Changes In ΔΨ Research Articles

Antibacterial Activity of Bifidobacterium breve Against Clostridioides difficile.

Bifidobacterium breve (YH68) is widely used in the fields of food fermentation and biomedicine. In this study, we explored the antibacterial activity of the cell free culture supernatant (CFCS) of YH68 against Clostridioides difficile ATCC 9689 (CD) by measuring multiple indexes, including the growth, spores production, toxin A/B production, and the expression levels of the tcdA and tcdB genes of CD. In addition, we examined the changes in major cellular functional groups, structures, permeability, integrity, and the proton motive force (PMF) of the cytoplasmic membrane. The results showed that double-dilution ratio of YH68-CFCS (3 × 109 CFU/mL) was the MIC value. The cell density, spores production, and the toxin production of CD treated with YH68-CFCS were lower than that of the control (p < 0.05). In addition, the gene expression levels of tcdA and tcdB in CD treated with YH68-CFCS were significant downregulated (p < 0.05). Marked differences were observed in the cell membrane and cell wall by a FT-IR spectroscopy and SEM. Analysis of the cell membrane permeability and integrity of the CD cells revealed that YH68-CFCS induced the leakage of a large amount of intracellular K+, inorganic phosphate, ATP, nucleic acids and proteinaceous substances. Furthermore, PMF analysis indicated that there was a significant change in Δψ and ΔpH. These findings demonstrated that the antibacterial activity of YH68-CFCS against CD involved the inhibition of growth, spore production, toxin production, and virulence genes expression; a consumption of PMF in the cytoplasmic membrane, the formation of pore in the cell membrane, together with the enhanced cell membrane permeability; and, eventually, cell completely disintegration.

P-123 - Interaction of NO with mitochondrial Complex III: functional consequences and mechanism

Previous results from our laboratory showed that NO inhibits complex III producing antimycin-like effects regarding to the enhancements of [UQH•]ss, [cyt. b2+], and O2•- and H2O2 productions. In this work, the effects of NO solution (obtained by bubbling NO gas; 10% in N2), on complex III enriched fraction -isolated from bovine heart- were studied. Complex III activity was hyperbolic inhibited by NO (IC50=225 nM). The absorbance spectra of complex III exposed to NO showed the characteristic peak of cyt. bH2+ (562 nm). In coupled mitochondria, 1 µM NO inhibited succinate-sustained state 3 O2 consumption (50%). Addition of HbO2 recovered O2 consumption by 90%; the remaining 10% was insensitive to NO scavenging, suggesting the blockage of complex III by NO. Moreover, mitochondria exposed to NO showed a less change in ΔΨ (30%) in the transition from state 4 to state 3 respiration, lowering ATP synthesis capacity. According to [UQH•]ss enhancement detected by EPR, H2O2 production rate was augmented (55%). Altogether, these results could be explained through a kinetic model which considers the interaction of NO with cyt. bH and, consequently, the inhibition of electron transfer between cytochromes b, the formation of UQH•, and the increase of O2•- and H2O2 production rates.

Erastin-Like Anti-Warburg Agents Prevent Mitochondrial Depolarization Induced by Free Tubulin and Decrease Lactate Formation in Cancer Cells

In Warburg metabolism, suppression of mitochondrial metabolism contributes to a low cytosolic ATP/ADP ratio favoring enhanced aerobic glycolysis. Flux of metabolites across the mitochondrial outer membrane occurs through voltage-dependent anion channels (VDAC). In cancer cells, free dimeric tubulin induces VDAC closure and dynamically regulates mitochondrial membrane potential (ΔΨ). Erastin, a small molecule that binds to VDAC, antagonizes the inhibitory effect of tubulin on VDAC and hyperpolarizes mitochondria in intact cells. Here, our aim was to identify novel compounds from the ChemBridge DIVERSet library that block the inhibitory effect of tubulin on ΔΨ using cell-based screening. HCC4006 cells were treated with nocodazole (NCZ) to increase free tubulin and decrease ΔΨ in the presence or absence of library compounds. Tetramethylrhodamine methylester (TMRM) fluorescence was assessed by high-content imaging to determine changes in ΔΨ. Compounds were considered positive if ΔΨ increased in the presence of NCZ. Using confocal microscopy, we identified and validated six lead molecules that antagonized the depolarizing effect of NCZ. Lead compounds and erastin did not promote microtubule stabilization, so changes in ΔΨ were independent of tubulin dynamics. The most potent lead compound also decreased lactate formation. These novel small molecules represent a potential new class of anti-Warburg drugs.

