scholarly journals Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts

2004 ◽  
Vol 380 (3) ◽  
pp. 919-928 ◽  
Author(s):  
Eveline HUTTER ◽  
Kathrin RENNER ◽  
Gerald PFISTER ◽  
Petra STÖCKL ◽  
Pidder JANSEN-DÜRR ◽  
...  
Keyword(s):  
Electron Transport ◽  
Oxidative Phosphorylation ◽  
Replicative Senescence ◽  
Citrate Synthase ◽  
Human Fibroblasts ◽  
Respiratory Control ◽  
Compensatory Mechanisms ◽  
Intact Cells ◽  
Atp Production ◽  
Human Diploid Fibroblasts

Limitation of lifespan in replicative senescence is related to oxidative stress, which is probably both the cause and consequence of impaired mitochondrial respiratory function. The respiration of senescent human diploid fibroblasts was analysed by highresolution respirometry. To rule out cell-cycle effects, proliferating and growth-arrested young fibroblasts were used as controls. Uncoupled respiration, as normalized to citrate synthase activity, remained unchanged, reflecting a constant capacity of the respiratory chain. Oligomycin-inhibited respiration, however, was significantly increased in mitochondria of senescent cells, indicating a lower coupling of electron transport with phosphorylation. In contrast, growth-arrested young fibroblasts exhibited a higher coupling state compared with proliferating controls. In intact cells, partial uncoupling may lead to either decreased oxidative ATP production or a compensatory increase in routine respiration. To distinguish between these alternatives, we subtracted oligomycin-inhibited respiration from routine respiration, which allowed us to determine the part of respiratory activity coupled with ATP production. Despite substantial differences in the respiratory control ratio, ranging from 4 to 11 in the different experimental groups, a fixed proportion of respiratory capacity was maintained for coupled oxidative phosphorylation in all the experimental groups. This finding indicates that the senescent cells fully compensate for increased proton leakage by enhanced electron-transport activity in the routine state. These results provide a new insight into age-associated defects in mitochondrial function and compensatory mechanisms in intact cells.

Dual Mutations Reveal Interactions Between Components of Oxidative Phosphorylation in Kluyveromyces lactis

Genetics ◽  
2001 ◽  
Vol 159 (3) ◽  
pp. 929-938
Author(s):  
G D Clark-Walker ◽  
X J Chen
Keyword(s):  
Electron Transport ◽  
Oxidative Phosphorylation ◽  
Atp Synthase ◽  
Kluyveromyces Lactis ◽  
Mitochondrial Protein ◽  
Proton Pumping ◽  
Nuclear Genes ◽  
Suppressor Activity ◽  
Inner Membrane ◽  
Atp Production

Abstract Loss of mtDNA or mitochondrial protein synthesis cannot be tolerated by wild-type Kluyveromyces lactis. The mitochondrial function responsible for ρ0-lethality has been identified by disruption of nuclear genes encoding electron transport and F0-ATP synthase components of oxidative phosphorylation. Sporulation of diploid strains heterozygous for disruptions in genes for the two components of oxidative phosphorylation results in the formation of nonviable spores inferred to contain both disruptions. Lethality of spores is thought to result from absence of a transmembrane potential, ΔΨ, across the mitochondrial inner membrane due to lack of proton pumping by the electron transport chain or reversal of F1F0-ATP synthase. Synergistic lethality, caused by disruption of nuclear genes, or ρ0-lethality can be suppressed by the atp2.1 mutation in the β-subunit of F1-ATPase. Suppression is viewed as occurring by an increased hydrolysis of ATP by mutant F1, allowing sufficient electrogenic exchange by the translocase of ADP in the matrix for ATP in the cytosol to maintain ΔΨ. In addition, lethality of haploid strains with a disruption of AAC encoding the ADP/ATP translocase can be suppressed by atp2.1. In this case suppression is considered to occur by mutant F1 acting in the forward direction to partially uncouple ATP production, thereby stimulating respiration and relieving detrimental hyperpolarization of the inner membrane. Participation of the ADP/ATP translocase in suppression of ρ0-lethality is supported by the observation that disruption of AAC abolishes suppressor activity of atp2.1.

