Changes In Delta Psi Research Articles

Neuroprotective Effect of KB-R7943 Against Glutamate Excitotoxicity is Related to Mild Mitochondrial Depolarization

KB-R7943, an inhibitor of a reversed Na(+)/Ca(2+) exchanger, exhibits neuroprotection against glutamate excitotoxicity. Taking into consideration that prolonged exposure of neurons to glutamate induces delayed calcium deregulation (DCD) and irreversible decrease of mitochondrial membrane potential (Deltapsi(mit)), we examined the effect of KB-R7943 on glutamate and kainate-induced [Ca(2+)](i) and on Deltapsi(mit) changes in rat cultured cerebellar granule neurons. 15 micromol/l KB-R7943 significantly delayed the onset of DCD in response to kainate but not in response to glutamate. In spite of [Ca(2+)](i) overload, KB-R7943 considerably improved the [Ca(2+)](i) recovery and restoration of Deltapsi(mit) after glutamate and kainate washout and increased cell viability after glutamate exposure. In resting neurons, KB-R7943 induced a statistically significant decrease in Deltapsi(mit). KB-R7943 also depolarized isolated brain mitochondria and slightly inhibited mitochondrial Ca(2+) uptake. These findings suggest that mild mitochondrial depolarization and diminution of Ca(2+) accumulation in the organelles might contribute to neuroprotective effect of KB-R7943.

The targeting of cyclophilin D by RNAi as a novel cardioprotective therapy: evidence from two-photon imaging

An opening of the mitochondrial permeability transition pore (MPTP), which leads to the loss of mitochondrial membrane potential (DeltaPsi(m)), is the earliest event that commits cells to death, and this process is potentially a prime target for therapeutic intervention against myocardial ischaemia/reperfusion. We aimed to investigate the protective effects of RNA interference (RNAi)-mediated gene silencing of cyclophilin D (CypD), one of the putative components of the MPTP, against myocardial ischaemia/reperfusion using two-photon laser scanning microscopy. We created an adenovirus carrying short-interfering RNA (siRNA) that inactivates CypD. Transduction of CypD-siRNA in rat cardiomyocytes achieved a 61% reduction in CypD mRNA and a 63% reduction in protein levels as well as protection against oxidant-induced DeltaPsi(m) loss and cytotoxicity. To further investigate the effects in vivo, we monitored the spatio-temporal changes of DeltaPsi(m) in perfused rat hearts subjected to ischaemia/reperfusion using two-photon imaging. Adult rats received direct intramyocardial injections of the adenovirus. Two to three days after injection, rat hearts were perfused by the Langendorff method and DeltaPsi(m) levels of individual cells were monitored. The progressive loss of DeltaPsi(m) during ischaemia/reperfusion was significantly suppressed in CypD-siRNA-transduced cells compared with non-transduced cells. Furthermore, the protective effect of CypD-siRNA was dose-dependent. Therapeutic interventions designed to inactivate CypD may be a promising strategy for reducing cardiac injury against myocardial ischaemia/reperfusion. The two-photon imaging technique provides deeper insight into cardioprotective therapy that targets mitochondria.

Cytoplasmic Na+‐dependent modulation of mitochondrial Ca2+ via electrogenic mitochondrial Na+–Ca2+ exchange

