Simultaneous evaluation of substrate-dependent oxygen consumption rates and mitochondrial membrane potential by TMRM and safranin in cortical mitochondria

Mitochondrial membrane potential (mtMP) is critical for maintaining the physiological function of the respiratory chain to generate ATP. The present study characterized the inter-relationship between mtMP, using safranin and tetramethyl rhodamine methyl ester (TMRM), and mitochondrial respiratory activity and established a protocol for functional analysis of mitochondrial bioenergetics in a multi-sensor system. Coupled respiration was decreased by 27 and 30–35% in the presence of TMRM and safranin respectively. Maximal respiration was higher than coupled with Complex I- and II-linked substrates in the presence of both dyes. Safranin showed decreased maximal respiration at a higher concentration of carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) compared with TMRM. FCCP titration revealed that maximal respiration in the presence of glutamate and malate was not sustainable at higher FCCP concentrations as compared with pyruvate and malate. Oxygen consumption rate (OCR) and mtMP in response to mitochondrial substrates were higher in isolated mitochondria compared with tissue homogenates. Safranin exhibited higher sensitivity to changes in mtMP than TMRM. This multi-sensor system measured mitochondrial parameters in the brain of transgenic mice that model Alzheimer''s disease (AD), because mitochondrial dysfunction is believed to be a primary event in the pathogenesis of AD. The coupled and maximal respiration of electron transport chain were decreased in the cortex of AD mice along with the mtMP compared with age-matched controls. Overall, these data demonstrate that safranin and TMRM are suitable for the simultaneous evaluation of mtMP and respiratory chain activity using isolated mitochondria and tissue homogenate. However, certain care should be taken concerning the selection of appropriate substrates and dyes for specific experimental circumstances.     

Decavanadate induces mitochondrial membrane depolarization and inhibits oxygen consumption

Decavanadate induced rat liver mitochondrial depolarization at very low concentrations, half-depolarization with 39 nM decavanadate, while it was needed a 130-fold higher concentration of monomeric vanadate (5 microM) to induce the same effect. Decavanadate also inhibits mitochondrial repolarization induced by reduced glutathione in vitro, with an inhibition constant of 1 microM, whereas no effect was observed up to 100 microM of monomeric vanadate. The oxygen consumption by mitochondria is also inhibited by lower decavanadate than monomeric vanadate concentrations, i.e. 50% inhibition is attained with 99 M decavanadate and 10 microM monomeric vanadate. Thus, decavanadate is stronger as mitochondrial depolarization agent than as inhibitor of mitochondrial oxygen consumption. Up to 5 microM, decavanadate does not alter mitochondrial NADH levels nor inhibit neither F(O)F(1)-ATPase nor cytochrome c oxidase activity, but it induces changes in the redox steady-state of mitochondrial b-type cytochromes (complex III). NMR spectra showed that decameric vanadate is the predominant vanadate species in decavanadate solutions. It is concluded that decavanadate is much more potent mitochondrial depolarization agent and a more potent inhibitor of mitochondrial oxygen consumption than monomeric vanadate, pointing out the importance to take into account the contribution of higher oligomeric species of vanadium for the biological effects of vanadate solutions.     

Spontaneous changes in mitochondrial membrane potential in single isolated brain mitochondria

In this study we measured DeltaPsim in single isolated brain mitochondria using rhodamine 123. Mitochondria were attached to coverslips and superfused with K(+)-based HEPES-buffer medium supplemented with malate and glutamate. In approximately 70% of energized mitochondria we observed large amplitude spontaneous fluctuations in DeltaPsim with a time course comparable to that observed previously in mitochondria of intact cells. The other 30% of mitochondria maintained a stable DeltaPsim. Some of the "stable" mitochondria began to fluctuate spontaneously during the recording period. However, none of the initially fluctuating mitochondria became stable. Upon the removal of substrates from the medium or application of small amounts of Ca(2+), rhodamine 123 fluorescence rapidly dropped to background values in fluctuating mitochondria, while nonfluctuating mitochondria depolarized with a delay and often began to fluctuate before complete depolarization. The changes in DeltaPsim were not connected to oxidant production since reducing illumination or the addition of antioxidants had no effect on DeltaPsim. Fluctuating mitochondria did not lose calcein, nor was there any effect of cyclosporin A on DeltaPsim, which ruled out a contribution of permeability transition. We conclude that the fluctuations in DeltaPsim reflect an intermediate, unstable state of mitochondria that may lead to or reflect mitochondrial dysfunction.     

Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential

Bax, a pro-apoptotic member of the Bcl-2 family, is a cytosolic protein that inserts into mitochondrial membranes upon induction of cell death. Using the green fluorescent protein fused to Bax (GFP-Bax) to quantitate mitochondrial binding in living cells we have investigated the cause of Bax association with mitochondria and the time course relative to endogenous and induced changes in mitochondrial membrane potential (DeltaPsi(m)). We have found that staurosporine (STS) induces a loss in DeltaPsi(m) before GFP-Bax translocation can be measured. The onset of the DeltaPsi(m) loss is followed by a rapid and complete collapse of DeltaPsi(m) which is followed by Bax association with mitochondria. The mitochondria uncoupler FCCP, in the presence of the F(1)-F(0) ATPase inhibitor oligomycin, can trigger Bax translocation to mitochondria suggesting that when ATP levels are maintained a collapse of DeltaPsi(m) induces Bax translocation. Neither FCCP nor oligomycin alone alters Bax location. Bax association with mitochondria is also triggered by inhibitors of the electron transport chain, antimycin and rotenone, compounds that collapse DeltaPsi(m) without inducing rapid ATP hydrolysis that typically occurs with uncouplers such as FCCP. Taken together, our results suggest that alterations in mitochondrial energization associated with apoptosis can initiate Bax docking to mitochondria.     

Zinc causes loss of membrane potential and elevates reactive oxygen species in rat brain mitochondria

Emerging evidence suggests that Zn2+ may impair neuronal metabolism. We examined how Zn2+ affects the activity of isolated brain mitochondria fueled with glutamate + malate, succinate or glycerol 3-phosphate. Submicromolar levels of Zn2+ dissipated membrane potential and inhibited oxygen utilization in all three substrate conditions. Zn(2+)-induced depolarization was reversed by the membrane-impermeant metal chelator, EGTA, and was inhibited by uniporter blockade. Cyclosporin A did not block Zn(2+)-induced depolarization. Added Zn2+ increased accumulation of reactive oxygen species (ROS) in glutamate + malate or glycerol 3-phosphate conditions, but inhibited succinate-supported ROS accumulation. These results show that Zn2+ blocks mitochondrial function in all physiologically relevant substrate conditions.     

Modulation of mitochondrial membrane potential and reactive oxygen species production by copper in astrocytes

In monolayers of cultured rat astrocytes a number of agents that induce oxidative stress act synergistically with exposure to copper leading to rapid depolarization of the mitochondrial membrane potential (Psi m) and increased reactive oxygen species (ROS) production. Copper sensitized astrocytes to the action of menadione, an intracellular generator of superoxide anion radical, exogenous hydrogen peroxide (H2O2) and rotenone, an inhibitor of mitochondrial electron transport chain complex I. However, significant differences were observed in the ability to modulate the copper-enhanced oxidative stress depending on which stressor was used. The inhibitor of mitochondrial permeability transition cyclosporin A attenuated the effect of copper and rotenone, but had no protective action in the case of H2O2/copper and menadione/copper combinations. The H2O2 scavenger pyruvate was effective at protecting mitochondria against damage associated with the combined exposure to H2O2/copper and menadione/copper but not to the rotenone/copper combination. The antioxidant Trolox was ineffective at protecting against any of these actions and indeed had a damaging effect when combined with copper. The membrane-permeable copper chelator neocuproine combined with sensitizing concentrations of menadione caused a decrease in Psi m, mimicking the action of copper. Penicillamine, a membrane-impermeable copper chelator, was effective at reducing copper sensitization. Endogenous copper, mobilized during periods of oxidative stress, may play a role in the pathophysiology of brain injury. Our results suggest that this might be particularly dangerous in dysfunctional conditions in which the mitochondrial electron transport chain is compromised.     

Salicylate-collapsed membrane potential in pea stem mitochondria

Salicylate, acetylsalicylate, benzoate and 3,5-diiodosalicylate were examined for their effects on pea ( Pisum sativum L. cv. Alaska) stem mitochondria and on a tonoplast-enriched fraction. Salicylate collapsed the transmembrane electrochemical potential of mitochondria and the ATP-dependent proton gradient of the tonoplast-enriched vesicle preparation. Benzoate and acetylsalicylate did not show any effect, while 3,5-diiodosalicylate inhibited both basal O₂ consumption and ATPase activity of pea mitochondria. Salicylate seems to act as a protonophore. However, its effect is evident only at concentrations higher than those required by classical protonophores and, in addition, can be abolished after removing salicylate from the incubation medium. The activity of salicylate appears linked to the presence of the free phenolic hydroxyl on the benzene ring.     