Assessing Mitochondrial Function in In Vitro and Ex Vivo Models of Huntington's Disease.

Mitochondrial dysfunction has gained a preponderant role in the pathogenesis of Huntington's disease (HD). Mutant huntingtin (mHTT) directly interacts with mitochondria in a deleterious manner. As the central hub of the cell, not only mitochondrial bioenergetics is affected but there is also diminished mitochondrial membrane potential (Δψ m) and altered production of reactive oxygen species (ROS). Restoration of mitochondrial function has proven to be a major player in the search and establishment of therapeutics for HD patients. As such, performing an overall study of mitochondrial function is crucial. In this chapter, we describe some methodologies used to study mitochondrial function by determining the oxygen consumption, changes in Δψ m, mitochondrial calcium handling, and levels of mitochondrial ROS. Here we focus on biological samples derived from HD versus control cells and/or animal models, namely functional isolated brain mitochondria, an ex vivo animal model, and cultured cells, including cell lines and primary neural cultures, as in vitro models.

Induction of apoptosis of liver cancer cells by nanosecond pulsed electric fields (nsPEFs).

The application of nanosecond pulsed electric fields (nsPEFs) is a novel method to induce the death of cancer cells. NsPEFs could directly function on the cell membrane and activate the apoptosis pathways, then induce apoptosis in various cell lines. However, the nsPEFs-inducing-apoptosis action sites and the exact pathways are not clear now. In this study, nsPEFs were applied to the human liver cancer cells HepG2 with different parameters. By apoptosis assay, morphological observation, detecting the mitochondrial membrane potential (ΔΨ m), intracellular calcium ion concentration ([Ca2+]i) and the expressions of key apoptosis factors, we demonstrated that nsPEFs could induce the morphology of cell apoptosis, the change in ΔΨ m, [Ca2+]i and the upregulation of some key apoptosis factors, which revealed the responses of liver cancer cells and indicated that cells may undergo apoptosis through the mitochondria-dependent pathway after nsPEFs were applied.

Identification and Characterization of Mitochondrial Subtypes in Caenorhabditis elegans via Analysis of Individual Mitochondria by Flow Cytometry.

Mitochondrial bioenergetics has been implicated in a number of vital cellular and physiological phenomena, including aging, metabolism, and stress resistance. Heterogeneity of the mitochondrial membrane potential (Δψ), which is central to organismal bioenergetics, has been successfully measured via flow cytometry in whole cells but rarely in isolated mitochondria from large animal models. Similar studies in small animal models, such as Caenorhabditis elegans (C. elegans), are critical to our understanding of human health and disease but lack analytical methodologies. Here we report on new methodological developments that make it possible to investigate the heterogeneity of Δψ in C. elegans during development and in tissue-specific studies. The flow cytometry methodology described here required an improved collagenase-3-based mitochondrial isolation procedure and labeling of mitochondria with the ratiometric fluorescent probe JC-9. To demonstrate feasibility of tissue-specific studies, we used C. elegans strains expressing blue-fluorescent muscle-specific proteins, which enabled identification of muscle mitochondria among mitochondria from other tissues. This methodology made it possible to observe, for the first time, critical changes in Δψ during C. elegans larval development and provided direct evidence of the elevated bioenergetic status of muscle mitochondria relative to their counterparts in the rest of the organism. Further application of these methodologies can help tease apart bioenergetics and other biological complexities in C. elegans and other small animal models used to investigate human disease and aging.

Moderate dependence of reactive oxygen species production on membrane potential in avian muscle mitochondria oxidizing glycerol 3-phosphate.