To clarify the role of mitochondrial Na(+)-Ca(2+) exchange (NCX(mito)) in regulating mitochondrial Ca(2+) (Ca(2+)(mito)) concentration at intact and depolarized mitochondrial membrane potential (DeltaPsi(mito)), we measured Ca(2+)(mito) and DeltaPsi(mito) using fluorescence probes Rhod-2 and TMRE, respectively, in the permeabilized rat ventricular cells. Applying 300 nm cytoplasmic Ca(2+) (Ca(2+)(c)) increased Ca(2+)(mito) and this increase was attenuated by cytoplasmic Na(+) (Na(+)(c)) with an IC(50) of 2.4 mm. To the contrary, when DeltaPsi(mito) was depolarized by FCCP, a mitochondrial uncoupler, Na(+)(c) enhanced the Ca(2+)(c)-induced increase in Ca(2+)(mito) with an EC(50) of about 4 mm. This increase was not significantly affected by ruthenium red or cyclosporin A. The inhibition of NCX(mito) by CGP-37157 further increased Ca(2+)(mito) when DeltaPsi(mito) was intact, while it suppressed the Ca(2+)(mito) increase when DeltaPsi(mito) was depolarized, suggesting that DeltaPsi(mito) depolarization changed the exchange mode from forward to reverse. Furthermore, DeltaPsi(mito) depolarization significantly reduced the Ca(2+)(mito) decrease via forward mode, and augmented the Ca(2+)(mito) increase via reverse mode. When the respiratory chain was attenuated, the induction of the reverse mode of NCX(mito) hyperpolarized DeltaPsi(mito), while DeltaPsi(mito) depolarized upon inducing the forward mode of NCX(mito). Both changes in DeltaPsi(mito) were remarkably inhibited by CGP-37157. The above experimental data indicated that NCX(mito) is voltage dependent and electrogenic. This notion was supported theoretically by computer simulation studies with an NCX(mito) model constructed based on present and previous studies, presuming a consecutive and electrogenic Na(+)-Ca(2+) exchange and a depolarization-induced increase in Na(+) flux. It is concluded that Ca(2+)(mito) concentration is dynamically modulated by Na(+)(c) and DeltaPsi(mito) via electrogenic NCX(mito).

Neuroprotectant FK506 inhibits glutamate‐induced apoptosis of astrocytes in vitro and in vivo

Neuron-astrocyte interactions are critical for signalling, energy metabolism, extracellular ion and glutamate homeostasis, volume regulation and neuroprotection in the CNS. Glutamate uptake by astrocytes may prevent excitotoxic glutamate elevation and determine neuronal survival. However, an excess of glutamate can cause the death of astrocytes. FK506, an inhibitor of calcineurin, and an immunosuppressive drug, is neuroprotective in animal models of neurologic diseases, including focal and global ischaemia. In the present work, we demonstrate that a single injection of FK506 60 min after a transient middle cerebral artery occlusion (MCAo) significantly decreases the number of terminal deoxynucleotidyl transferase nick-end labelling (TUNEL)-positive cells in the ischaemic cortex and striatum. Using 3-D confocal microscopy we found that, 24 h after MCAo, many TUNEL-positive cells in the ischaemic striatum and cortex are astrocytes. Furthermore, we demonstrate that exposure of cultured cortical astrocytes to 50-100 mM Glu for 24 h induces apoptotic alterations in nuclear morphology, DNA fragmentation, dissipation of mitochondrial transmembrane potential (DeltaPsi) and caspase activation. FK506 (1 muM) efficiently inhibits Glu-induced apoptosis of cultured astrocytes, DNA fragmentation and changes in mitochondrial DeltaPsi. Our findings suggest that modulation of glutamate-induced astrocyte death early after reperfusion may be a novel mechanism of FK506-mediated neuroprotection in ischaemia.

Mg2+ release coupled to Ca2+ uptake: A novel Ca2+ accumulation mechanism in rat liver

Isolated hepatocytes release 2-3 nmol Mg2+/mg protein or approximately 10% of the total cellular Mg2+ content within 2 minutes from the addition of agonists that increase cellular cAMP, for example, isoproterenol (ISO). During Mg2+ release, a quantitatively similar amount of Ca2+ enters the hepatocyte, thus suggesting a stoichiometric exchange ratio of 1 Mg2+:1Ca2+. Calcium induced Mg2+ extrusion is also observed in apical liver plasma membranes (aLPM), in which the process presents the same 1 Mg2+:1Ca2+ exchange ratio. The uptake of Ca2+ for the release of Mg2+ occurs in the absence of significant changes in Deltapsi as evidenced by electroneutral exchange measurements with a tetraphenylphosphonium (TPP+) electrode or 3H-TPP+. Collapsing the Deltapsi by high concentrations of TPP+ or protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) does not inhibit the Ca2+-induced Mg2+ extrusion in cells or aLPM. Further, the process is strictly unidirectional, serving only in Ca2+ uptake and Mg2+ release. These data demonstrate the operation of an electroneutral Ca2+/Mg2+ exchanger which represents a novel pathway for Ca2+ accumulation in liver cells following adrenergic receptor stimulation.

