Purification and characterization of glutamate dehydrogenase as another isoprotein binding to the membrane of rough endoplasmic reticulum

Glutamate dehydrogenase (GDH) was purified from rough endoplasmic reticulum (RER) in rat liver using anion‐exchange and affinity chromatography. As GDH has been known as an enzyme that exists mainly in the matrix of mitochondria, the properties of purified GDH were compared with those of mitochondrial GDH. The GDH activity in 0.1% Triton X‐100‐treated RER subcellular fraction was nearly the same as intact RER, whereas that of the mitochondrial fraction increased by 50% after the detergent treatment. In kinetic values, in addition, mitochondrial GDH had a higher K_m value for NADP⁺ than NAD⁺, whereas the K_m value for NAD⁺ was higher than that for NADP⁺ in the case of GDH of RER, which showed a difference in specificity to cofactors. Moreover, when two GDH isoproteins were incubated at 42°C or treated with trypsin, GDH from RER was more stable against heat inactivation and less susceptible to proteolysis than mitochondrial GDH in both cases. In addition, GDH of RER had at least five amino acids different from mitochondrial GDH when sequences of N‐terminal and several internal peptide fragments were analyzed. These results showed that GDH of RER is another isoprotein of GDH, of whose properties are different from those of mitochondrial GDH. J. Cell. Biochem. 76:244–253, 1999. © 1999 Wiley‐Liss, Inc.     

Flux ratio of valinomycin-mediated K+ fluxes across the human red cell membrane in the presence of the protonophore CCCP

Summary The ratio of valinomycin-mediated unidirectional K⁺ fluxes across the human red cell membrane, has been determined in the presence of the protonophore carbonylcyanidem-chlorophenylhydrazone, CCCP, using the K⁺ net efflux and⁴²K influx. The driving force for the net efflux (V _m –E _K ⁺) has been calculated from the membrane potential, estimated by the CCCP-mediated proton distribution and the Nernst potential for potassium ions across the membrane. An apparent driving potential for the K⁺ net efflux has been calculated from the K⁺ flux ratio, determined in experiments where the valinomycin and CCCP concentrations were varied systematically. This apparent driving force, in conjunction with the actual driving force calculated on basis of the CCCP estimated membrane potential, is used to calculate a flux ratio exponent, which represents an estimate of the deviation of valinomycin-mediated K⁺ transport from unrestricted electrodiffusion, when protonophore is present.In the present work, the flux ratio exponent is found to be 0.90 when the CCCP concentration is 5.0 m and above, while the exponent decreases to about 0.50 when no CCCP is present. The influence of CCCP upon the rate constants in the valinomycin transport cycle is discussed. The significance of this result is that red cell membrane potentials are overestimated, when calculated from valinomycin-mediated potassium isotope fluxes, using a constant field equation.     

10H‐3,6‐Diazaphenothiazines triggered the mitochondrial‐dependent and cell death receptor‐dependent apoptosis pathways and further increased the chemosensitivity of MCF‐7 breast cancer cells via inhibition of AKT1 pathways

Breast cancer is one of the leading causes of death in cancer categories, followed by lung, colorectal, and ovarian among the female gender across the world. 10H‐3,6‐diazaphenothiazine (PTZ) is a thiazine derivative compound that exhibits many pharmacological activities. Herein, we proceed to investigate the pharmacological activities of PTZ toward breast cancer MCF‐7 cells as a representative in vitro breast cancer cell model. The PTZ exhibited a proliferation inhibition (IC₅₀ = 0.895 µM) toward MCF‐7 cells. Further, cell cycle analysis illustrated that the S‐phase checkpoint was activated to achieve proliferation inhibition. In vitro cytotoxicity test on three normal cell lines (HEK293 normal kidney cells, MCF‐10A normal breast cells, and H9C2 normal heart cells) demonstrated that PTZ was more potent toward cancer cells. Increase in the levels of reactive oxygen species results in polarization of mitochondrial membrane potential (ΔΨm), together with suppression of mitochondrial thioredoxin reductase enzymatic activity suggested that PTZ induced oxidative damages toward mitochondria and contributed to improved drug efficacy toward treatment. The RT² PCR Profiler Array (human apoptosis pathways) proved that PTZ induced cell death via mitochondria‐dependent and cell death receptor‐dependent pathways, through a series of modulation of caspases, and the respective morphology of apoptosis was observed. Mechanistic studies of apoptosis suggested that PTZ inhibited AKT1 pathways resulting in enhanced drug efficacy despite it preventing invasion of cancer cells. These results showed the effectiveness of PTZ in initiation of apoptosis, programmed cell death, toward highly chemoresistant MCF‐7 cells, thus suggesting its potential as a chemotherapeutic drug.     

