Repetitive transient depolarizations of the inner mitochondrial membrane induced by proton pumping.

Single mitochondria show the spontaneous fluctuations of DeltaPsim. In this study, to examine the mechanism of the fluctuations, we observed DeltaPsim in single isolated heart mitochondria using time-resolved fluorescence microscopy. Addition of malate, succinate, or ascorbate plus TMPD to mitochondria induced polarization of the inner membrane followed by repeated cycles of rapid depolarizations and immediate repolarizations. ADP significantly decreased the frequency of the rapid depolarizations, but the ADP effect was counteracted by oligomycin. On the other hand, the rapid depolarizations did not occur when mitochondria were polarized by the efflux of K(+) from the matrix. The rapid depolarizations became frequent with the increase in the substrate concentration or pH of the buffer. These results suggest that the rapid depolarizations depend on the net translocation of protons from the matrix. The frequency of the rapid depolarizations was not affected by ROS scavengers, Ca(2+), CsA, or BA. In addition, the obvious increase in the permeability of the inner membrane to calcein (MW 623) that was entrapped in the matrix was not observed upon the transient depolarization. The mechanisms of the spontaneous oscillations of DeltaPsim are discussed in relation to the matrix pH and the permeability transitions.

[1]  G. Steele,et al.  Intracellular heterogeneity in mitochondrial membrane potentials revealed by a J-aggregate-forming lipophilic cation JC-1. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[2]  C. Hoppel,et al.  Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle. , 1977, The Journal of biological chemistry.

[3]  G. Rutter,et al.  Integrating cytosolic calcium signals into mitochondrial metabolic responses , 1998, The EMBO journal.

[4]  P. R. Gardner,et al.  Superoxide scavenging by Mn(II/III) tetrakis (1-methyl-4-pyridyl) porphyrin in mammalian cells. , 1996, Archives of biochemistry and biophysics.

[5]  G. Fiskum,et al.  Cyclosporin A-insensitive Permeability Transition in Brain Mitochondria , 2003, Journal of Biological Chemistry.

[6]  Yoshihiro Ohta,et al.  Fluorescence imaging of metabolic responses in single mitochondria. , 2002, Biochemical and biophysical research communications.

[7]  A. Beavis,et al.  On the regulation of the mitochondrial inner membrane anion channel by magnesium and protons. , 1989, The Journal of biological chemistry.

[8]  M. Duchen,et al.  Mitochondria Exert a Negative Feedback on the Propagation of Intracellular Ca2+ Waves in Rat Cortical Astrocytes , 1999, The Journal of cell biology.

[9]  L. Blatter,et al.  Imaging the permeability pore transition in single mitochondria. , 1998, Biophysical journal.

[10]  P. M. Sokolove,et al.  Palmitic acid opens a novel cyclosporin A-insensitive pore in the inner mitochondrial membrane. , 2001, Archives of biochemistry and biophysics.

[11]  Peter Lipp,et al.  Mitochondria are morphologically and functionally heterogeneous within cells , 2002, The EMBO journal.

[12]  R. Haworth,et al.  The Ca2+-induced membrane transition in mitochondria. III. Transitional Ca2+ release. , 1979, Archives of biochemistry and biophysics.

[13]  B. Miroux,et al.  Disruption of the uncoupling protein-2 gene in mice reveals a role in immunity and reactive oxygen species production , 2000, Nature Genetics.

[14]  Steven J. Sollott,et al.  Reactive Oxygen Species (Ros-Induced) Ros Release , 2000, The Journal of experimental medicine.

[15]  A. Lundin,et al.  A sensitive method for measuring ATP-formation in rat muscle mitochondria. , 1990, Scandinavian journal of clinical and laboratory investigation.

[16]  B Chance,et al.  Sustained oscillations of ionic constituents of mitochondria. , 1966, Archives of biochemistry and biophysics.

[17]  L. Packer,et al.  Oscillatory systems in mitochondria. , 1974, Biochimica et biophysica acta.

[18]  Lawrence M. Lifshitz,et al.  Close contacts with the endoplasmic reticulum as determinants of mitochondrial Ca2+ responses. , 1998, Science.

[19]  Brian O'Rourke,et al.  Synchronized Whole Cell Oscillations in Mitochondrial Metabolism Triggered by a Local Release of Reactive Oxygen Species in Cardiac Myocytes* , 2003, Journal of Biological Chemistry.

[20]  Y. Kaneda,et al.  Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage , 2000, Nature.