scholarly journals Uncoupling between Phenotypic Senescence and Cell Cycle Arrest in Aging p21-Deficient Fibroblasts

2000 ◽  
Vol 20 (18) ◽  
pp. 6741-6754 ◽  
Author(s):  
Vjekoslav Dulić ◽  
Georges-Edouard Beney ◽  
Guillaume Frebourg ◽  
Linda F. Drullinger ◽  
Gretchen H. Stein
Keyword(s):  
Cell Cycle ◽  
Replicative Senescence ◽  
Cyclin E ◽  
Human Fibroblasts ◽  
Population Expansion ◽  
Cell Cycle Checkpoints ◽  
Critical Event ◽  
Human Diploid Fibroblasts ◽  
Senescent Phenotype ◽  
Molecular Events

ABSTRACT Irreversible G1 arrest in senescent human fibroblasts is mediated by two inhibitors of cyclin-dependent kinases (Cdks), p21Cip1/SDI1/WAF1 and p16Ink4A. To determine the physiological and molecular events that specifically require p21, we studied senescence in human diploid fibroblasts expressing the human papillomavirus type 16 E6 oncogene, which confers low p21 levels via enhanced p53 degradation. We show that in late-passage E6 cells, high Cdk activity drives the cell cycle, but population expansion is slowed down by crisis-like events, probably owing to defective cell cycle checkpoints. At the end of lifespan, terminal-passage E6 cells exhibited several aspects of the senescent phenotype and accumulated unphosphorylated pRb and p16. However, both replication and cyclin-Cdk2 kinase activity were still not blocked, demonstrating that phenotypic and replicative senescence are uncoupled in the absence of normal p21 levels. At this stage, E6 cells also failed to upregulate p27 and inactivate cyclin-Cdk complexes in response to serum deprivation. Eventually, irreversible G1 arrest occurred coincident with inactivation of cyclin E-Cdk2 owing to association with p21. Similarly, when p21−/− mouse embryo fibroblasts reached the end of their lifespan, they had the appearance of senescent cells yet, in contrast to their wild-type counterparts, they were deficient in downregulating bromodeoxyuridine incorporation, cyclin E- and cyclin A-Cdk2 activity, and inhibiting pRb hyperphosphorylation. These data support the model that the critical event ensuring G1arrest in senescence is p21-dependent Cdk inactivation, while other aspects of senescent phenotype appear to occur independently of p21.

scholarly journals Alteration of Pseudomonas aeruginosa respiration by 4-(1-adamantyl)-phenol derivative

Biologija ◽  
2018 ◽  
Vol 64 (3) ◽  
Author(s):  
Daria M. Dudikova ◽  
Nina O. Vrynchanu ◽  
Valentyna I. Nosar
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Oxygen Consumption ◽  
Oxidative Phosphorylation ◽  
Respiratory Chain ◽  
Structural Integrity ◽  
Respiratory Control ◽  
Endogenous Respiration ◽  
Phenol Derivative ◽  
Succinate Oxidation ◽  
Intact Cells

