Measurement of mitochondrial respiration in permeabilized murine neuroblastoma (N-2α) cells, a simple and rapid in situ assay to investigate mitochondrial toxins

Steyn, Stefanus J.; Pieterse, Daniël J.; Mienie, Lodewyk J.; Schyf, Cornelis J. Van der

doi:10.1016/j.jbbm.2004.07.002

Cited by 6 publications

(6 citation statements)

References 53 publications

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“…To determine whether altered respiration may be the cause of the higher ROS levels in unstable cells, we measured respiratory rates of mitochondria in whole cells with plasma membranes permeabilized by digitonin. Digitonin can be used to permeabilize cell membranes, whereas intact mitochondria respiration is measured in situ in a physiologically relevant environment (38). In contrast to intact cells where metabolic demand normally limits the rate of respiration, the rate of O 2 consumption by permeabilized cells can be measured at maximum and minimum rates using high levels of ADP and oligomycin, respectively.…”

Section: Resultsmentioning

confidence: 99%

A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability

Fiskum¹,

Morgan

2006

Cancer Research

142

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Radiation-induced genomic instability (RIGI) manifests as a heritable increased rate of genetic alterations in the progeny of irradiated cells generations after the initial insult. The progeny can show an increased frequency of chromosomal translocations, deletions, mutations, micronuclei, and decreased plating efficiency. What perpetuates RIGI is unclear; however, persistently increased levels of reactive oxygen species (ROS) are frequently associated with genomically unstable clones. Furthermore, addition of free radical scavengers (e.g., DMSO, glycerol, and cationic thiol cysteamine) reduces the incidence of instability after irradiation, implicating a ROS-mediated role in RIGI induction. Because mitochondria are a major natural cellular source of ROS, we tested the hypothesis that mitochondrial dysfunction has a role in maintaining the elevated ROS levels in our irradiated, genetically unstable GM10115 Chinese hamster ovary cells. Amplex Red fluorometry measurements indicate that the relative contribution of uncoupler-sensitive mitochondrial hydrogen peroxide production to total cellular hydrogen peroxide generation is greater in unstable cells. Measurements of mitochondrial DNA levels and cell cytometric fluorescent measurements of Mitotracker Green FM indicate that differences in mitochondrial ROS production are not due to varying mitochondrial levels. However, mitochondrial respiration measured in digitonin-permeabilized cells is impaired in unstable clones. In addition, manganese superoxide dismutase, a major mitochondrial antioxidant enzyme, exhibits increased immunoreactivity but decreased enzyme activity in unstable clones, which along with decreased respiration rates may explain the increased levels of cellular ROS. These studies show that mitochondria from unstable cells are abnormal and likely contribute to the persistent oxidative stress in the unstable clones. (Cancer Res 2006; 66(21): 10377-83)

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Section: Resultsmentioning

confidence: 99%

A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability

Fiskum¹,

Morgan

2006

Cancer Research

142

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“…ATP production rate from mitochondrial respiratory chain was measured as reported before (35) with some modifications. After the glucose/virus treatments, NCM were washed with phosphate-buffered saline (PBS, 150 mM NaCl, 10 mM potassium phosphate, pH 7.4) for two times, trypsinized, and pelleted by centrifugation for 5 min at 400 g. Cells were resuspended in PBS and incubated at 37°C for 15 min to deplete residual intracellular ATP stores and sources.…”

Section: Methodsmentioning

confidence: 99%

Alterations in mitochondrial function and cytosolic calcium induced by hyperglycemia are restored by mitochondrial transcription factor A in cardiomyocytes

Suárez

Makino

et al. 2008

American Journal of Physiology-Cell Physiology

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Mitochondrial transcription factor A (TFAM) is essential for mitochondrial DNA transcription and replication. TFAM transcriptional activity is decreased in diabetic cardiomyopathy; however, the functional implications are unknown. We hypothesized that a reduced TFAM activity may be responsible for some of the alterations caused by hyperglycemia. Therefore, we investigated the effect of TFAM overexpression on hyperglycemia-induced cytosolic calcium handling and mitochondrial abnormalities. Neonatal rat cardiomyocytes were exposed to high glucose (30 mM) for 48 h, and we examined whether TFAM overexpression, by protecting mitochondrial DNA, could reestablish calcium fluxes and mitochondrial alterations toward normal. Our results shown that TFAM overexpression increased to more than twofold mitochondria copy number in cells treated either with normal (5.5 mM) or high glucose. ATP content was reduced by 30% and mitochondrial calcium decreased by 40% after high glucose. TFAM overexpression returned these parameters to even higher than control values. Calcium transients were prolonged by 70% after high glucose, which was associated with diminished sarco(endo)plasmic reticulum Ca(2+)-ATPase 2a and cytochrome-c oxidase subunit 1 expression. These parameters were returned to control values after TFAM overexpression. High glucose-induced protein oxidation was reduced by TFAM overexpression, indicating a reduction of the high glucose-induced oxidative stress. In addition, we found that TFAM activity can be modulated by O-linked beta-N-acetylglucosamine glycosylation. In conclusion, TFAM overexpression protected cell function against the damage induced by high glucose in cardiomyocytes.

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“…Mitochondrial complex I‐driven ATP synthesis was measured as described by Steyn et al 8 . with minor modifications.…”

Section: Methodsmentioning

confidence: 99%

“…Mitochondrial complex I-driven ATP synthesis was measured as described by Steyn et al 8 with minor modifications. SH-SY5Y cells (1 × 10 5 per well in 24-well plates) seeded overnight were treated with PC (20 g/mL) for 24 h at 37 • C. The cells were washed twice with phosphate-buffered solution, followed by incubation with reaction mixture (150 mM KCl, 75 mM Tris-HCl, 2 mM EDTA, 10 mM KH 2 PO 4 , 0.1% bovine serum albumin, pH 7.4) containing 25 M digitonin in the presence of mitochondrial complex I substrates (glutamate and malate, 10 mM each) for 15 min at 37 • C. Equal volumes of trichloroacetic acid (TCA, 8%) were added to each well for 5 min to stop the synthesis of ATP.…”

Section: Mitochondrial Complex I-driven Atp Synthesismentioning

confidence: 99%

Psoralea corylifolia L. Inhibits Mitochondrial Complex I and Proteasome Activities in SH‐SY5Y Cells

Tang¹,

Gruber²,

Wong³

et al. 2007

Annals of the New York Academy of Sciences

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The growing interest in alternative medicines, including traditional medicinal plants, has caused some health concerns due to poor awareness in the general population of the possible side effects from inappropriate practices. Psoralea corylifolia L. has been used in Chinese and Indian traditional medicine for the treatment of various inflammatory diseases of the skin and to improve vitality. Our data show that the extract obtained from the seeds of Psoralea corylifolia L. decreased mitochondrial complex I and proteasome activities; and oxidative stress might be an early event.

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Measurement of mitochondrial respiration in permeabilized murine neuroblastoma (N-2α) cells, a simple and rapid in situ assay to investigate mitochondrial toxins

Cited by 6 publications

References 53 publications

A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability

A Role for Mitochondrial Dysfunction in Perpetuating Radiation-Induced Genomic Instability

Alterations in mitochondrial function and cytosolic calcium induced by hyperglycemia are restored by mitochondrial transcription factor A in cardiomyocytes

Psoralea corylifolia L. Inhibits Mitochondrial Complex I and Proteasome Activities in SH‐SY5Y Cells

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