Normal Human Fibroblasts Exposed to High- or Low-Dose Ionizing Radiation: Differential Effects on Mitochondrial Protein Import and Membrane Potential

How oxidative metabolism modulates effects of ionizing radiation is incompletely understood. Because mitochondria participate in oxidative metabolism, we investigated the modulation of mitochondrial protein import and membrane potential (DeltaPsi) in irradiated cells. Our data show that effects at low dose cannot be predicted from effects at high dose. When density-inhibited normal human fibroblasts were exposed to a toxic dose of 4 Gy, protein import into mitochondria isolated from these cells was decreased. In contrast, protein import into mitochondria isolated from low-dose-irradiated (10 cGy) cells was enhanced, suggesting that mitochondria may play a crucial role in low-dose-induced adaptive responses. At high dose, import defects were not solely due to changes in mitochondrial DeltaPsi, and modulation of import was not tightly linked to the cellular capacity to repair radiation damage. Another striking observation is that in proliferating nonirradiated cells, mitochondrial protein import and DeltaPsi were regulated in a cell cycle-dependent manner, being lower in S phase than in G (1). Interestingly, when quiescent G (0)/G (1) phase cells exposed to high-dose radiation were stimulated to proliferate, events associated with S phase, but not G (1), significantly affected import. The strategy described here may serve as novel end points to study radiation-induced effects.

Mitochondrial membrane potential and reactive oxygen species content of endothelial and smooth muscle cells cultured on poly(ε-caprolactone) films

A transitory but significant stimulation of mitochondrial activity, increase of reactive oxygen species (ROS) and oxidative stress were previously observed in L929 fibroblasts cultured on poly(epsilon-caprolactone) (PCL) films. ROS, mainly formed in mitochondria, play a physiological role but an excessive production can promote endothelial dysfunction, cause oxidative injury to vascular cells, oxidize lipoproteins and accelerate atherothrombogenesis. On the other hand, mitochondria have a crucial position in programmed cell death control and are responsible for ATP synthesis through the coupling of oxidative phosphorylation to respiration. This coupling requires the existence of a mitochondrial membrane potential (Deltapsi(m)). The aim of the present study was to evaluate by flow cytometry the ROS content and Deltapsi(m) of both endothelial (EC) and smooth muscle cells (SMC) cultured on PCL films as a potential substrate for vascular graft development. Cell size, internal complexity and cell cycle were also analyzed to detect the possible appearance of the subG(1) cell fraction, characteristic of apoptotic cells. The effect of treating PCL films with NaOH before culture was also studied. PCL decreases the ROS content of EC during the culture but produces an increase of these levels in SMC after 7 days. PCL also induces variations of Deltapsi(m) which show a significant parallelism with the changes observed in ROS levels proving the importance and sensitivity of these measurements as indicators of the mitochondrial function. The treatment of PCL with NaOH decreases these effects demonstrating the benefits of increasing the surface hydrophilicity before cell culture which improves cell adhesion and proliferation and reduces oxidative stress. Since no important changes have been detected in subG(1) fraction of EC and SMC cultured on either PCL or PCL-NaOH, the changes of Deltapsi(m) observed in the present study cannot be related to apoptosis. These results confirm the potential utility of PCL as a suitable scaffold in Vascular Tissue Engineering.

Cytochrome c is released in a single step during apoptosis

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7 degrees C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (DeltaPsi(m)). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in DeltaPsi(m) preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in DeltaPsi(m).