Derivatives of 4-(1-adamantyl)-phenol are a promising class of antimicrobials affecting the structural integrity and functions of the bacterial cell membrane. The functioning of Pseudomonas aeruginosa respiratory chain and related system of oxidative phosphorylation was investigated before and after treatment with a derivative of 4-(1-adamantyl)-phenol (compound KVM-97). Oxygen consumption was measured polarographically with a Clark-type oxygen electrode. KVM-97 was tested at 0.5× and 1.0× MIC (minimum inhibitory concentration). Specific substrates of the respiratory chain (either 3.0 mM glutamate with 2.0 mM malonate or 3.0 mM succinate with 5.0 μM rotenone) were used. All reactions were stimulated by addition of ADP (0.2 mmol). It was found that at tested concentrations, KVM-97 inhibited the endogenous respiration and substrate oxidation in P. aeruginosa cells. The inhibiting effect was dose-dependent and more pronounced with succinate oxidation rather than glutamate oxidation. The respiratory control index value (RCI) in compound-treated cells was in average 1.5 times lower compared to the intact cells. The decrease in the RCI was related to changing the oxygen uptake rates in state 3 and state 4, which indicate the uncoupling of respiration and oxidative phosphorylation. The data obtained showed that 4-(1-adamantyl)-phenol derivative inhibits oxygen consumption and has uncoupling effects in P. aeruginosa cells.

Sulphide oxidation and oxidative phosphorylation in the mitochondria of the lugworm

1997 ◽  
Vol 200 (1) ◽  
pp. 83-92 ◽  
Author(s):  
S Vökel ◽  
M K Grieshaber
Keyword(s):  
Oxygen Consumption ◽  
Cytochrome C ◽  
Oxidative Phosphorylation ◽  
Cytochrome C Oxidase ◽  
Electron Flow ◽  
Respiratory Control ◽  
Sulphide Oxidation ◽  
Atp Production ◽  
Sulphide Concentration ◽  
Flow Through

Oxygen consumption, ATP production and cytochrome c oxidase activity of isolated mitochondria from body-wall tissue of Arenicola marina were measured as a function of sulphide concentration, and the effect of inhibitors of the respiratory complexes on these processes was determined. Concentrations of sulphide between 6 and 9 µmol l-1 induced oxygen consumption with a respiratory control ratio of 1.7. Production of ATP was stimulated by the addition of sulphide, reaching a maximal value of 67 nmol min-1 mg-1 protein at a sulphide concentration of 8 µmol l-1. Under these conditions, 1 mole of ATP was formed per mole of sulphide consumed. Higher concentrations of sulphide led to a decrease in ATP production until complete inhibition occurred at approximately 50 µmol l-1. The production of ATP with malate and succinate was stimulated by approximately 15 % in the presence of 4 µmol l-1 sulphide, but decreased at sulphide concentrations higher than 15­20 µmol l-1. Cytochrome c oxidase was also inhibited by sulphide, showing half-maximal inhibition at 1.5 µmol l-1 sulphide. Sulphide-induced ATP production was inhibited by antimycin, cyanide and oligomycin but not by rotenone or salicylhydroxamic acid. The present data indicate that sulphide oxidation is coupled to oxidative phosphorylation solely by electron flow through cytochrome c oxidase, whereas the alternative oxidase does not serve as a coupling site. At sulphide concentrations higher than 20 µmol l-1, oxidation of sulphide serves mainly as a detoxification process rather than as a source of energy.

Oxidative Phosphorylation in Hypoxic Airway Smooth Muscle

1974 ◽  
Vol 52 (1) ◽  
pp. 84-89 ◽  
Author(s):  
N. L. Stephens ◽  
J. Vogel
Keyword(s):  
Smooth Muscle ◽  
Electron Transport ◽  
Oxidative Phosphorylation ◽  
Energy Production ◽  
Respiratory Control ◽  
Mechanical Function ◽  
Loose Coupling ◽  
Experimental Conditions ◽  
Death Studies ◽  
Significant Difference

