Raffaele Colonna

Transient and Long-Lasting Openings of the Mitochondrial Permeability Transition Pore Can Be Monitored Directly in Intact Cells by Changes in Mitochondrial Calcein Fluorescence

The occurrence and the mode of opening of the mitochondrial permeability transition pore (MTP) were investigated directly in intact cells by monitoring the fluorescence of mitochondrial entrapped calcein. When MH1C1 cells and hepatocytes were loaded with calcein AM, calcein was also present within mitochondria, because (i) its mitochondrial signal was quenched by the addition of tetramethylrhodamine methyl ester and (ii) calcein-loaded mitochondria could be visualized after digitonin permeabilization. Under the latter condition, the addition of Ca2+ induced a prompt and massive release of the accumulated calcein, which was prevented by CsA, indicating that calcein release could, in principle, probe MTP opening in intact cells as well. To study this process, we developed a procedure by which the cytosolic calcein signal was quenched by Co2+. In hepatocytes and MH1C1 cells coloaded with Co2+ and calcein AM, treatment with MTP inducers caused a rapid, though limited, decrease in mitochondrial calcein fluorescence, which was significantly reduced by CsA. We also observed a constant and spontaneous decrease in mitochondrial calcein fluorescence, which was completely prevented by CsA. Thus MTP likely fluctuates rapidly between open and closed states in intact cells.

On the effects of paraquat on isolated mitochondria. Evidence that paraquat causes opening of the cyclosporin A-sensitive permeability transition pore synergistically with nitric oxide

This paper reports an investigation on the effects of the bipyridylium herbicide, paraquat, on rat liver mitochondria in vitro. We show that paraquat induces a Ca(2+)-dependent permeability increase of the inner mitochondrial membrane leading to membrane depolarization, uncoupling and matrix swelling. The permeability increase is not observed in the absence of Ca2+ accumulation, and is not due to a direct effect of paraquat on the membrane energy level, as assessed by measurements of membrane potential, respiration and mitochondrial permeability to solutes at high concentrations of paraquat in the presence of excess ethylene-bis(oxoethylenenitrilo)tetraacetic acid (EGTA), a Ca2+ chelator. The Ca(2+)-dependent permeability increase is due to inappropriate opening of the endogenous permeability transition pore (MTP), a regulated, voltage-dependent channel of the inner mitochondrial membrane. The pore is primarily affected by paraquat through a shift of the gating potential to more negative values, allowing pore opening at physiological membrane potential. This effect apparently involves oxidation of a critical dithiol in the pore voltage sensor, while other regulatory aspects of the MTP (matrix pH and Ca2+) are unaffected by paraquat, which is not transported inside the mitochondrial matrix. The effects of paraquat on MTP opening depend on inhibition of electron transfer at Site I by rotenone, or by respiratory chain inhibition by nitric oxide, one of the proposed endogenous mediators of paraquat toxicity to the lung (Berisha, H.I., Hedayatollah, P., Absood, A., and Said, S.I. (1994) Proc. Natl. Acad. Sci. USA 91, 7445-7449). Taken together, these data provide an additional biochemical mechanism by which paraquat may affect cell function, and support the idea that mitochondrial damage is an important determinant in paraquat toxicity (Hirai, K.-I., Ikeda, K., and Wang, G.-Y. (1992) Toxicology 72, 1-16).

Chloromethyltetramethylrosamine (Mitotracker OrangeTM) Induces the Mitochondrial Permeability Transition and Inhibits Respiratory Complex I IMPLICATIONS FOR THE MECHANISM OF CYTOCHROME cRELEASE

We have investigated the interactions with isolated mitochondria and intact cells of chloromethyltetramethylrosamine (CMTMRos), a probe (Mitotracker OrangeTM) that is increasingly used to monitor the mitochondrial membrane potential (Δψm) in situ. CMTMRos binds to isolated mitochondria and undergoes a large fluorescence quenching. Most of the binding is energy-independent and can be substantially reduced by sulfhydryl reagents. A smaller fraction of the probe is able to redistribute across the inner membrane in response to a membrane potential, with further fluorescence quenching. Within minutes, however, this energy-dependent fluorescence quenching spontaneously reverts to the same level obtained by treating mitochondria with the uncoupler carbonylcyanide-p-trifluoromethoxyphenyl hydrazone. We show that this event depends on inhibition of the mitochondrial respiratory chain at complex I and on induction of the permeability transition pore by CMTMRos, with concomitant depolarization, swelling, and release of cytochrome c. After staining cells with CMTMRos, depolarization of mitochondriain situ with protonophores is accompanied by changes of CMTMRos fluorescence that range between small and undetectable, depending on the probe concentration. A lasting decrease of cellular CMTMRos fluorescence associated with mitochondria only results from treatment with thiol reagents, suggesting that CMTMRos binding to mitochondria in living cells largely occurs at SH groups via the probe chloromethyl moiety irrespective of the magnitude of Δψm. Induction of the permeability transition precludes the use of CMTMRos as a reliable probe of Δψm in situ and demands a reassessment of the conclusion that cytochromec release can occur without membrane depolarization and/or onset of the permeability transition.