[21]  M. Zoratti,et al.  Electrophysiology of the inner mitochondrial membrane , 1994, Journal of bioenergetics and biomembranes.

[22]  J. Dubinsky,et al.  Dual Responses of CNS Mitochondria to Elevated Calcium , 2000, The Journal of Neuroscience.

[23]  J. Mazat,et al.  Mitochondria Are Excitable Organelles Capable of Generating and Conveying Electrical and Calcium Signals , 1997, Cell.

[24]  A. Baracca,et al.  Rhodamine 123 as a probe of mitochondrial membrane potential: evaluation of proton flux through F(0) during ATP synthesis. , 2003, Biochimica et biophysica acta.

[25]  K. Shimamoto,et al.  Modulation of the mitochondrial permeability transition pore complex in GSK-3beta-mediated myocardial protection. , 2007, Journal of molecular and cellular cardiology.

[26]  D. Ricquier,et al.  Mitochondrial uncoupling proteins: from mitochondria to the regulation of energy balance , 2000, The Journal of physiology.

[27]  F S Fay,et al.  Imaging in five dimensions: time-dependent membrane potentials in individual mitochondria. , 1993, Biophysical journal.

[28]  Y. Kushnareva,et al.  Effect of butylhydroxytoluene and related compounds on permeability of the inner mitochondrial membrane. , 1997, Archives of biochemistry and biophysics.

[29]  B. Lowell,et al.  A significant portion of mitochondrial proton leak in intact thymocytes depends on expression of UCP2 , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[30]  P. Bernardi.  Modulation of the mitochondrial cyclosporin A-sensitive permeability transition pore by the proton electrochemical gradient. Evidence that the pore can be opened by membrane depolarization. , 1992, The Journal of biological chemistry.

[31]  S. Lowen.  The Biophysical Journal , 1960, Nature.

[32]  A. Halestrap,et al.  The permeability transition pore complex: another view. , 2002, Biochimie.

[33]  Dean P. Jones,et al.  The ADP/ATP translocator is not essential for the mitochondrial permeability transition pore , 2004, Nature.

[34]  E. Hultman,et al.  ATP production rate in mitochondria isolated from microsamples of human muscle. , 1990, The American journal of physiology.

[35]  J. Mazat,et al.  A model of mitochondrial Ca(2+)-induced Ca2+ release simulating the Ca2+ oscillations and spikes generated by mitochondria. , 1998, Biophysical chemistry.

[36]  L. Blatter,et al.  Fluctuations in mitochondrial membrane potential caused by repetitive gating of the permeability transition pore. , 1999, The Biochemical journal.

[37]  J. Mazat,et al.  From calcium signaling to cell death: two conformations for the mitochondrial permeability transition pore. Switching from low- to high-conductance state. , 1998, Biochimica et biophysica acta.

[38]  I. Reynolds,et al.  Spontaneous changes in mitochondrial membrane potential in single isolated brain mitochondria. , 2003, Biophysical journal.

[39]  I. Fridovich,et al.  Stable Mn(III) porphyrins mimic superoxide dismutase in vitro and substitute for it in vivo. , 1994, The Journal of biological chemistry.

[40]  M. Zoratti,et al.  Modulation of the mitochondrial permeability transition pore. Effect of protons and divalent cations. , 1992, The Journal of biological chemistry.

[41]  G. Miotto,et al.  Transient and long-lasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. , 1999, Biophysical journal.

[42]  B. Herman,et al.  Distribution of electrical potential, pH, free Ca2+, and volume inside cultured adult rabbit cardiac myocytes during chemical hypoxia: a multiparameter digitized confocal microscopic study. , 1994, Biophysical journal.

[43]  A. Beavis,et al.  The mitochondrial inner membrane anion channel is inhibited by DIDS , 1996, Journal of bioenergetics and biomembranes.

[44]  F. Ichas,et al.  Electrical coupling and plasticity of the mitochondrial network. , 2000, Cell calcium.

[45]  M. Duchen,et al.  Mitochondrial oxidative stress and cell death in astrocytes--requirement for stored Ca2+ and sustained opening of the permeability transition pore. , 2002, Journal of cell science.

[46]  Michael R. Duchen,et al.  Transient Mitochondrial Depolarizations Reflect Focal Sarcoplasmic Reticular Calcium Release in Single Rat Cardiomyocytes , 1998, The Journal of cell biology.

[47]  L. Loew,et al.  Physiological cytosolic Ca2+ transients evoke concurrent mitochondrial depolarizations. , 1994, Proceedings of the National Academy of Sciences of the United States of America.