Mode of Action of Lactocin 160, a Bacteriocin From Vaginal Lactobacillus rhamnosus

OBJECTIVES: To determine the mechanism of antimicrobial action of lactocin 160, a bacteriocin produced by the healthy vaginal strain of Lactobacillus rhamnosus, using an established model, with Micrococcus luteus ATCC 10420 as a test organism. METHODS: Sensitivity of M. luteus to lactocin 160 was determined by the diffusion assay. Loss of cellular ATP in the lactocin-treated cells was elucidated using a commercially available ATP determination kit (luciferin-luciferase bioluminescence assay). Luminescence intensity as a reflection of ATP quantity was determined using a luminometer. Dissipation of membrane potential (Deltapsi) was studied using fluorophore DiSC3(5) with the fluorescence spectrum sensitive to changes in Deltapsi. RESULTS: Lactocin 160 inhibited growth of M. luteus ATCC 10420 at a concentration of 5 microg/ml. There were no significant changes in the intracellular ATP level of M. luteus upon the addition of 20 microg/ml of lactocin 160. However, the extracellular ATP level increased significantly. This means that the treatment of cells with lactocin 160 resulted in an efflux of ATP from inside the cells. Therefore, a partially purified lactocin 160 preparation (16 microg /ml of the bacteriocin in the sample) killed sensitive cells and dissipated 3.12 +/- 0.36% of Deltapsi. CONCLUSION: Lactocin 160 has a mode of action typical for bacteriocins. It disturbs the cellular membrane (Deltapsi dissipation) and induces ATP efflux, most likely because of the pore formation, which is a common mechanism of action for many bacteriocins.

Alkylphosphocholine-induced glioma cell death is BCL-X(L)-sensitive, caspase-independent and characterized by massive cytoplasmic vacuole formation.

Alkylphosphocholines (APC) are candidate anticancer agents. We here report that APC induce the formation of large vacuoles and typical features of apoptosis in human glioma cell lines, but not in immortalized astrocytes. APC promote caspase activation, poly(ADP-ribose)-polymerase (PARP) processing and cytochrome c release from mitochondria. Adenoviral X-linked inhibitor of apoptosis (XIAP) gene transfer, or exposure to the caspase inhibitor, benzyloxycarbonyl-Val-Ala-DL-Asp-fluoro-methylketone zVAD-fmk, blocks caspase-7 and PARP processing, but not cell death, whereas BCL-X(L) blocks not only caspase-7 and PARP processing but also cell death. APC induce changes in Delta Psi m in sensitive glioma cells, but not in resistant astrocytes. The changes in Delta Psi m are unaffected by crm-A (cowpox serpin-cytokine response modifier protein A), XIAP or zVAD-fmk, but blocked by BCL-X(L), and are thus a strong predictor of cell death in response to APC. Free radicals are induced, but not responsible for cell death. APC thus induce a characteristic morphological, BCL-X(L)-sensitive, apparently caspase-independent cell death involving mitochondrial alterations selectively in neoplastic astrocytic cells.

Insulin-like growth factor-I regulates glucose-induced mitochondrial depolarization and apoptosis in human neuroblastoma.

Neuroblastoma, a pediatric peripheral nervous system tumor, frequently contains alterations in apoptotic pathways, producing chemoresistant disease. Insulin-like growth factor (IGF) system components are highly expressed in neuroblastoma, further protecting these cells from apoptosis. This study investigates IGF-I regulation of apoptosis at the mitochondrial level. Elevated extracellular glucose causes rapid mitochondrial enlargement coupled with an increase in the mitochondrial membrane potential (Delta Psi(M)) followed by mitochondrial membrane depolarization (MMD), uncoupling protein 3 (UCP3) downregulation, caspase-3 activation and decreased Bcl-2. MMD inhibition by Bongkrekic acid prevents high-glucose-induced loss of UCP3 and apoptosis. Glucose exposure induces caspase-9 cleavage within 30 min, and caspase-9 inhibition prevents glucose-mediated apoptosis. IGF-I prevents caspase activation and mitochondrial events leading to apoptosis. These results suggest that elevated glucose produces early initiator caspase activation, followed by Delta Psi(M) changes, in neuroblastoma cells; in turn, IGF-I prevents apoptosis by preventing downstream caspase activation, maintaining Delta Psi(M) and regulating Bcl proteins.

The cardioprotective and mitochondrial depolarising properties of exogenous nitric oxide in mouse heart.