Induction of the mitochondrial permeability transition by N-ethylmaleimide depends on secondary oxidation of critical thiol groups. Potentiation by copper-ortho-phenanthroline without dimerization of the adenine nucleotide translocase

Addition to energized rat liver mitochondria of low micromolar concentrations of the thiol oxidant, copper-o-phenanthroline [Cu(OP)2], causes opening of the permeability transition pore, a cyclosporin A-sensitive channel. The effects of Cu(OP)2 can be reversed by reduction with dithiothreitol (DTT), suggesting that a dithiol-disulfide interconversion is involved. However, at variance with all pore inducers known to act through dithiol oxidation, the effects of Cu(OP)2 are not prevented by treatment of mitochondria with low (10-20 microM) concentrations of N-ethylmaleimide (NEM). Rather, these concentrations of NEM potentiate the inducing effects of Cu(OP)2. We show that this enhancing effect of NEM is blocked by the subsequent addition of DTT, indicating that potentiation by NEM is mediated by an oxidative event rather than by substitution as such. We find that also pore induction by high (0.5-1.0 mM) concentrations of NEM in the absence of oxidants is completely blocked by reduction with DTT or beta-mercaptoethanol. These results underscore the unexpected importance of oxidative events in pore opening by substituting agents. Since we find that pore opening by Cu(OP)2 or by high concentrations of NEM is not accompanied by dimerization of the adenine nucleotide translocase, we conclude that the translocase itself is not the target of the pore-inducing oxidative events triggered by Cu(OP)2 and NEM.

Effects of fatty acids on mitochondria: implications for cell death

Fatty acids have prominent effects on mitochondrial energy coupling through at least three mechanisms: (i) increase of the proton conductance of the inner mitochondrial membrane; (ii) respiratory inhibition; (iii) opening of the permeability transition pore (PTP). Furthermore, fatty acids physically interact with membranes and possess the potential to alter their permeability; and they are also excellent respiratory substrates that feed electrons into the respiratory chain. Due to the complexity of their actions, the effects of fatty acids on mitochondrial function in situ are difficult to predict. We have investigated the mitochondrial and cellular effects of fatty acids of increasing chain length and degree of unsaturation in relation to their potential to affect mitochondrial function in situ and to cause cell death. We show that saturated fatty acids have little effect on the mitochondrial membrane potential in situ, and display negligible short-term cytotoxicity for Morris Hepatoma 1C1 cells. The presence of double bonds increases both the depolarizing effects and the cytotoxicity, but these effects are offset by the hydrocarbon chain length, so that more unsaturations are required to observe an effect as the hydrocarbon chain length is increased. With few exceptions, depolarization and cell death are due to opening of the PTP rather than to the direct effects of fatty acids on energy coupling.

The Mitochondrial Effects of Small Organic Ligands of BCL-2 SENSITIZATION OF BCL-2-OVEREXPRESSING CELLS TO APOPTOSIS BY A PYRIMIDINE-2,4,6-TRIONE DERIVATIVE

We have investigated the mitochondrial effects of BH3I-2′, Chelerythrine, and HA14-1, small organic molecules that share the ability to bind the BH3 domain of BCL-2. All compounds displayed a biphasic effect on mitochondrial respiration with uncoupling at low concentrations and respiratory inhibition at higher concentrations, the relative uncoupling potency being BH3I-2′ (half-maximal uncoupling at about 80 nm) > Chelerythrine (half-maximal uncoupling at about 2 μm) > HA14-1 (half-maximal uncoupling at about 20 μm). At concentrations lower than required for uncoupling all compounds sensitized the permeability transition pore (PTP) to opening both in isolated mitochondria and intact cells. To assess whether the effects on BCL-2 binding, PTP induction and respiration could be due to different structural determinants we have tested a set of HA14-1 analogs from the Hoffmann-La Roche chemical library. We have identified 5-(6-chloro-2,4-dioxo-1,3,4,10-tetrahydro-2H-9-oxa-1,3-diaza-anthracen-10-yl)-pyrimidine-2,4,6-trione (EM20-25) as a molecule devoid of effects on respiration that is able to induce PTP opening, to disrupt the BCL-2/BAX interactions in situ and to activate caspase-9 in BCL-2-overexpressing cells. EM20-25 neutralized the antiapoptotic activity of overexpressed BCL-2 toward staurosporine and sensitized BCL-2-expressing cells from leukemic patients to the killing effects of staurosporine, chlorambucil, and fludarabine. These results provide a proof of principle that the potentially toxic effects of BCL-2 ligands on mitochondrial respiration are not essential for their antiapoptotic activity and represent an important step forward in the development of tumor-selective drugs acting on BCL-2.

