[41 ] Use of polarography to detect respiration defects in cell cultures

In the present work, a large scale investigation was done regarding the capacity of cultured human cell lines (carrying in homoplasmic form either the mitochondrial tRNA Lys A8344G mutation associated with the myoclonic epilepsy and ragged red fiber (MERRF) encephalomyopathy or a frameshift mutation, isolated in vitro, in the gene for the ND4 subunit of NADH dehydrogenase) to undergo transcomplementation of their recessive mitochondrial DNA (mtDNA) mutations after cell fusion. The presence of appropriate nuclear drug resistance markers in the two cell lines allowed measurements of the frequency of cell fusion in glucose-containing medium, non-selective for respiratory capacity, whereas the frequency of transcomplementation of the two mtDNA mutations was determined by growing the same cell fusion mixture in galactose-containing medium, selective for respiratory competence. Transcomplementation of the two mutations was revealed by the re-establishment of normal mitochondrial protein synthesis and respiratory activity and by the relative rates synthesis of two isoforms of the ND3 subunit of NADH dehydrogenase. The results of several experiments showed a cell fusion frequency between 1.4 and 3.4% and an absolute transcomplementation frequency that varied between 1.2 ؋ 10 ؊5 and 5.5 ؋ 10 ؊4 . Thus, only 0.3-1.6% of the fusion products exhibited transcomplementation of the two mutations. These rare transcomplementing clones were very sluggish in developing, grew very slowly thereafter, and showed a substantial rate of cell death (22-28%). The present results strongly support the conclusion that the capacity of mitochondria to fuse and mix their contents is not a general intrinsic property of these organelles in mammalian cells, although it may become activated in some developmental or physiological situations.

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“…It is known, in fact, that Complex I-dependent O 2 consumption is usually the rate-limiting step in respiration (47,48).…”

Section: Discussionmentioning

confidence: 99%

Very Rare Complementation between Mitochondria Carrying Different Mitochondrial DNA Mutations Points to Intrinsic Genetic Autonomy of the Organelles in Cultured Human Cells

Enríquez

Cabezas‐Herrera

Bayona‐Bafaluy

et al. 2000

Journal of Biological Chemistry

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“…Substrate oxidation rates and phosphorylating capacities of isolated synaptosomes were determined polarographically and were performed essentially according to the procedure of Hofhaus et al (1996). For respiration studies, synaptosomes were permeabilized with 0.007% digitonin.…”

Section: Methodsmentioning

confidence: 99%

Ischemic Preconditioning Targets the Respiration of Synaptic Mitochondria via Protein Kinase Cε

Dave¹,

DeFazio²,

Raval³

et al. 2008

J. Neurosci.

100

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In the brain, ischemic preconditioning (IPC) diminishes mitochondrial dysfunction after ischemia and confers neuroprotection. Activation of protein kinase C (PKC) has been proposed to be a key neuroprotective pathway during IPC. We tested the hypothesis that IPC increases the levels of PKC in synaptosomes from rat hippocampus, resulting in improved synaptic mitochondrial respiration. Preconditioning significantly increased the level of hippocampal synaptosomal PKC to 152% of sham-operated animals at 2 d of reperfusion, the time of peak neuroprotection. We tested the effect of PKC activation on hippocampal synaptic mitochondrial respiration 2 d after preconditioning. Treatment with the specific PKC activating peptide, tat-RACK (tat--receptor for activated C kinase), increased the rate of oxygen consumption in the presence of substrates for complexes I, II, and IV to 157, 153, and 131% of control (tat peptide alone). In parallel, we found that PKC activation in synaptosomes from preconditioned animals resulted in altered levels of phosphorylated mitochondrial respiratory chain proteins: increased serine and tyrosine phosphorylation of 18 kDa subunit of complex I, decreased serine phosphorylation of FeS protein in complex III, increased threonine phosphorylation of COX IV (cytochrome oxidase IV), increased mitochondrial membrane potential, and decreased H 2 O 2 production. In brief, ischemic preconditioning promoted significant increases in the level of synaptosomal PKC. Activation of PKC increased synaptosomal mitochondrial respiration and phosphorylation of mitochondrial respiratory chain proteins. We propose that, at 48 h of reperfusion after ischemic preconditioning, PKC is poised at synaptic mitochondria to respond to ischemia either by direct phosphorylation or activation of the PKC signaling pathway.

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“…Mitochondrial Respiration Measurements-While determining the conditions for blocking the electron transfer through the respiratory chain, the complex-specific oxygen consumption activities were measured (20). About 5 ϫ 10 6 cells were treated with digitonin.…”

Section: Methodsmentioning

confidence: 99%

An Assembled Complex IV Maintains the Stability and Activity of Complex I in Mammalian Mitochondria

D'Aurelio

Deng

et al. 2007

Journal of Biological Chemistry

125

100

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In the mammalian mitochondrial electron transfer system, the majority of electrons enter at complex I, go through complexes III and IV, and are finally delivered to oxygen. Previously we generated several mouse cell lines with suppressed expression of the nuclearly encoded subunit 4 of complex IV. This led to a loss of assembly of complex IV and its defective function. Interestingly, we found that the level of assembled complex I and its activity were also significantly reduced, whereas levels and activity of complex III were normal or up-regulated. The structural and functional dependence of complex I on complex IV was verified using a human cell line carrying a nonsense mutation in the mitochondrially encoded complex IV subunit 1 gene. Our work documents that, although there is no direct electron transfer between them, an assembled complex IV helps to maintain complex I in mammalian cells.

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[41 ] Use of polarography to detect respiration defects in cell cultures

Cited by 129 publications

References 8 publications

Very Rare Complementation between Mitochondria Carrying Different Mitochondrial DNA Mutations Points to Intrinsic Genetic Autonomy of the Organelles in Cultured Human Cells

Very Rare Complementation between Mitochondria Carrying Different Mitochondrial DNA Mutations Points to Intrinsic Genetic Autonomy of the Organelles in Cultured Human Cells

Ischemic Preconditioning Targets the Respiration of Synaptic Mitochondria via Protein Kinase Cε

An Assembled Complex IV Maintains the Stability and Activity of Complex I in Mammalian Mitochondria

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