Nitric oxide (NO) is reported to be both protective and detrimental in models of myocardial ischaemia/reperfusion injury, which may be concentration dependent. Our objective was to characterise this dichotomy using the nitric oxide donor, S-nitroso N-acetyl penicillamine (SNAP) in isolated perfused mouse heart and isolated mouse cardiac mitochondria. To determine the effect of nitric oxide concentration on myocardial viability, isolated mouse hearts were subjected to 35 min global ischaemia and 30 min reperfusion in the presence of SNAP (0.02-20 microM). To determine whether NO mediated protection was via opening of the putative mitochondrial K(ATP) channel and/or free radical synthesis, SNAP perfused hearts were also treated with the mitochondrial K(ATP) channel blocker, 5-hydroxy decanoate (5-HD) and the free-radical scavenger, N-(2-mercaptopropionyl)-glycine (MPG). This data was correlated with mitochondrial membrane potential (Delta Psi(m)), measured with the potentiometric dye, tetra-methyl rhodium methyl ester (TMRM), in isolated mitochondria,by flow cytometry. SNAP dose-dependently attenuated infarct size, with maximal protection observed at 2 microM (17+/-4% versus controls 32+/-3%, P<0.01). At greater concentrations however, protection was lost with infarct sizes tending towards control at 20 microM (29+/-3%). These results were paralleled by changes in Delta Psi(m) in the isolated mitochondria: Delta Psi(m) depolarisation peaking with 1 microM SNAP (26+/-4% shift in TMRM fluorescence, P<0.01); at greater concentrations, this relationship was lost. The mitochondrial K(ATP) channel blocker, 5-HD, resulted in both abrogation of SNAP infarct size reduction and concomitant loss of Delta Psi(m) depolarisation in the mitochondria. MPG however did not influence the cardioprotective properties of SNAP. We demonstrate that nitric oxide can mediate cardioprotection in a dose-dependent fashion by an effect that may be related to Delta Psi(m). Both cardioprotection and Delta Psi(m) changes are sensitive to 5-HD and the cardioprotection appears independent of free-radical synthesis.

Oxygen radical generation and endosulfan toxicity in Jurkat T-cells.

The mechanism by which endosulfan exposure causes cellular dysfunction in experimental animals and humans is not clear. In the present study, we provide experimental evidence in support of the role of oxidative stress in endosulfan toxicity. Using both cell free system and Jurkat cells as in vitro models, we demonstrate that endosulfan can generate oxygen radicals that is inhibitable by superoxide dismutase (SOD) and glutathione (GSH). In the cell culture model, oxygen radical generation in response to endosulfan was dose- (0-100 microM) and time-dependent (0-12 h). Two-color flowcytometric analysis showed that endosulfan mediated changes in delta psi(m) and generation of superoxide radicals do occur simultaneously in the affected cell population. It was hypothesized that endosulfan exerts a severe oxidative stress in Jurkat cells and this could be prevented or minimized by an antioxidant. To test this hypothesis, GSH was added exogenously and endosulfan toxicity was evaluated by alamarblue assay. In conclusion, our results demonstrate a role for reactive oxygen species (ROS) in endosulfan toxicity and that supplementation of antioxidants such as GSH may be useful in individuals who are at risk to endosulfan toxicity.

The mitochondrial membrane potential (deltapsi(m)) in apoptosis; an update.

Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and has even been suggested to be central to the apoptotic pathway. Indeed, opening of the mitochondrial permeability transition pore has been demonstrated to induce depolarization of the transmembrane potential (deltapsi(m)), release of apoptogenic factors and loss of oxidative phosphorylation. In some apoptotic systems, loss of deltapsi(m) may be an early event in the apoptotic process. However, there are emerging data suggesting that, depending on the model of apoptosis, the loss of deltapsi(m) may not be an early requirement for apoptosis, but on the contrary may be a consequence of the apoptotic-signaling pathway. Furthermore, to add to these conflicting data, loss of deltapsi(m) has been demonstrated to not be required for cytochrome c release, whereas release of apoptosis inducing factor AIF is dependent upon disruption of deltapsi(m) early in the apoptotic pathway. Together, the existing literature suggests that depending on the cell system under investigation and the apoptotic stimuli used, dissipation of deltapsi(m) may or may not be an early event in the apoptotic pathway. Discrepancies in this area of apoptosis research may be attributed to the fluorochromes used to detect deltapsi(m). Differential degrees of sensitivity of these fluorochromes exist, and there are also important factors that contribute to their ability to accurately discriminate changes in deltapsi(m).

