liver cancer. At present, only sorafenib is approved to treat HCC. In this study, we found that a 23-hydroxybetulinic acid derivative, B4G2, exhibited potent antiproliferative activity in HCC cell lines. Methods: We used four HCC cell lines (HepG2, HepG2/ADM, Hep3B and Bel-7402) to evaluate the anti-tumour activity and explore underlying mechanisms by which B4G2 induces apoptosis. Results: Among these cell lines, HepG2 showed the highest sensitivity to B4G2. HepG2 cells treated with B4G2 showed a depolarized mitochondrial membrane potential, released cytochrome c, activated caspase-9 and caspase-3 and cleaved poly ADP-ribose polymerase (PARP). However, Z-VAD-FMK, a pan-caspase inhibitor, did not attenuate B4G2-induced apoptosis, implying that the induction of mitochondrial apoptosis by B4G2 may be independent of caspases. Moreover, pre-treatment with MgCl₂, a blocker of Ca²⁺-dependent permeability transition (PT) pores, attenuated the depolarization of the mitochondrial potential and decreased the population of apoptotic cells, indicating that B4G2-induced apoptosis was partly dependent on the opening of the Ca²⁺-dependent PT pores. B4G2 also increased the levels of intracellular calcium and reactive oxygen species (ROS). Furthermore, an ROS scavenger, N-acetyl-cysteine (NAC), markedly decreased the accumulation of intracellular calcium and apoptosis. Conclusion: This is the first demonstration that B4G2 inhibits the growth of HCC cells and induces mitochondrial apoptosis in hepatocellular carcinoma cells by the ROS-mediated opening of Ca²⁺-dependent permeability transition pores.

The influence of spermine on Ca2+-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,ω-hexadecanedioic acids

The effect of spermine on Ca(2+)-dependent permeability transition in mitochondria and liposomes induced by palmitic and α,Ω-hexadecanedioic acid was studied. It has been shown that spermine inhibited the cyclosporin A-insensitive mitochondrial swelling induced by palmitic acid and Ca2+ and α,Ω-hexadecanedioic acid and Ca2+. 100 μM spermine did not influence the mitochondrial respiration in state V2 and the respiration stimulated by palmitic acid, α,Ω-hexadecanedioic acid and Ca2+. Pre-incubation of liposomes with 100 μM spermine resulted in the inhibition of palmitic acid/Ca(2+)- and α,Ω-hexadecanedioic acid/Ca(2+)-induced release of the fluorescent dye sulforhodamine B from liposomes. At the same time, spermine added to fatty acids-contained membranes of liposomes stimulated Ca(2+)-dependent release of sulforhodamine B from liposomes. It was shown that an addition of spermine to liposomes resulted in a significant increase in z-potential of liposomal membranes (from -39.8 mV to -18.6 mV). A possible mechanism of spermine influence on palmitic acid/Ca(2+)- and α,Ω-hexadecanedioic acid/Ca(2+)-induced permeability transition in mitochondria and liposomes is discussed.

Bioenergetic and Antiapoptotic Properties of Mitochondria from Cultured Human Prostate Cancer Cell Lines PC-3, DU145 and LNCaP

The purpose of this work was to reveal the metabolic features of mitochondria that might be essential for inhibition of apoptotic potential in prostate cancer cells. We studied mitochondria isolated from normal prostate epithelial cells (PrEC), metastatic prostate cancer cell lines LNCaP, PC-3, DU145; and non-prostate cancer cells - human fibrosarcoma HT1080 cells; and normal human lymphoblastoid cells. PrEC cells contained 2 to 4 times less mitochondria per gram of cells than the three PC cell lines. Respiratory activities of PrEC cell mitochondria were 5-20-fold lower than PC mitochondria, depending on substrates and the metabolic state, due to lower content and lower activity of the respiratory enzyme complexes. Mitochondria from the three metastatic prostate cancer cell lines revealed several features that are distinctive only to these cells: low affinity of Complex I for NADH, 20-30 mV higher electrical membrane potential (ΔΨ). Unprotected with cyclosporine A (CsA) the PC-3 mitochondria required 4 times more Ca2+ to open the permeability transition pore (mPTP) when compared with the PrEC mitochondria, and they did not undergo swelling even in the presence of alamethicin, a large pore forming antibiotic. In the presence of CsA, the PC-3 mitochondria did not open spontaneously the mPTP. We conclude that the low apoptotic potential of the metastatic PC cells may arise from inhibition of the Ca2+-dependent permeability transition due to a very high ΔΨ and higher capacity to sequester Ca2+. We suggest that due to the high ΔΨ, mitochondrial metabolism of the metastatic prostate cancer cells is predominantly based on utilization of glutamate and glutamine, which may promote development of cachexia.

