Biofactors - Volume 8, issue 3-4

Authors: Wallimann, Theo | Dolder, Max | Schlattner, Uwe | Eder, Michael | Hornemann, Thorsten | O’Gorman, Eddie | Rück, Alex | Brdiczka, Dieter

Article Type: Research Article

Abstract: Creatine kinase (CK) isoenzymes, specifically located at places of energy demand and energy production, are linked by a phosphocreatine/creatine (PCr/Cr) circuit, found in cells with intermittently high energy demands. Cytosolic CKs, in close conjunction with Ca^{2+} ‐pumps, play a crucial role for the energetics of Ca^{2+} ‐homeostasis. Mitochondrial Mi‐CK, a cuboidal‐shaped octamer with a central channel, binds and crosslinks mitochondrial membranes and forms a functionally coupled microcompartment with porin and adenine nucleotide translocase for vectorial export of PCr into the cytosol. The CK system is regulated by AMP‐activated protein kinase via PCr/Cr and ATP/AMP ratios. Mi‐CK stabilizes and …cross‐links cristae‐ or inner/outer membranes to form parallel membrane stacks and, if overexpressed due to creatine depletion or cellular energy stress, forms those crystalline intramitochondrial inclusions seen in some mitochondrial cytopathy patients. Mi‐CK is a prime target for free radical damage by peroxynitrite. Mi‐CK octamers, together with CK substrates have a marked stabilizing and protective effect against mitochondrial permeability transition pore opening, thus providing a rationale for creatine supplementation of patients with neuromuscular and neurodegenerative diseases. Show more

Keywords: Creatine kinase (CK), phosphocreatine shuttle, energetics of Ca^{2+}‐homeostasis, CK null mutant transgenic mice, mitochondrial creatine kinase (Mi‐CK), intramitochondrial inclusions, mitochondrial myopathies, AMP‐activated protein kinase, mitochondrial permeability transition, peroxynitrite, porin, adenine nucleotide translocase (ANT), cell‐ and neuroprotective effects of creatine, creatine supplementation, neuromuscular diseases

Citation: Biofactors, vol. 8, no. 3-4, pp. 229-234, 1998

Authors: Brdiczka, Dieter | Beutner, Gisela | Rück, Alexander | Dolder, Max | Wallimann, Theo

Article Type: Research Article

Abstract: Contact sites between the outer and peripheral inner membrane of mitochondria are involved in protein precursor uptake and energy transfer. Hexokinase and mitochondrial creatine kinase could be attributed by different techniques to the energy transfer contacts. Kinetic analyses suggested a functional interaction between the kinases, outer membrane pore protein, and inner membrane adenylate translocator (ANT). This suggestion was strongly supported by isolation of hexokinase and creatine kinase complexes that were constituted of kinase oligomers, porin and ANT. Phospholipid vesicles carrying reconstituted kinase–porin–ANT complexes enclosed internal ATP in contrast to vesicles containing free porin only. This indicated that unspecific transport through …porin was regulated by its interaction with a specific antiporter, ANT. A direct interaction between porin and ANT in the hexokinase complex conferred the reconstituted system with permeability properties reminiscent of the mitochondrial permeability transition (PT) pore. In the creatine kinase complex this interaction between porin and ANT was replaced by contact of both with the kinase octamer. Thus PT‐pore‐like functions were not observed unless the creatine kinase octamer was dissociated, suggesting that the ANT was locked in the antiporter state by interaction with the octamer. Indeed, reconstituted pure ANT showed PT‐pore‐like properties concerning Ca^{2+} sensitivity. However, as cyclophilin was missing, sensitivity against cyclosporin was not observed. Show more

Citation: Biofactors, vol. 8, no. 3-4, pp. 235-242, 1998

Authors: Pinton, Paolo | Brini, Marisa | Bastianutto, Carlo | Tuft, Richard A. | Pozzan, Tullio | Rizzuto, Rosario