Induction of apoptosis by nitric oxide in macrophages is independent of apoptotic volume decrease.

Apoptosis occurs through a sequence of specific biochemical and morphological alterations that define the progress of cell death. The changes of the mitochondrial inner membrane potential (DeltaPsi(m)), the release of cytochrome c to the cytosol, the apoptotic volume decrease (AVD) and the activation of caspases have been measured in RAW 264.7, HeLa and Jurkat T cells incubated with molecules that induce apoptosis through the mitochondrial pathway. Our data show that NO, staurosporine, etoposide and camptothecin increased DeltaPsi(m) in macrophages but not in HeLa and Jurkat cells, that exhibited a DeltaPsi(m) decrease. Moreover, the apoptosis induced by NO in macrophages, but not that promoted by staurosporine, might occur in the absence of AVD. Analysis of the sequence of apoptotic manifestations shows that DeltaPsi(m) precedes AVD and caspase activation in RAW 264.7 cells. Inhibition of AVD abrogates apoptosis in HeLa and Jurkat T cells regardless of the stimuli used. These data suggest that the changes of DeltaPsi(m) are cell-type dependent and that AVD is dispensable for apoptosis in macrophages.

Diazoxide opens the mitochondrial permeability transition pore and alters Ca2+ transients in rat ventricular myocytes.

The mitochondrial K(ATP) channel (mitoK(ATP)) has been implicated as an end effector or trigger of ischemic preconditioning (IP). Although a mitoK(ATP) opener, diazoxide, mimics IP, mechanisms for the cardioprotective action remain unclear. We measured Ca2+ transients (CaTs) and mitochondrial inner membrane potential (Deltapsi(m)) with confocal microscopy and the fluorescent probes fluo-4 and tetramethylrhodamine ethyl ester perchlorate in rat ventricular myocytes. Diazoxide increased the amplitudes and diastolic levels of CaTs dose dependently. The effects of diazoxide on CaTs were inhibited by the mitoK(ATP) antagonist sodium 5-hydroxydecanoic acid (100 micromol/L), whereas application of diazoxide caused little change in Deltapsi(m). After sarcoplasmic reticulum function was disabled with ryanodine and thapsigargin, the effects of diazoxide on CaTs were still observed. The opening of the mitochondrial permeability transition pore was monitored with fluorescent calcein. Diazoxide accelerated the leakage of calcein from mitochondrial matrix (16% of control; P<0.05), and this effect was inhibited by cyclosporin A (2 micromol/L). Cyclosporin A also abolished the effects of diazoxide on CaTs. Diazoxide oxidized flavoprotein fluorescence reversibly, and this effect was partially blunted by cyclosporin A (by 24%; P<0.05). We conclude that in rat ventricular myocytes, diazoxide modulates the opening of the mitochondrial permeability transition pore, resulting in an increase in CaTs independent of the changes in Deltapsi(m). The action of diazoxide on the mitochondrial permeability transition pore also affects the mitochondrial redox state.

Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm.

Sperm cell death appears to be a cause of male infertility. The objective of this study was to determine the most reliable method for the evaluation of sperm quality in semen samples during sperm preparation for IVF. Conventional analysis of semen samples was compared with several cytofluorometric methods detecting death-associated changes. Neat semen from infertile patients and sperm prepared by PureSperm gradient were studied by conventional microscopy and analysed for mitochondrial membrane potential (Delta Psi(m)), generation of reactive oxygen species, DNA fragmentation and cell viability. In neat semen, a positive correlation was found between the percentage of Delta Psi(m)(high) sperm cells and standard semen parameters (concentration/motility). Sperm cells depicting Delta Psi(m)(high) and cells with low DNA fragmentation displayed high fertilization rate after IVF. The only changes that could be detected in prepared sperm were changes in Delta Psi(m), with Delta Psi(m)(high) sperm positively correlated with forward motility and also with high fertilization rates after IVF. Analysis of mitochondrial membrane potential is the most sensitive test by which to determine sperm quality. These findings promise development of a test that may help to predict successful IVF.