The permeability transition pore as a Ca2+ release channel: New answers to an old question

Mitochondria possess a sophisticated array of Ca2+ transport systems reflecting their key role in physiological Ca2+ homeostasis. With the exception of most yeast strains, energized organelles are endowed with a very fast and efficient mechanism for Ca2+ uptake, the ruthenium red (RR)-sensitive mitochondrial Ca2+ uniporter (MCU); and one main mechanism for Ca2+ release, the RR-insensitive 3Na+–Ca2+ antiporter. An additional mechanism for Ca2+ release is provided by a Na+ and RR-insensitive release mechanism, the putative 3H+–Ca2+ antiporter. A potential kinetic imbalance is present, however, because the Vmax of the MCU is of the order of 1400nmol Ca2+mg−1 proteinmin−1 while the combined Vmax of the efflux pathways is about 20nmol Ca2+mg−1 proteinmin−1. This arrangement exposes mitochondria to the hazards of Ca2+ overload when the rate of Ca2+ uptake exceeds that of the combined efflux pathways, e.g. for sharp increases of cytosolic [Ca2+]. In this short review we discuss the hypothesis that transient opening of the Ca2+-dependent permeability transition pore may provide mitocondria with a fast Ca2+ release channel preventing Ca2+ overload. We also address the relevance of a mitochondrial Ca2+ release channel recently discovered in Drosophila melanogaster, which possesses intermediate features between the permeability transition pore of yeast and mammals.

Mitochondria from Dipodascus (Endomyces) magnusii and Yarrowia lipolytica yeasts did not undergo a Ca2+-dependent permeability transition even under anaerobic conditions

In this study we used tightly-coupled mitochondria from Yarrowia lipolytica and Dipodascus (Endomyces) magnusii yeasts. The two yeast strains are good alternatives to Saccharomyces cerevisiae, being aerobes containing well structured mitochondria (thus ensuring less structural limitation to observe their appreciable swelling) and fully competent respiratory chain with three invariantly functioning energy conservation points, including Complex I, that can be involved in induction of the canonical Ca²⁺/P(i)-dependent mitochondrial permeability transition (mPTP pore) with an increased open probability when electron flux increases(Fontaine et al. J Biol Chem 273: 25734–25740, 1998; Bernardi et al. FEBS J 273:2077–2099, 2006). High amplitude swelling and collapse of the membrane potential were used as parameters for demonstrating pore opening. Previously (Kovaleva et al. J Bioenerg Biomembr 41:239–249, 2009; Kovaleva et al. Biochemistry (Moscow) 75: 297–303, 2010) we have shown that mitochondria from Y.lipolytica and D. magnusii were very resistant to the Ca²⁺overload combined with varying concentrations of P(i),palmitic acid, SH-reagents, carboxyatractyloside (an inhibitor of ADP/ATP translocator), as well as depletion of intramitochondrial adenine nucleotide pools, deenergization of mitochondria, and shifting to acidic pH values in the presence of high [P(i)]. Here we subjected yeast mitochondria to other conditions known to induce an mPTP in animal and plant mitochondria, namely to Ca²⁺ overload under hypoxic conditions (anaerobiosis). We were unable to observe Ca²⁺-induced high permeability of the inner membrane of D. magnusii and Y. lipolytica yeast mitochondria under anaerobic conditions, thus suggesting that an mPTP-like pore, if it ever occurs in yeast mitochondria, is not coupled with the Ca²⁺ uptake. The results provide the first demonstration of ATP-dependent energization of yeast mitochondria under conditions of anaerobiosis.

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

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

Mitochondrial Ca2+ transport and permeability transition in zebrafish (Danio rerio)

We have studied mitochondrial Ca2+ transport and the permeability transition (PT) in the teleost zebrafish (Danio rerio), a key model system for human diseases. Permeabilized zebrafish embryo cells displayed a mitochondrial energy-dependent Ca2+ uptake system that, like the Ca2+ uniporter of mammals, was inhibited by ruthenium red. Zebrafish mitochondria underwent a Ca2+-dependent PT that displayed Pi-dependent desensitization by cyclosporin A, and responded appropriately to key modulators of the mammalian PT pore (voltage, pH, ubiquinone 0, dithiol oxidants and cross linkers, ligands of the adenine nucleotide translocator, arachidonic acid). Opening of the pore was documented in intact cells, where it led to death that could largely be prevented by cyclosporin A. Our results represent a necessary step toward the use of zebrafish for the screening and validation of PTP inhibitors of potential use in human diseases, as recently shown for collagen VI muscular dystrophy [Telfer et al., 2010].