Article Type: Research Article

Abstract: The possibility of specifically addressing recombinant probes to mitochondria is a novel, powerful way to study these organelles within living cells. We first showed that the Ca^{2+} ‐sensitive photoprotein aequorin, modified by the addition of a mitochondrial targeting sequence, allows to monitor specifically the Ca^{ 2+} concentration in the mitochondrial matrix ([Ca^{2+} ]_\mathrm{m} ) of living cells. With this tool, we could show that, upon physiological stimulation, mitochondria undergo a major rise in [Ca^{2+} ]_\mathrm{m} , well in the range of the Ca^{2+} sensitivity of the matrix dehydrogenases, in a wide …variety of cell types, ranging from non excitable, e.g., HeLa and CHO, and excitable, e.g., cell lines to primary cultures of various embryological origin, such as myocytes and neurons. This phenomenon, while providing an obvious mechanism for tuning mitochondrial activity to cell needs, appeared at first in striking contrast with the low affinity of mitochondrial Ca^{2+} uptake mechanisms. Based on indirect evidence, we proposed that the mitochondria might be close to the source of the Ca^{2+} signal and thus exposed to microdomains of high [Ca^{2+} ], hence allowing the rapid accumulation of Ca^{2+} into the organelle. In order to verify this intriguing possibility, we followed two approaches. In the first, we constructed a novel aequorin chimera, targeted to the mitochondrial intermembrane space (MIMS), i.e., the region sensed by the low‐affinity Ca^{2+} uptake systems of the inner mitochondrial membrane. With this probe, we observed that, upon agonist stimulation, a portion of the MIMS is exposed to saturating Ca^{2+} concentrations, thus confirming the occurrence of microdomains of high [Ca^{ 2+} ] next to mitochondria. In the second approach, we directly investigated the spatial relationship of the mitochondria and the ER, the source of agonist‐releasable Ca^{2+} in non‐excitable cells. For this purpose, we constructed GFP‐based probes of organelle structure; namely, by targeting to these organelles GFP mutants with different spectral properties, we could label them simultaneously in living cells. By using an imaging system endowed with high speed and sensitivity, which allows to obtain high‐resolution 3D images, we could demonstrate that close contacts (< 80 nm) occur in vivo between mitochondria and the ER. Show more

Citation: Biofactors, vol. 8, no. 3-4, pp. 243-253, 1998

Authors: Maechler, Pierre | Wollheim, Claes B.

Article Type: Research Article

Abstract: The control of insulin secretion from the pancreatic \beta ‐cell involves a complex cascade of events in which the mitochondria play a central role. In the consensus model this role is essentially restricted to the production of ATP promoting membrane depolarisation and a rise in cytosolic [Ca^{2+} ]. Evidence for the generation of an additional mitochondrial factor implicated in metabolism‐secretion coupling is provided in this review. Although not yet identified, the formation of this putative factor requires an increase of [Ca^{2+} ] in the mitochondrial matrix together with a supply of carbons to the tricarboxylic acid (TCA) …cycle. In this model, calcium activates matrix dehydrogenases, in particular those of the TCA cycle. This enables the synthesis of the mitochondrial factor from the TCA cycle intermediates. Experimental evidence gathered in permeabilised cells largely supports this model. Show more

Citation: Biofactors, vol. 8, no. 3-4, pp. 255-262, 1998

Authors: Petronilli, Valeria | Miotto, Giovanni | Canton, Marcella | Colonna, Raffaele | Bernardi, Paolo | Di Lisa, Fabio