[5] Preparation of bovine heart mitochondria in high yield

Publisher Summary Heart muscle has two distinct advantages with respect to liver as a source of mitochondria. The first advantage is that the heart muscle tissue can be obtained in large amounts, so that several grams of mitochondrial proteins can be isolated during a single isolation procedure. Second, heart muscle mitochondria are considerably more resistant than liver mitochondria to aging. The reason for this resistance is not completely clear but is presumably related to the higher degree of release of fatty acids during storage in the case of liver mitochondria. Heart muscle mitochondria are preferred for the preparation of submitochondrial particles and coupling factors. This chapter discusses the procedure for preparing heart muscle mitochondria. . The equipment required for the procedures includes an electric meat grinder, the plate holes of which are 4–5 mm (250 rpm), a Braun blender, which possesses three speeds (5,000; 8,000; 10,000 rpm), and a one liter glass container: power 400 W, and a glass–Teflon homogenizer.

Perspectives on the mitochondrial permeability transition

Abstract The permeability transition, a sudden permeability increase of the inner mitochondrial membrane that is greatly favored by Ca2+ accumulation, has puzzled mitochondrial scientists for more than 40 years. It is now recognized that this phenomenon is mediated by opening a high conductance channel (the mitochondrial permeability transition pore) whose open-closed transitions are highly regulated. Through the pore mitochondria may participate in intracellular signalling, and release proteins involved in amplification of the cell death cascade triggered by a variety of physiological and pathological stimuli. Yet, the basic questions of the molecular nature of the permeability transition pore, its physiological role and its very occurrence in vivo remain a matter of intense debate. This short review is meant to summarize our current views on the mitochondrial permeability transition, its perspectives, and our strategies to resolve at least some of the outstanding issues about its nature and function.

Lipid interaction of diphtheria toxin and mutants with altered fragment B

The interaction of diphtheria toxin and its cross-reacting mutants crm 45,228 and 1001 with small unilamellar vesicles has been followed by a turbidity assay, electron microscopy, fluorescence energy transfer and membrane permeability. All toxins at pH lower than 6 induce the aggregation and fusion of liposomes containing negatively charged phospholipids; crm 45 and crm 1001 are less potent than diphtheria toxin. Isolated diphtheria toxin fragment B is very effective while isolated fragment A is ineffective. Liposome fusion induced by the toxins at low pH occurs without release of the internal content implying that fusion does not involve vesicle breakage and resealing. The pH dependence of the membrane interaction of diphtheria toxin monitored by turbidity is in close agreement with that monitored by fluorescence energy transfer. It shows that diphtheria toxin can alter the lipid bilayer structure in the pH interval 5-6. This pH range occurs in endosomes and suggests that histidyl and carboxyl residues are likely to be involved in the conformational change of diphtheria toxin triggered by acidic pH.

Covalent binding of arylazido derivatives of cytochrome c to cytochrome oxidase.

The interaction among proteins in biological membranes is an important phenomenon responsible for a number of membrane catalyzed reactions. Crosslinking reagents and spectroscopic techniques have been employed to study nearest neighbor interactions and distances between proteins in membranes [l-3]. Cross-linking of proteins, while largely employed in membrane studies, suffers from the disadvantage that the reactive groups of the reagents select the target groups of the proteins on the basis of their chemical nature and reactivity [2]. Consequently to this, cross-linking of membrane proteins is a function of two parameters, i.e. vicinity and availability of specific chemical groups. In contrast photoactivation of arylazido derivatives yields nitrenes of very broad specificity [4,5]. This in turn allows one to circumvent the problem of specific group availability at the surface of neighbouring proteins and to define their interactions with less uncertainty. The proteins chosen in this study were horse-heart cytochrome c, and beef-heart cytochrome oxidase. The data suggest that cytochrome c labeled at lysyl 13 residue with a 4-nitrophenylazide group forms a covalent complex with the polypeptide of mol. wt 23 700 (band II) of cytochrome oxidase. When the label is located at lysyl 22 residue, no covalent protein binding is observed. 2. Materials and methods