Mitochondria are a major source of reactive oxygen species production in cells, and the production level is sensitive to the magnitude of the membrane potential (ΔΨ). The present study investigated the level of superoxide production in mitochondria oxidizing glycerol 3-phosphate (GP) and its dependence on ΔΨ in isolated avian muscle mitochondria. The levels of superoxide produced in mitochondria oxidizing GP were lower than those obtained with succinate and were similar to those obtained with NADH-linked substrates (glutamate/malate/pyruvate). The dependence of superoxide production on ΔΨ in mitochondria oxidizing GP was lower than that of mitochondria oxidizing succinate, and a weak dependence of GP-supported superoxide production on ΔΨ was observed in the presence of NADH-linked substrates or succinate. These results suggest that the levels of superoxide generated in response to GP are quantitatively low, but they are unsusceptible to changes in ΔΨ in avian muscle mitochondria.

Evidence against a Ca(2+)-induced potentiation of dehydrogenase activity in pancreatic beta-cells.

Pancreatic beta-cells respond to an unchanging stimulatory glucose concentration with oscillations in membrane potential (Vm), cytosolic Ca(2+) concentration ([Ca(2+)]c), and insulin secretion. The underlying mechanisms are largely ascertained. Some particular details, however, are still in debate. Stimulus-secretion coupling (SSC) of beta-cells comprises glucose-induced Ca(2+) influx into the cytosol and thus into mitochondria. It is suggested that this activates (mitochondrial) dehydrogenases leading to an increase in reduction equivalents and ATP production. According to SSC, a glucose-induced increase in ATP production would thus further augment ATP production, i.e. induce a feed-forward loop that is hardly compatible with oscillations. Consistently, other studies favour a feedback mechanism that drives oscillatory mitochondrial ATP production. If Ca(2+) influx activates dehydrogenases, a change in [Ca(2+)]c should increase the concentration of reduction equivalents. We measured changes in flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) autofluorescence in response to changes in glucose concentration or glucose-independent changes in [Ca(2+)]c. The FAD signal was altered by glucose but not by alterations in [Ca(2+)]c. NAD(P)H was increased by glucose but even decreased by Ca(2+) influx evoked by tolbutamide. The mitochondrial membrane potential ΔΨ was hyperpolarized by 4mM glucose. As adding tolbutamide then depolarized ΔΨ, we deduce that Ca(2+) does not activate mitochondrial activity but by contrast even inhibits it by reducing the driving force for ATP production. Inhibition of Ca(2+) influx reversed the Ca(2+)-induced changes in ΔΨ and NAD(P)H. The results are consistent with a feedback mechanism which transiently and repeatedly reduces ATP production and explain the oscillatory activity of pancreatic beta-cells at increased glucose concentrations.

Toward high-content screening of mitochondrial morphology and membrane potential in living cells

Mitochondria are double membrane organelles involved in various key cellular processes. Governed by dedicated protein machinery, mitochondria move and continuously fuse and divide. These “mitochondrial dynamics” are bi-directionally linked to mitochondrial and cell functional state in space and time. Due to the action of the electron transport chain (ETC), the mitochondrial inner membrane displays a inside-negative membrane potential (Δψ). The latter is considered a functional readout of mitochondrial “health” and required to sustain normal mitochondrial ATP production and mitochondrial fusion. During the last decade, live-cell microscopy strategies were developed for simultaneous quantification of Δψ and mitochondrial morphology. This revealed that ETC dysfunction, changes in Δψ and aberrations in mitochondrial structure often occur in parallel, suggesting they are linked potential targets for therapeutic intervention. Here we discuss how combining high-content and high-throughput strategies can be used for analysis of genetic and/or drug-induced effects at the level of individual organelles, cells and cell populations.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.

Advances around technologies investigating mitochondrial function and insights gained by their applications.

Diabetes mellitus is defined as a state of persistently high levels of blood glucose and insulin deficiency, and resistance to it is considered as the cause(s). However, the metabolic abnormalities of diabetes are not limited to glucose, but encompass the entire metabolism. Insulin controls not only glucose, but fat and protein metabolism as well. Furthermore, body structures of patients with this disease become abnormal, termed complications, which eventually lead to the death of patients.