The impairment of mechanical function that we have previously reported in tracheal smooth muscle under conditions of hypoxia and substrate depletion may be due to a defect in the utilization of the energy produced, to a decrease in energy production in the muscle after the hypoxic inhibition of mitochondria, or to a combination of these. To test the second hypothesis mitochondria were isolated from the trachealis smooth muscle of dogs whose [Formula: see text] had been maintained at 30 mm Hg ± 7 (S.E.), for 1 h before death. Studies of oxidative phosphorylation revealed no significant differences in ADP/O ratios in these mitochondria as compared with normal nor was there any significant difference in rate of O2 uptake as long as ADP was available (state 3 respiration). However, there was a significant difference in respiratory control ratios (R.C.R.), generally regarded as the most sensitive biochemical index of the function integrity of mitochondria, as well as a significant difference between control and experimental conditions in the rate of O2 uptake when ADP was not available (state 4 respiration). These findings suggested loose coupling between electron transport and oxidative phosphorylation.

scholarly journals Studies on the Energy Metabolism of Human Leukocytes

Blood ◽  
1967 ◽  
Vol 30 (2) ◽  
pp. 168-175 ◽  
Author(s):  
JOHN M. FOSTER ◽  
MARY L. TERRY ◽  
Harriet Gunther
Keyword(s):  
Energy Metabolism ◽  
Electron Transport ◽  
Oxidative Phosphorylation ◽  
Respiratory Control ◽  
Endogenous Respiration ◽  
Mitochondrial Oxidative Phosphorylation ◽  
Human Leukocytes ◽  
Endogenous Activity ◽  
Stimulation Of

Abstract 1. Oxidative phosphorylation has been studied in mitochondrial preparations from human leukocytes, using recently developed methods for homogenization, measuring respiration, and assaying for ATP. 2. Appreciable stimulation of both respiration and phosphorylation was limited to 3 substrates: succinate, malate, and α-glycerophosphate. The effects of other substrates were minimal. 3. The stimulating effects of these 3 substrates responded to inhibitors in a manner typical of mitochondrial oxidative phosphorylation. There was also considerable endogenous activity which, however, was insensitive to inhibitors. It is concluded the endogenous respiration and phosphorylation are not associated with electron transport. Subtracting their values from the data, P/O ratios consistent with good phosphorylation with the 3 substrates are obtained. 4. Studies with oligomycin and dinitrophenol suggest the presence of respiratory control. This indicates the mitochondria are intact. It is concluded that in the intact leukocyte the mitochondria are a major source of ATP.

scholarly journals Iron-citrate can induce cell senescent phenotype in human fibroblasts in vitro

2009 ◽  
Vol 15 (S3) ◽  
pp. 15-16
Author(s):  
L. Matos ◽  
H. Almeida
Keyword(s):  
Cell Injury ◽  
Replicative Senescence ◽  
Human Fibroblasts ◽  
Human Diploid Fibroblasts ◽  
Senescent Phenotype ◽  
Redox Active ◽  
Stress Induced Premature Senescence ◽  
Beta Galactosidase

AbstractAfter a number of replications, human diploid fibroblasts (HDFs) in culture lose the ability to divide, become insensitive to further proliferation and enter a state of replicative senescence (RS). Subcytotoxic doses of several stressful agents such as hydrogen peroxide, tertbutylhydroperoxide or ethanol, are able to cause stress-induced premature senescence (SIPS) in HDFs in vitro. Such senescent cells display many features of RS as growth arrest, senescence associated beta-galactosidase (SA beta-gal), cell enlargement and overexpression of several genes (e.g., p21, TGF beta-1,IGFBP3). During ageing, iron accumulates in several tissues in vivo, and also in senescent HDFs in vitro. Due to its redox-active properties, it promotes hydroxyl radical production (Fenton reaction) and eventually leads to cell injury. Free radical reactions are known to cause the accumulation of intracellular damage resulting in ageing. Iron may thus be able to cause SIPS. The main objective of the present study was to investigate whether the exposure of HDFs to a subcytotoxic concentration of iron is able to cause SIPS.

scholarly journals Assessing mitochondrial dysfunction in cells

2011 ◽  
Vol 435 (2) ◽  
pp. 297-312 ◽  
Author(s):  
Martin D. Brand ◽  
David G. Nicholls
Keyword(s):  
Mitochondrial Dysfunction ◽  
Coupling Efficiency ◽  
Respiratory Control ◽  
Information Measurement ◽  
Intact Cells ◽  
Atp Production ◽  
Additional Information ◽  
Isolated Mitochondria ◽  
In Cells

