The mitochondrial permeability transition.

It has been firmly established that the rapid uptake of Ca2+ by mitochondria from a wide range of sources is mediated by a uniporter which permits transport of the ion down its electrochemical gradient. Several mechanisms of Ca2+ efflux from mitochondria have also been extensively discussed in the literature. Energized mitochondria must expend a significant amount of energy to transport Ca2+ against its electrochemical gradient from the matrix space to the external space. Two separate mechanisms have been found to mediate this outward transport: a Ca2+/nNa+ exchanger and a Na(+)-independent efflux mechanism. These efflux mechanisms are considered from the perspective of available energy. In addition, a reversible Ca2(+)-induced increase in inner membrane permeability can also occur. The induction of this permeability transition is characterized by swelling of the mitochondria, leakiness to small ions such as K+, Mg2+, and Ca2+, and loss of the mitochondrial membrane potential. It has been suggested that the permeability transition and its reversal may also function as a mitochondrial Ca2+ efflux mechanism under some conditions. The characteristics of each of these mechanisms are discussed, as well as their possible physiological functions.

Mechanisms by which mitochondria transport calcium.

Programmed cell death (PCD) is a physiological process commonly defined by alterations in nuclear morphology (apoptosis) and/or characteristic stepwise degradation of chromosomal DNA occurring before cytolysis. However, determined characteristics of PCD such as loss in mitochondrial reductase activity or cytolysis can be induced in enucleated cells, indicating cytoplasmic PCD control. Here we report a sequential disregulation of mitochondrial function that precedes cell shrinkage and nuclear fragmentation. A first cyclosporin A-inhibitable step of ongoing PCD is characterized by a reduction of mitochondrial transmembrane potential, as determined by specific fluorochromes (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine++ + iodide; 3,3'dihexyloxacarbocyanine iodide). Cytofluorometrically purified cells with reduced mitochondrial transmembrane potential are initially incapable of oxidizing hydroethidine (HE) into ethidium. Upon short-term in vitro culture, such cells acquire the capacity of HE oxidation, thus revealing a second step of PCD marked by mitochondrial generation of reactive oxygen species (ROS). This step can be selectively inhibited by rotenone and ruthenium red yet is not affected by cyclosporin A. Finally, cells reduce their volume, a step that is delayed by radical scavengers, indicating the implication of ROS in the apoptotic process. This sequence of alterations accompanying early PCD is found in very different models of apoptosis induction: glucocorticoid-induced death of lymphocytes, activation-induced PCD of T cell hybridomas, and tumor necrosis factor-induced death of U937 cells. Transfection with the antiapoptotic protooncogene Bcl-2 simultaneously inhibits mitochondrial alterations and apoptotic cell death triggered by steroids or ceramide. In vivo injection of fluorochromes such as 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide; 3,3'dihexyloxacarbocyanine iodide; or HE allows for the detection of cells that are programmed for death but still lack nuclear DNA fragmentation. In particular, assessment of mitochondrial ROS generation provides an accurate picture of PCD-mediated lymphocyte depletion. In conclusion, alterations of mitochondrial function constitute an important feature of early PCD.

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Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death.

This review provides a selective history of how studies of mitochondrial cation transport (K+, Na+, Ca2+) developed in relation to the major themes of research in bioenergetics. It then covers in some detail specific transport pathways for these cations, and it introduces and discusses open problems about their nature and physiological function, particularly in relation to volume regulation and Ca2+homeostasis. The review should provide the basic elements needed to understand both earlier mitochondrial literature and current problems associated with mitochondrial transport of cations and hopefully will foster new interest in the molecular definition of mitochondrial cation channels and exchangers as well as their roles in cell physiology.

http://agrino.org/skalifourta/Mitochondrial%20Transport%20of%20Cations-%20Channels,%20Exchangers,%20and%20Permeability%20Transition.pdf

Mitochondrial Transport of Cations: Channels, Exchangers, and Permeability Transition

Anucleate cells can be induced to undergo programmed cell death (PCD), indicating the existence of a cytoplasmic PCD pathway that functions independently from the nucleus. Cytoplasmic structures including mitochondria have been shown to participate in the control of apoptotic nuclear disintegration. Before cells exhibit common signs of nuclear apoptosis (chromatin condensation and endonuclease-mediated DNA fragmentation), they undergo a reduction of the mitochondrial transmembrane potential (delta psi m) that may be due to the opening of mitochondrial permeability transition (PT) pores. Here, we present direct evidence indicating that mitochondrial PT constitutes a critical early event of the apoptotic process. In a cell-free system combining purified mitochondria and nuclei, mitochondria undergoing PT suffice to induce chromatin condensation and DNA fragmentation. Induction of PT by pharmacological agents augments the apoptosis-inducing potential of mitochondria. In contrast, prevention of PT by pharmacological agents impedes nuclear apoptosis, both in vitro and in vivo. Mitochondria from hepatocytes or lymphoid cells undergoing apoptosis, but not those from normal cells, induce disintegration of isolated Hela nuclei. A specific ligand of the mitochondrial adenine nucleotide translocator (ANT), bongkreik acid, inhibits PT and reduces apoptosis induction by mitochondria in a cell-free system. Moreover, it inhibits the induction of apoptosis in intact cells. Several pieces of evidence suggest that the proto-oncogene product Bcl-2 inhibits apoptosis by preventing mitochondrial PT. First, to inhibit nuclear apoptosis, Bcl-2 must be localized in mitochondrial but not nuclear membranes. Second, transfection-enforced hyperexpression of Bcl-2 directly abolishes the induction of mitochondrial PT in response to a protonophore, a pro-oxidant, as well as to the ANT ligand atractyloside, correlating with its apoptosis-inhibitory effect. In conclusion, mitochondrial PT appears to be a critical step of the apoptotic cascade.

https://rupress.org/jem/article-pdf/183/4/1533/1108103/1533.pdf

Mitochondrial control of nuclear apoptosis

The capacity of cyclosporin A to inhibit opening of a Ca2+-dependent pore in the inner membrane of heart mitochondria was investigated. Whereas in the presence of 25 nmol of Ca2+/mg of mitochondrial protein and 5 mM-Pi mitochondria were unable to maintain accumulated Ca2+, inner-membrane potential and sucrose impermeability, all three parameters were preserved when cyclosporin was included. Pore opening was assayed directly by [14C]sucrose entry and entrapment in the matrix space. [14C]Sucrose entry induced by both Ca2+ plus Pi and Ca2+ plus t-butyl hydroperoxide was almost completely inhibited by 60 pmol of cyclosporin/mg of mitochondrial protein. It is concluded that cyclosporin A is a potent inhibitor of the pore.

A Costi

Papers

Inhibition by cyclosporin A of a Ca2+-dependent pore in heart mitochondria activated by inorganic phosphate and oxidative stress.