Astrocytic Mitochondrial Membrane Hyperpolarization following Extended Oxygen and Glucose Deprivation

Astrocytes can tolerate longer periods of oxygen and glucose deprivation (OGD) as compared to neurons. The reasons for this reduced vulnerability are not well understood. Particularly, changes in mitochondrial membrane potential (Δψm) in astrocytes, an indicator of the cellular redox state, have not been investigated during reperfusion after extended OGD exposure. Here, we subjected primary mouse astrocytes to glucose deprivation (GD), OGD and combinations of both conditions varying in duration and sequence. Changes in Δψm, visualized by change in the fluorescence of JC-1, were investigated within one hour after reconstitution of oxygen and glucose supply, intended to model in vivo reperfusion. In all experiments, astrocytes showed resilience to extended periods of OGD, which had little effect on Δψm during reperfusion, whereas GD caused a robust Δψm negativation. In case no Δψm negativation was observed after OGD, subsequent chemical oxygen deprivation (OD) induced by sodium azide caused depolarization, which, however, was significantly delayed as compared to normoxic group. When GD preceded OD for 12 h, Δψm hyperpolarization was induced by both GD and subsequent OD, but significant interaction between these conditions was not detected. However, when GD was extended to 48 h preceding OGD, hyperpolarization enhanced during reperfusion. This implicates synergistic effects of both conditions in that sequence. These findings provide novel information regarding the role of the two main substrates of electron transport chain (glucose and oxygen) and their hyperpolarizing effect on Δψm during substrate deprivation, thus shedding new light on mechanisms of astrocyte resilience to prolonged ischemic injury.

Role of the LytSR Two-Component Regulatory System in Adaptation to Cationic Antimicrobial Peptides in Staphylococcus aureus

Many host defense cationic antimicrobial peptides (HDPs) perturb the staphylococcal cell membrane (CM) and alter transmembrane potential (ΔΨ) as key parts of their lethal mechanism. Thus, a sense-response system for detecting and mediating adaptive responses to such stresses could impact organism survival; the Staphylococcus aureus LytSR two-component regulatory system (TCRS) may serve as such a ΔΨ sensor. One well-known target of this system is the lrgAB operon, which, along with the related cidABC operon, has been shown to be a regulator in the control of programmed cell death and lysis. We used an isogenic set of S. aureus strains: (i) UAMS-1, (ii) its isogenic ΔlytS and ΔlrgAB mutants, and (iii) plasmid-complemented ΔlytSR and ΔlrgAB mutants. The ΔlytS strain displayed significantly increased in vitro susceptibilities to all HDPs tested (neutrophil-derived human neutrophil peptide 1 [hNP-1], platelet-derived thrombin-induced platelet microbicidal proteins [tPMPs], and the tPMP-mimetic peptide RP-1), as well as to calcium-daptomycin (DAP), a cationic antimicrobial peptide (CAP). In contrast, the ΔlrgAB strain exhibited no significant changes in susceptibilities to these cationic peptides, indicating that although lytSR positively regulates transcription of lrgAB, increased HDP/CAP susceptibilities in the ΔlytS mutant were lrgAB independent. Further, parental UAMS-1 (but not the ΔlytS mutant) became more resistant to hNP-1 and DAP following pretreatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) (a CM-depolarizing agent). Of note, lytSR-dependent survival against CAP/HDP killing was not associated with changes in either surface positive charge, expression of mprF and dlt, or CM fluidity. The ΔlytS strain (but not the ΔlrgAB mutant) displayed a significant reduction in target tissue survival in an endocarditis model during DAP treatment. Collectively, these results suggest that the lytSR TCRS plays an important role in adaptive responses of S. aureus to CM-perturbing HDPs/CAPs, likely by functioning as a sense-response system for detecting subtle changes in ΔΨ.