Ca2+-dependent permeability transition regulation in rat brain mitochondria by 2′,3′-cyclic nucleotides and 2′,3′-cyclic nucleotide 3′-phosphodiesterase

Recent evidence indicates that 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP), a marker enzyme of myelin and oligodendrocytes, is also present in neural and nonneural mitochondria. However, its role in mitochondria is still completely unclear. We found CNP in rat brain mitochondria and studied the effects of CNP substrates, 2',3'-cyclic nucleotides, on functional parameters of rat brain mitochondria. 2',3'-cAMP and 2',3'-cNADP stimulated Ca(2+) overload-induced Ca(2+) release from mitochondrial matrix. This Ca(2+) release under threshold Ca(2+) load correlated with membrane potential dissipation and mitochondrial swelling. The effects of 2',3'-cyclic nucleotides were suppressed by cyclosporin A, a potent inhibitor of permeability transition (PT). PT development is a key stage in initiation of apoptotic mitochondria-induced cell death. 2',3'-cAMP effects were observed on the functions of rat brain mitochondria only when PT was developed. This demonstrates involvement of 2',3'-cAMP in PT regulation in rat brain mitochondria. We also discovered that, under PT development, the specific enzymatic activity of CNP was reduced. Thus we hypothesize that suppression of CNP activity under threshold Ca(2+) load leads to elevation of 2',3'-cAMP levels that, in turn, promote PT development in rat brain mitochondria. Similar effects of 2',3'-cyclic nucleotides were observed in rat liver mitochondria. Involvement of CNP in PT regulation was confirmed in experiments using mitochondria from CNP-knockdown oligodendrocytes (OLN93 cells). CNP reduction in these mitochondria correlated with lowering the threshold for Ca(2+) overload-induced Ca(2+) release. Thus our results reveal a new function for CNP and 2',3'-cAMP in mitochondria, being a regulator/promotor of mitochondrial PT.

Quantitative evaluation of the effects of mitochondrial permeability transition pore modifiers on accumulation of calcium phosphate: comparison of rat liver and brain mitochondria

Mitochondria play a critical role in some forms of apoptosis, and the Ca 2+-dependent permeability transition (PT) is a key initiator of this process. We quantitatively examined major control mechanisms of PT in rat brain (RBM) and liver (RLM) mitochondria. Compared with RLM, RBM were less sensitive to cyclosporin A (CsA), but the combined action of CsA + ADP was much more pronounced in RBM. Carboxyatractyloside abrogated the effects of all mPTP inhibitors in RBM but not in RLM, where the effects of CsA were not reduced. Estimated H +/Ca 2+ ratios were 0.81 ± 0.01 for RLM and 0.84 − 0.93 for RBM, suggesting that Ca 2+ and Pi were sequestered in the matrix as CaHPO 4 and Ca 3(PO 4) 2 salts, and that RBM sequester more CaPi as the least soluble salt. We conclude that: (1) RBM and RLM differ in their baseline behavior of the PT and in their responses to PT modifiers, and (2) PT modifiers can be functionally divided into those which directly affect the mitochondrial PT pore and are not energy-dependent (CsA, free Ca 2+, ADP ex, and Mg 2+), and those which affect the energy-dependent calcium phosphate sequestration process (ADP mt, CATR, local anesthetics). We also conclude that ANT affects PT by changing mitochondrial capacity for energization.

Ca(2+)-dependent permeability transition and complex I activity in lymphoblast mitochondria from normal individuals and patients with Huntington's or Alzheimer's disease.

Annals of the New York Academy of SciencesVolume 893, Issue 1 p. 365-368 Ca2+-Dependent Permeability Transition and Complex I Activity in Lymphoblast Mitochondria from Normal Individuals and Patients with Huntington's or Alzheimer's Disease ALEXANDER PANOV, Corresponding Author ALEXANDER PANOV Dpartment of Neurology, Emory University, Atlanta, Georgia 30322, USA Address for correspondence: Alexander Panov, Department of Neurology, Emory University, 1639 Pierce Drive, WMB Suite 6000, Atlanta, GA 30322. Phone: 404-727-9799; fax: 404-727-3157. e-mail: apanov@emory.eduSearch for more papers by this authorTRACY OBERTONE, TRACY OBERTONE Dpartment of Neurology, Emory University, Atlanta, Georgia 30322, USASearch for more papers by this authorJULIE BENNETT-DESMELIK, JULIE BENNETT-DESMELIK Dpartment of Neurology, Emory University, Atlanta, Georgia 30322, USASearch for more papers by this authorJ. TIMOTHY GREENAMYRE, J. TIMOTHY GREENAMYRE Dpartment of Neurology, Emory University, Atlanta, Georgia 30322, USASearch for more papers by this author ALEXANDER PANOV, Corresponding Author ALEXANDER PANOV Dpartment of Neurology, Emory University, Atlanta, Georgia 30322, USA Address for correspondence: Alexander Panov, Department of Neurology, Emory University, 1639 Pierce Drive, WMB Suite 6000, Atlanta, GA 30322. Phone: 404-727-9799; fax: 404-727-3157. e-mail: apanov@emory.eduSearch for more papers by this authorTRACY OBERTONE, TRACY OBERTONE Dpartment of Neurology, Emory University, Atlanta, Georgia 30322, USASearch for more papers by this authorJULIE BENNETT-DESMELIK, JULIE BENNETT-DESMELIK Dpartment of Neurology, Emory University, Atlanta, Georgia 30322, USASearch for more papers by this authorJ. TIMOTHY GREENAMYRE, J. TIMOTHY GREENAMYRE Dpartment of Neurology, Emory University, Atlanta, Georgia 30322, USASearch for more papers by this author First published: 06 February 2006 https://doi.org/10.1111/j.1749-6632.1999.tb07856.xCitations: 18Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume893, Issue1OXIDATIVE/ENERGY METABOLISM IN NEURODEGENERATIVE DISORDERSNovember 1999Pages 365-368 RelatedInformation