Assessing mitochondrial dysfunction requires definition of the dysfunction to be investigated. Usually, it is the ability of the mitochondria to make ATP appropriately in response to energy demands. Where other functions are of interest, tailored solutions are required. Dysfunction can be assessed in isolated mitochondria, in cells or in vivo, with different balances between precise experimental control and physiological relevance. There are many methods to measure mitochondrial function and dysfunction in these systems. Generally, measurements of fluxes give more information about the ability to make ATP than do measurements of intermediates and potentials. For isolated mitochondria, the best assay is mitochondrial respiratory control: the increase in respiration rate in response to ADP. For intact cells, the best assay is the equivalent measurement of cell respiratory control, which reports the rate of ATP production, the proton leak rate, the coupling efficiency, the maximum respiratory rate, the respiratory control ratio and the spare respiratory capacity. Measurements of membrane potential provide useful additional information. Measurement of both respiration and potential during appropriate titrations enables the identification of the primary sites of effectors and the distribution of control, allowing deeper quantitative analyses. Many other measurements in current use can be more problematic, as discussed in the present review.

scholarly journals Cardiac, skeletal, and smooth muscle mitochondrial respiration: are all mitochondria created equal?

2014 ◽  
Vol 307 (3) ◽  
pp. H346-H352 ◽  
Author(s):  
Song-Young Park ◽  
Jayson R. Gifford ◽  
Robert H. I. Andtbacka ◽  
Joel D. Trinity ◽  
John R. Hyngstrom ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Oxidative Phosphorylation ◽  
Mitochondrial Respiration ◽  
Complex I ◽  
Citrate Synthase ◽  
Smooth Muscles ◽  
Respiratory Control ◽  
Respiratory Control Ratio ◽  
Mitochondrial Content ◽  
Healthy Human

Unlike cardiac and skeletal muscle, little is known about vascular smooth muscle mitochondrial respiration. Therefore, the present study examined mitochondrial respiratory rates in smooth muscle of healthy human feed arteries and compared with that of healthy cardiac and skeletal muscles. Cardiac, skeletal, and smooth muscles were harvested from a total of 22 subjects (53 ± 6 yr), and mitochondrial respiration was assessed in permeabilized fibers. Complex I + II, state 3 respiration, an index of oxidative phosphorylation capacity, fell progressively from cardiac to skeletal to smooth muscles (54 ± 1, 39 ± 4, and 15 ± 1 pmol·s−1·mg−1, P < 0.05, respectively). Citrate synthase (CS) activity, an index of mitochondrial density, also fell progressively from cardiac to skeletal to smooth muscles (222 ± 13, 115 ± 2, and 48 ± 2 μmol·g−1·min−1, P < 0.05, respectively). Thus, when respiration rates were normalized by CS (respiration per mitochondrial content), oxidative phosphorylation capacity was no longer different between the three muscle types. Interestingly, complex I state 2 normalized for CS activity, an index of nonphosphorylating respiration per mitochondrial content, increased progressively from cardiac to skeletal to smooth muscles, such that the respiratory control ratio, state 3/state 2 respiration, fell progressively from cardiac to skeletal to smooth muscles (5.3 ± 0.7, 3.2 ± 0.4, and 1.6 ± 0.3 pmol·s−1·mg−1, P < 0.05, respectively). Thus, although oxidative phosphorylation capacity per mitochondrial content in cardiac, skeletal, and smooth muscles suggest all mitochondria are created equal, the contrasting respiratory control ratio and nonphosphorylating respiration highlight the existence of intrinsic functional differences between these muscle mitochondria. This likely influences the efficiency of oxidative phosphorylation and could potentially alter ROS production.

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