Ca2+‐induced mitochondrial permeability transition pore opening is substrate‐dependent

Mitochondrial permeability transition pore (mPTP) opening is a key event in ischemia and reperfusion (I/R) injury. Several factors modulate mPTP opening, of which the increase in mitochondrial free Ca2+ plays a critical role. During I/R, there is a substrate switch in mitochondria and it is not known with certainty if/how substrate conditions influence mPTP opening. Hence, we investigated Ca2+‐induced mPTP opening by monitoring Ca2+ retention capacity (CRC) under different substrate conditions. Guinea pig heart mitochondria were suspended in respiration media containing rhodamine 123 or indo‐1 to monitor changes in ΔΨ or extra matrix [Ca2+], respectively. Either pyruvate or succinate was added first, followed by continuous infusion of CaCl2. ΔΨ became depolarized at 275±25 s or at 687±37 s with pyruvate or succinate, respectively. Indo‐1 experiments showed mitochondrial Ca2+ release in the buffer that paralleled ΔΨ depolarization. Adding cyclosporine A prolonged CRC with both substrates indicating that ΔΨ depolarization and Ca2+ release were due to mPTP opening. It is widely accepted that mPTP opening occurs upon reperfusion due to normalization of cytosolic pH. However, our results suggest a role for the complex I substrate pyruvate in Ca2+‐induced mPTP opening and thus, mitochondrial utilization of succinate during reperfusion may prove beneficial against mPTP opening.

SIRT3 protects from hypoxia and staurosporine-mediated cell death by maintaining mitochondrial membrane potential and intracellular pH

Mitochondrial sirtuin 3 (SIRT3) mediates cellular resistance toward various forms of stress. Here, we show that in mammalian cells subjected to hypoxia and staurosporine treatment SIRT3 prevents loss of mitochondrial membrane potential (ΔΨ(mt)), intracellular acidification and reactive oxygen species accumulation. Our results indicate that: (i) SIRT3 inhibits mitochondrial permeability transition and loss of membrane potential by preventing HKII binding to the mitochondria, (ii) SIRT3 increases catalytic activity of the mitochondrial carbonic anhydrase VB, thereby preventing intracellular acidification, Bax activation and apoptotic cell death. In conclusion we propose that, in mammalian cells, SIRT3 has a central role in connecting changes in ΔΨ(mt), intracellular pH and mitochondrial-regulated apoptotic pathways.

Optical imaging of mitochondrial function uncovers actively propagating waves of mitochondrial membrane potential collapse across intact heart

Polarization of the mitochondrial membrane potential (ΔΨ m) is critical for normal mitochondrial function and cellular energetics. Mitochondrial dysfunction, manifesting as disrupted ΔΨ m polarization (i.e. depolarization or hyperpolarization), underlies several important and highly prevalent diseases, including a variety of cardiac and neurological disorders. As such, ΔΨ m instability might form a unifying mechanism for a class of metabolic disorders affecting excitable tissues. Here, we measured the spatio-temporal kinetics of ΔΨ m changes across the intact heart using high-resolution optical ΔΨ m imaging and uncovered surprisingly complex spatial patterns and dynamically fluctuating changes in ΔΨ m that developed into actively propagating waves of mitochondrial depolarization during global ischemia. Our data further indicated that the recovery of ΔΨ m upon reperfusion is dictated by the duration of the preceding ischemic insult. Post-ischemic electrical and functional recovery was dependent on early ΔΨ m recovery but independent of overall cellular injury measured using a standard assay of lactate dehydrogenase release. These findings reveal a novel mechanism by which instabilities in cellular energetic properties that are independent of irreversible cellular injury can scale to the level of the intact organ via an organized process of active conduction involving the multi-cellular network. This highlights the importance of investigating cellular metabolic properties in the context of the intact organ.

Sap velocity as an indicator of diurnal and long-term hydraulic resistance changes in mature ‘Walker’ hybrid poplar treesThis article is one of a selection of papers published in the Special Issue on Poplar Research in Canada.