Article Type: Research Article

Abstract: The involvement of mitochondrial permeability transition pore (MTP) in cellular processes is generally investigated by indirect means, such as changes in mitochondrial membrane potential or pharmacological inhibition. However, such effects could not be related univocally to MTP. In addition, source of errors could be represented by the increased retention of membrane potential probes induced by cyclosporin A (CsA) and the interactions between fluorescent probes. We developed a direct technique for monitoring MTP. Cells were co‐loaded with calcein‐AM and CoCl_2 , resulting in the quenching of the cytosolic signal without affecting the mitochondrial fluorescence. MTP inducers caused a rapid …decrease in mitochondrial calcein fluorescence which, however, was not completely prevented by CsA. Besides the large and rapid efflux of calcein induced by MTP agonists, we also observed a constant and spontaneous decrease of mitochondrial calcein which was completely prevented by CsA. Thus, MTP likely fluctuates between open and closed states in intact cells. Show more

Keywords: Mitochondria, calcium, calcein, permeability transition pore, cyclosporin

Citation: Biofactors, vol. 8, no. 3-4, pp. 263-272, 1998

Authors: Bernardi, Paolo | Colonna, Raffaele | Costantini, Paola | Eriksson, Ove | Fontaine, Eric | Ichas, François | Massari, Stefano | Nicolli, Annamaria | Petronilli, Valeria | Scorrano, Luca

Article Type: Research Article

Abstract: This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A‐sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.

Citation: Biofactors, vol. 8, no. 3-4, pp. 273-281, 1998

Authors: Lemasters, John J. | Qian, Ting | Elmore, Steven P. | Trost, Lawrence C. | Nishimura, Yoshiya | Herman, Brian | Bradham, Cynthia A. | Brenner, David A. | Nieminen, Anna‐Liisa

Article Type: Research Article

Abstract: Onset of the cyclosporin‐A‐sensitive mitochondrial permeability transition (MPT) in individual mitochondria within living cells can be visualized by laser scanning confocal microscopy. The MPT is a causative event in many types of necrotic and apoptotic cell death, including oxidative stress, ischemia/reperfusion injury, Ca^{2+} ionophore toxicity and tumor necrosis factor alpha (TNF\alpha ) induced apoptosis, and may contribute to Reye’s‐related drug toxicity. Pyridine nucleotide oxidation, mitochondrial generation of reactive oxygen species, and increased mitochondrial Ca^{2+} and pH can each promote onset of the MPT in situ. The MPT can also be directly visualized during TNF\alpha …‐induced apoptosis to hepatocytes. Mitochondria spontaneously depolarize in situ after nutrient deprivation before entering an acidic lysosomal compartment, suggesting that the MPT precedes the normal process of mitochondrial autophagy. We propose a model in which onset of the MPT to increasing numbers of mitochondria leads progressively to autophagy, apoptosis and necrotic cell death. Show more

Citation: Biofactors, vol. 8, no. 3-4, pp. 283-285, 1998

Authors: Nicholls, David G. | Budd, Samantha L.

Article Type: Research Article

Abstract: Chronic activation of NMDA receptors by glutamate is toxic to cultured neurons. The extensive Ca^{2+} entry accompanying receptor activation is largely accumulated by the intracellular mitochondria, with resultant effects on mitochondrial membrane potential, ATP synthesis, glycolysis, reactive oxygen species generation and ultimately failure of cytoplasmic Ca^{2+} homeostasis and cell death. Each of these parameters is inter‐related and in this review we describe attempts to separate out each factor to establish the sequence of events following NMDA receptor activation. The conclusion is that mitochondrial Ca^{2+} accumulation is a key event in glutamate excitotoxicity, and that …cells maintained by glycolysis in the absence of a mitochondrial membrane potential are highly resistant to glutamate excitotoxicity. Show more

Citation: Biofactors, vol. 8, no. 3-4, pp. 287-299, 1998

Authors: Di Virgilio, Francesco

Article Type: Research Article

Citation: Biofactors, vol. 8, no. 3-4, pp. 301-303, 1998

Authors: Azzone, Giovanni Felice

Article Type: Research Article

Citation: Biofactors, vol. 8, no. 3-4, pp. 305-316, 1998