Reoxygenation-Induced Mitochondrial Damage is Caused by the Ca 2-Dependent Mitochondrial Inner Membrane Permeability Transition

Anoxia/reoxygenation injury of isolated rat liver mitochondria was investigated. During anoxia of up to 60 min, the membrane potential was largely preserved and mitochondrial swelling was not observed. Reoxygenation of anoxic mitochondria rapidly caused swelling, cyclosporin A-sensitive Ca 2+ efflux, [ 14C]sucrose trapping, and loss of the membrane potential along with increased generation of reactive oxygen intermediates (ROI). Although pretreatment with catalase and superoxide dismutase completely abolished reoxygenation-induced generation of ROI, mitochondrial damage was not prevented, as indicated by swelling, loss of the membrane potential, a decrease of the ATP content, and cyclosporin A-sensitive Ca 2+ efflux. However, addition of the immunosuppressant cyclosporin A or addition of ADP completely prevented the mitochondrial damage induced by reoxygenation. The same protective effect was noted when Ca 2+ cycling was prevented, either by chelating Ca 2+ with EGTA or by inhibiting Ca 2+ reuptake with ruthenium red. These findings indicate that mitochondrial anoxia/reoxygenation injury is caused by the cyclosporin A-sensitive and Ca 2+-dependent membrane permeability transition. In contrast, reoxygenation injury does not appear to be triggered by the enhanced production of ROI.

The Permeability Transition Pore Opening in Intact Mitochondria and Submitochondrial Particles

The mitochondrial permeability transition was investigated under both oxidative and nonoxidative conditions. It was observed that dithiothreitol (DTT) was able to inhibit the permeability transition only when an oxidant, t -butylhydroperoxide, was used. Although cyclosporin A (CsA) showed also a partial protective effect under these conditions, it progressively lost its ability as the oxidant concentration was increased. Indeed, CsA and ADP were very effective under nonoxidative conditions where Ca2+ and Pi were used to induce the permeability transition, and no effect of DTT was observed. These results suggest that the Ca2+-dependent permeability transition pore opening is not directly dependent of dithiol oxidation. It was also shown here that CsA, independent of the presence of ADP, was able to restore the mitochondrial membrane electrical potential (Δψ) after the Ca2+-induced collapse. Moreover, carboxyatractyloside (CAT) did not prevent the effect of CsA, even when previously added, although it completely abolished the protective effect of ADP, indicating the participation of the ADP/ATP carrier on this process. The data with submitochondrial particles, besides providing further support to the existence of two distinct binding sites for Ca2+ in the mitochondrial inner membrane, with opposite effects on the pore opening probability, demonstrated, for the first time, that very low Ca2+ concentrations induced the permeability transition pore (PTP) opening in submitochondrial particles, an event fully prevented by CsA. The existence of such CsA-sensitive Ca2+-induced pore in submitochondrial particles also suggests that matrix cyclophilin is probably not the mediator of this process.

Thyroxine induces cyclosporin A-insensitive, Ca 2+-dependent reversible permeability transition pore in rat liver mitochondria

The effect of thyroxine on Ca 2+-dependent mitochondrial permeability transition has been examined. It is shown that 40 μM thyroxine induces high amplitude swelling and decrease in membrane potential in Ca 2+-loaded rat liver mitochondria, both in the presence and absence of cyclosporin A. Thyroxine-induced decrease in membrane potential is partially or completely reversed by addition of EGTA into the incubation medium. Nigericin and ADP are shown to prevent, or significantly delay, the effects of thyroxine on both mitochondrial swelling and membrane potential, whereas nicotinamide potentiates the permeabilisation of mitochondria. It is suggested that thyroxine induced reversible, cyclosporin A-insensitive permeability transition pore (PTP) opening in the inner mitochondrial membrane.