Hydraulic resistance directly limits sap flow in trees so that understanding water-use patterns in poplar trees requires knowledge about diurnal and long-term changes in resistances in the soil–plant–atmosphere continuum. Granier thermal dissipation probes were used for continuous measurements of sap velocity (V) changes over time and whole-tree water-use patterns in the main stems of ‘Walker’ hybrid poplar trees. When atmospheric demand was low, V depended only on solar radiation (Rg) and vapour pressure difference (VPD). However, on warm, sunny days, midday V decreased, despite a constant leaf water potential (Ψl) of –1.5 MPa. Because bulk soil water potential (Ψs) did not change significantly, the constant Ψl implied that the water potential difference (ΔΨ) remained diurnally constant. Although stomatal control would explain changes in V when VPD was low, an increase in hydraulic resistance was required to explain the decrease in V when VPD was high. Xylem cavitation was ruled out as a cause because the apparent increase in resistance reversed in late afternoon, while VPD was still high. Mid-August irrigation immediately increased daily V, which was attributed to a change in ΔΨ because of less negative Ψs. A gradual increase in sap flow over two weeks following irrigation implied a decrease in plant hydraulic resistance, most likely attributable to new root growth. It was concluded that hydraulic resistance changes, both diurnal and in the longer term, occurred primarily at the rhizosphere or root level.

β-Cell Mitochondria Exhibit Membrane Potential Heterogeneity That Can Be Altered by Stimulatory or Toxic Fuel Levels

beta-Cell response to glucose is characterized by mitochondrial membrane potential (Delta Psi) hyperpolarization and the production of metabolites that serve as insulin secretory signals. We have previously shown that glucose-induced mitochondrial hyperpolarization accompanies the concentration-dependent increase in insulin secretion within a wide range of glucose concentrations. This observation represents the integrated response of a large number of mitochondria within each individual cell. However, it is currently unclear whether all mitochondria within a single beta-cell represent a metabolically homogenous population and whether fuel or other stimuli can recruit or silence sizable subpopulations of mitochondria. This study offers insight into the different metabolic states of beta-cell mitochondria. We show that mitochondria display a wide heterogeneity in Delta Psi and a millivolt range that is considerably larger than the change in millivolts induced by fuel challenge. Increasing glucose concentration recruits mitochondria into higher levels of homogeneity, while an in vitro diabetes model results in increased Delta Psi heterogeneity. Exploration of the mechanism behind heterogeneity revealed that temporary changes in Delta Psi of individual mitochondria, ATP-hydrolyzing mitochondria, and uncoupling protein 2 are not significant contributors to Delta Psi heterogeneity. We identified BAD, a proapoptotic BCL-2 family member previously implicated in mitochondrial recruitment of glucokinase, as a significant factor influencing the level of heterogeneity. We suggest that mitochondrial Delta Psi heterogeneity in beta-cells reflects a metabolic reservoir recruited by an increased level of fuels and therefore may serve as a therapeutic target.

Inhibition of K + efflux prevents mitochondrial dysfunction, and suppresses caspase-3-, apoptosis-inducing factor-, and endonuclease G-mediated constitutive apoptosis in human neutrophils

Neutrophils die rapidly via apoptosis and their survival is contingent upon rescue from constitutive programmed cell death by signals from the microenvironment. In these experiments, we investigated whether prevention of K + efflux could affect the apoptotic machinery in human neutrophils. Disruption of the natural K + electrochemical gradient suppressed neutrophil apoptosis (assessed by annexin V binding, nuclear DNA content and nucleosomal DNA fragmentation) and prolonged cell survival within 24–48 h of culture. High extracellular K + (10–100 mM) did not activate extracellular signal-regulated kinase (ERK) and Akt, nor affected phosphorylation of p38 MAPK associated with constitutive apoptosis. Consistently, pharmacological blockade of ERK kinase or phosphatidylinositol 3-kinase (PI 3-kinase) did not affect the anti-apoptotic action of KCl. Inhibition of K + efflux effectively reduced, though never completely inhibited, decreases in mitochondrial transmembrane potential (ΔΨ m) that preceded development of apoptotic morphology. Changes in ΔΨ m resulted in attenuation of cytochrome c release from mitochondria into the cytosol and decreases in caspase-3 activity. Culture of neutrophils in medium containing 80 mM KCl with the pan-caspase inhibitor Z-VAD-FMK resulted in slightly greater suppression of apoptosis than KCl alone. High extracellular KCl also attenuated translocation of apoptosis-inducing factor (AIF) and endonuclease G (EndoG) from mitochondria to nuclei. The DNase inhibitor, aurintricarboxylic acid (ATA) partially inhibited nucleosomal DNA fragmentation, and the effects of ATA and 80 mM KCl were not additive. These results show that prevention of K + efflux promotes neutrophil survival by suppressing apoptosis through preventing mitochondrial dysfunction and release of the pro-apoptotic proteins cytochrome c, AIF and EndoG independent of ERK, PI 3-kinase and p38 MAPK. Thus, K + released locally from damaged cells may function as a survival signal for neutrophils.