Dopamine quinone modifies and decreases the abundance of the mitochondrial selenoprotein glutathione peroxidase 4

Oxidative stress and mitochondrial dysfunction are known to contribute to the pathogenesis of Parkinson's disease. Dopaminergic neurons may be more sensitive to these stressors because they contain dopamine (DA), a molecule that oxidizes to the electrophilic dopamine quinone (DAQ) which can covalently bind nucleophilic amino acid residues such as cysteine. The identification of proteins that are sensitive to covalent modification and functional alteration by DAQ is of great interest. We have hypothesized that selenoproteins, which contain a highly nucleophilic selenocysteine residue and often play vital roles in the maintenance of neuronal viability, are likely targets for the DAQ. Here we report the findings of our studies on the effect of DA oxidation and DAQ on the mitochondrial antioxidant selenoprotein glutathione peroxidase 4 (GPx4). Purified GPx4 could be covalently modified by DAQ, and the addition of DAQ to rat testes lysate resulted in dose-dependent decreases in GPx4 activity and monomeric protein levels. Exposing intact rat brain mitochondria to DAQ resulted in similar decreases in GPx4 activity and monomeric protein levels as well as detection of multiple forms of DA-conjugated GPx4 protein. Evidence of both GPx4 degradation and polymerization was observed following DAQ exposure. Finally, we observed a dose-dependent loss of mitochondrial GPx4 in differentiated PC12 cells treated with dopamine. Our findings suggest that a decrease in mitochondrial GPx4 monomer and a functional loss of activity may be a contributing factor to the vulnerability of dopaminergic neurons in Parkinson's disease.     

Human mitochondrial variants influence on oxygen consumption

This work investigates if human mitochondrial variants influence on maximal oxygen consumption (VO(2max)). With this purpose we recruited, as a uniform population in term of nutritional habits and life style, 114 healthy male Spanish subjects that practiced fitness exercises 3-4 times a week. Once mtDNA haplogroups were determined, we found that J presents with lower VO(2max) (P=0.02) than nonJ variants. J has been related with a lower efficiency of electron transport chain (ETC), diminished ATP and ROS production. Thus, the difficult to compensate the mitochondrial energetic deficiency could explain the accumulation of J haplogroup in LHON and multiple sclerosis. Furthermore, the lower ROS production associated to J could also account for the accrual of this variant in elderly people consequent to a decreased oxidative damage.     

Hepatitis B virus HBx protein localizes to mitochondria in primary rat hepatocytes and modulates mitochondrial membrane potential

Over 350 million people are chronically infected with hepatitis B virus (HBV), and a significant number of chronically infected individuals develop primary liver cancer. HBV encodes seven viral proteins, including the nonstructural X (HBx) protein. The results of studies with immortalized or transformed cells and with HBx-transgenic mice demonstrated that HBx can interact with mitochondria. However, no studies with normal hepatocytes have characterized the precise mitochondrial localization of HBx or the effect of HBx on mitochondrial physiology. We have used cultured primary rat hepatocytes as a model system to characterize the mitochondrial localization of HBx and the effect of HBx expression on mitochondrial physiology. We now show that a fraction of HBx colocalizes with density-gradient-purified mitochondria and associates with the outer mitochondrial membrane. We also demonstrate that HBx regulates mitochondrial membrane potential in hepatocytes and that this function of HBx varies depending on the status of NF-kappaB activity. In primary rat hepatocytes, HBx activation of NF-kappaB prevented mitochondrial membrane depolarization; however, when NF-kappaB activity was inhibited, HBx induced membrane depolarization through modulation of the mitochondrial permeability transition pore. Collectively, these results define potential pathways through which HBx may act in order to modulate mitochondrial physiology, thereby altering many cellular activities and ultimately contributing to the development of HBV-associated liver cancer.     

Fluctuations in mitochondrial membrane potential in single isolated brain mitochondria: modulation by adenine nucleotides and Ca2+

In this study we investigated fluctuations in mitochondrial membrane potential (DeltaPsim) in single isolated brain mitochondria using fluorescence imaging. Mitochondria were attached to coverslips and perfused with K+-based buffer containing 20 microM EDTA, supplemented with malate and glutamate, and rhodamine 123 for DeltaPsim determination. DeltaPsim fluctuations were triggered by mitochondrial Ca2+ uptake since they were inhibited by both ruthenium red, a Ca2+-uniporter blocker, and by high concentrations of EGTA. A very low concentration of Ca2+ (approximately 30 nM) was required to initiate the fluctuations. Both ATP and ADP reversibly inhibited DeltaPsim fluctuations, with maximal effects occurring at 100 microM. The effect of nucleotides could not be explained by the reversed mode of mitochondrial ATP-synthase, since oligomycin was not effective and nonhydrolysable analogs of ATP and ADP did not stop the fluctuations. The effects of adenine nucleotides were abolished by blockade of the adenine nucleotide translocator with carboxyatractyloside, but were insensitive to another inhibitor, bongkrekic acid. ATP-sensitive K+-channels are not involved in the mechanism of DeltaPsim fluctuations, since the inhibitor 5-hydroxydecanoate or the activator diazoxide did not affect dynamics of DeltaPsim. We suggest DeltaPsim fluctuations in brain mitochondria are not spontaneous, but are triggered by Ca2+ and are modulated by adenine nucleotides, possibly from the matrix side of the inner mitochondrial membrane.     