DeltaPsi(m)-Dependent and -independent production of reactive oxygen species by rat brain mitochondria.

Mitochondria are widely believed to be the source of reactive oxygen species (ROS) in a number of neurodegenerative disease states. However, conditions associated with neuronal injury are accompanied by other alterations in mitochondrial physiology, including profound changes in the mitochondrial membrane potential DeltaPsi(m). In this study we have investigated the effects of DeltaPsi(m) on ROS production by rat brain mitochondria using the fluorescent peroxidase substrates scopoletin and Amplex red. The highest rates of mitochondrial ROS generation were observed while mitochondria were respiring on the complex II substrate succinate. Under this condition, the majority of the ROS signal was derived from reverse electron transport to complex I, because it was inhibited by rotenone. This mode of ROS generation is very sensitive to depolarization of DeltaPsi(m), and even the depolarization associated with ATP generation was sufficient to inhibit ROS production. Mitochondria respiring on the complex I substrates, glutamate and malate, produce very little ROS until complex I is inhibited with rotenone, which is also consistent with complex I being the major site of ROS generation. This mode of oxidant production is insensitive to changes in DeltaPsi(m). With both substrates, ubiquinone-derived ROS can be detected, but they represent a more minor component of the overall oxidant signal. These studies demonstrate that rat brain mitochondria can be effective producers of ROS. However, the optimal conditions for ROS generation require either a hyperpolarized membrane potential or a substantial level of complex I inhibition.

Mitochondrial transmembrane potential changes support the concept of mitochondrial heterogeneity during apoptosis.

Dissipation of mitochondrial membrane potential (DeltaPsi(m)) and release of cytochrome c from mitochondria appear to be key events during apoptosis. The precise relationship (cause or consequence) between both is currently unclear. We previously showed in a model of serum-free cultured granulosa explants that cytochrome c is retained in a subset of respiring mitochondria until late in the apoptotic process. In this study we further investigated the issue of heterogeneity by using the DeltaPsi(m)-sensitive probe CM-H2TMRos in combination with a DNA fluorochrome. Changes of DeltaPsi(m) were assessed qualitatively by epifluorescence microscopy and were quantified using digital imaging microscopy. This approach yielded the following results: (a) CM-H2TMRos staining is a reliable and specific procedure to detect DeltaPsi(m) changes in granulosa cells explants; (b) dissipation of transmembrane potential is an early event during apoptosis preceding nuclear changes but is confined to a subpopulation of mitochondria within an individual cell; (c) in frankly apoptotic cells a few polarized mitochondria can be detected. These findings support the hypothesis that ATP needed for completion of the apoptotic cascade can be generated during apoptosis in a subset of respiring mitochondria and is not necessarily derived from anaerobic glycolysis.

Resting membrane potential as a marker of apoptosis: studies on Xenopus oocytes microinjected with cytochrome c.

Observation of the electrical potential difference across the cell membrane is described as a new method for monitoring apoptosis of a single cell. The resting membrane potential (DeltaPsi) of Xenopus oocytes has been recorded in real time following microinjection of cytochrome c. Soon after microinjection, DeltaPsi becomes less negative and attains a new constant value with a half time, t(m), of about 35 (+ /- 5) min at all cytochrome c concentrations greater than 1 microM. The cytosol extract of cytochrome c-injected oocytes shows DEVD proteolytic activity characteristic of aspartate specific proteases, implicating an apoptotic death pathway. In response to the delivery of cytochrome c into the cytosol, caspases are activated within 7 min while the changes in DeltaPsi begin to occur after about 30 min. The method described here will be potentially useful to assess the effectiveness of cell death regulators and modulators of synthetic and biological origin, and the results presented shed light on the currently debated issue of the importance of the redox state of cytochrome c in the initiation of apoptosis.