Effect of Bcl-X L overexpression on reactive oxygen species, intracellular calcium, and mitochondrial membrane potential following injury in astrocytes

Many studies have demonstrated the protective effects of Bcl-x L against both apoptotic and necrotic cell death, but the mode of action of Bcl-x L remains unclear. This work analyzed effects of Bcl-x L overexpression on cellular levels of reactive oxygen species (ROS), intracellular calcium ([Ca 2+] i), and mitochondrial membrane potential (ΔΨ m) in cultured mouse primary astrocytes after exposure to glucose deprivation (GD) or hydrogen peroxide (H 2O 2). Upon exposure to GD or H 2O 2, uninfected and Lac-Z-expressing astrocytes showed an immediate, rapid increase in ROS accumulation that was slowed and or reduced by Bcl-x L. Changes in ΔΨ m in response to the two insults differed. H 2O 2 induced a decrease in ΔΨ m that was initially greater in Bcl-x L cells, but then held stable. ΔΨ m in control and Lac-Z-expressing cells initially declined more slowly, but after about 20 min showed rapid deterioration. Five hours of GD caused mitochondrial membrane hyperpolarization followed by a decrease in ΔΨ m, which was not observed with Bcl-x L overexpression. Bcl-x L failed to inhibit the calcium dysregulation seen in control cells exposed to 400 μM H 2O 2, but still improved cell survival. There was no increase in [Ca 2+] i with 5 h of GD. These data thus dissociate the effect of Bcl-x L on calcium homeostasis from effects on ROS, ΔΨ m, and for H 2O 2 exposure, cell survival.

Simulation of cardiac work transitions, in vitro: effects of simultaneous Ca2+and ATPase additions on isolated porcine heart mitochondria

During increases in cardiac work there are net increases in cytosolic [Ca2+] and ATP hydrolysis by myofiliments and ion transport ATPases. However, it is still unclear what role Ca2+or the ATP hydrolysis products, ADP and Pi, have on the regulation of mitochondrial ATP production. In this study, work jumps were simulated by simultaneous additions of Ca2+and ATPase to porcine heart mitochondria. The net effects on the mitochondrial ATP production were monitored by simultaneously monitoring respiration (mVo2), [NADH], [ADP] and membrane potential (δc) at 37°C. Addition of exogenous ATPase (300mlU.ml−1)]ATP (3.4mM) was used to generate a 'resting' background production of ADP. This resting metabolic rate was 200% higher than the quiescent rate while [NADH] and δψ were reduced. Subsequent ATPase additions (1.3IU.ml−) were made with varying amounts of Ca2+(0 to 535nM) to simulate step increases in cardiac work. Ca2+additions increased mVo2and depolarized δψ, and were consistent with an activation of Fo/F1ATPase. In contrast, Ca2+reduced the [NADH] response to the ATPase addition, consistent with Ca2+-sensitive dehydrogenase activity (CaDH). The calculated free ADP response to ATPase decreased \\2-fold in the presence of Ca2+. The addition of 172nM free Ca2+] ATPase increased mVo2by 300% (P<0.05, n=8) while δψ decreased by 14.9±0.1mV without changes in [NADH] (P≥0.05, n=8), consistent with working heart preparations. The addition of Ca2+and ATPase combined increased the mitochondrial ATP production rate with changes in δψ, NADH and [ADP], consistent with an activation of CaDH and F o /F1ATPase activity. These balancing effects of ATPase activity and [Ca2+] may explain several aspects of metabolic regulation in the heart during work transitions in vivo.