Dopamine covalently modifies and functionally inactivates parkin

Inherited mutations in PARK2, the gene encoding parkin, cause selective degeneration of catecholaminergic neurons in the substantia nigra and locus coeruleus of the brainstem, resulting in early-onset parkinsonism. But the role of parkin in common, sporadic forms of Parkinson disease remains unclear. Here we report that the neurotransmitter dopamine covalently modifies parkin in living dopaminergic cells, a process that increases parkin insolubility and inactivates its E3 ubiquitin ligase function. In the brains of individuals with sporadic Parkinson disease, we observed decreases in parkin solubility consistent with its functional inactivation. Using a new biochemical method, we detected catechol-modified parkin in the substantia nigra but not other regions of normal human brain. These findings show a vulnerability of parkin to modification by dopamine, the principal transmitter lost in Parkinson disease, suggesting a mechanism for the progressive loss of parkin function in dopaminergic neurons during aging and sporadic Parkinson disease.     

Probing the outer mitochondrial membrane in cardiac mitochondria with nanoparticles

The outer mitochondrial membrane (OMM) is the last barrier between the mitochondrion and the cytoplasm. Breaches of OMM integrity result in the release of cytochrome c oxidase, triggering apoptosis. In this study, we used calibrated gold nanoparticles to probe the OMM in rat permeabilized ventricular cells and in isolated cardiac mitochondria under quasi-physiological ionic conditions and during permeability transition. Our experiments showed that under control conditions, the OMM is not permeable to 6-nm particles. However, 3-nm particles could enter the mitochondrial intermembrane space in mitochondria of permeabilized cells and isolated cardiac mitochondria. Known inhibitors of the voltage-dependent anion channel (VDAC), K?nig polyanion, and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid inhibited this entrance. Thus, 3-nm particles must have entered the mitochondrial intermembrane space through the VDAC. The permeation of the isolated cardiac mitochondria OMM for 3-nm particles was approximately 20 times that in permeabilized cells, suggesting low availability of VDAC pores within the cell. Experiments with expressed green fluorescent protein showed the existence of intracellular barriers restricting the VDAC pore availability in vivo. Thus, our data showed that 1), the physical diameter of VDAC pores in cardiac mitochondria is >or=3 nm but 相似文献   

Reactive oxygen species and mitochondrial membrane potential are modulated during CDDP-induced apoptosis in EC-109 cells.

cis-Diamminedichloroplatinum (CDDP), commonly know as cisplatin, is a well known DNA-damaging agent, which is highly active in suppressing the proliferation of tumor cells. However, it is not clear that CDDP can induce growth inhibition of esophagus cancer cells. Using the cell line EC-109 from the esophagus, we found that CDDP would induce apoptotic responses. The addition of CDDP to cells led to the inhibition of growth in a time- and dose-dependent manner. CDDP generated reactive oxygen species (ROSs) in cells, which brought about a reduction in the intracellular mitochondrial transmembrane potential (Deltapsim), leading to apoptosis. Our findings demonstrate that ROSs, and the resulting oxidative stress, play a pivotal role in apoptosis. Preincubation of EC-109 cells with the hydrogen-peroxide-scavenging enzyme catalase partially inhibited the following: (i) the production of ROS; (ii) the disruption of the Deltapsim; and (iii) apoptosis. These results indicate that the enhancement of the generation of ROS and the disruption of Deltapsim are events involved in the apoptotic pathway of EC-109 induced by CDDP.     

Changes in mitochondrial membrane potential and accumulation of reactive oxygen species precede ultrastructural changes during ovule abortion

In many species, environmental stress reduces plant fertility. In Arabidopsis thaliana, a significant fraction of this reduction in plant fertility results from ovule abortion and embryo senescence. In this species, environmental conditions were identified that induced 94% of the developing ovules to either undergo stress-induced ovule abortion or embryo senescence (Sun et al. Plant Physiol 135:2358–2367, 2004). Following salt stress, physiological and anatomical changes were first detected in the female gametophyte of an aborting ovule. Two to four hours after a period of salt stress that induces most ovules to abort, the mitochondrial membrane potential dissipated. Subsequently, cells in the gametophyte accumulated reactive oxygen species, which are known to be molecules that promote programmed cell death (PCD). Because mitochondria often play an important role in PCD, these organelles were closely examined for changes in structure. Although the anatomy of mitochondria varied, reproducible changes in mitochondria structure were not observed. Nonetheless, other changes in ultrastructure were found. In some aborting gametophytes, concentric rings of endoplasmic reticulum were formed. In a fraction of the aborting ovules, cytoplasmic contents and organelles were invaginated into the vacuole. Even in cryofixed sections, many of these bodies appeared indistinct, which is consistent with the degradation of their contents.