Availability of the key metabolic substrates dictates the respiratory response of cancer cells to the mitochondrial uncoupling

Active glycolysis and glutaminolysis provide bioenergetic stability of cancer cells in physiological conditions. Under hypoxia, metabolic and mitochondrial disorders, or pharmacological treatment, a deficit of key metabolic substrates may become life-threatening to cancer cells. We analysed the effects of mitochondrial uncoupling by FCCP on the respiration of cells fed by different combinations of Glc, Gal, Gln and Pyr. In cancer PC12 and HCT116 cells, a large increase in O₂ consumption rate (OCR) upon uncoupling was only seen when Gln was combined with either Glc or Pyr. Inhibition of glutaminolysis with BPTES abolished this effect. Despite the key role of Gln, addition of FCCP inhibited respiration and induced apoptosis in cells supplied with Gln alone or Gal/Gln. For all substrate combinations, amplitude of respiratory responses to FCCP did not correlate with Akt, Erk and AMPK phosphorylation, cellular ATP, and resting OCR, mitochondrial Ca^2 + or membrane potential. However, we propose that proton motive force could modulate respiratory response to FCCP by regulating mitochondrial transport of Gln and Pyr, which decreases upon mitochondrial depolarisation. As a result, an increase in respiration upon uncoupling is abolished in cells, deprived of Gln or Pyr (Glc). Unlike PC12 or HCT116 cells, mouse embryonic fibroblasts were capable of generating pronounced response to FCCP when deprived of Gln, thus exhibiting lower dependence on glutaminolysis. Overall, the differential regulation of the respiratory response to FCCP by metabolic environment suggests that mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function, and can be explored for selective cancer treatment.     

Probing the metabolic phenotype of breast cancer cells by multiple tracer stable isotope resolved metabolomics

Breast cancers vary by their origin and specific set of genetic lesions, which gives rise to distinct phenotypes and differential response to targeted and untargeted chemotherapies. To explore the functional differences of different breast cell types, we performed Stable Isotope Resolved Metabolomics (SIRM) studies of one primary breast (HMEC) and three breast cancer cells (MCF-7, MDAMB-231, and ZR75-1) having distinct genotypes and growth characteristics, using ¹³C₆-glucose, ¹³C-1+2-glucose, ¹³C₅,¹⁵N₂-Gln, ¹³C₃-glycerol, and ¹³C₈-octanoate as tracers. These tracers were designed to probe the central energy producing and anabolic pathways (glycolysis, pentose phosphate pathway, Krebs Cycle, glutaminolysis, nucleotide synthesis and lipid turnover). We found that glycolysis was not associated with the rate of breast cancer cell proliferation, glutaminolysis did not support lipid synthesis in primary breast or breast cancer cells, but was a major contributor to pyrimidine ring synthesis in all cell types; anaplerotic pyruvate carboxylation was activated in breast cancer versus primary cells. We also found that glucose metabolism in individual breast cancer cell lines differed between in vitro cultures and tumor xenografts, but not the metabolic distinctions between cell lines, which may reflect the influence of tumor architecture/microenvironment.     

Mitochondrial uncoupler FCCP activates proton conductance but does not block store-operated Ca current in liver cells

Uncouplers of mitochondrial oxidative phosphorylation, including carbonilcyanide p-triflouromethoxyphenylhydrazone (FCCP) and carbonilcyanide m-cholorophenylhydrazone (CCCP), are widely used in experimental research to investigate the role of mitochondria in cellular function. Unfortunately, it is very difficult to interpret the results obtained in intact cells using FCCP and CCCP, as these agents not only inhibit mitochondrial potential, but may also affect membrane potential and cell volume. Here we show by whole-cell patch clamping that in primary rat hepatocytes and H4IIE liver cells, FCCP induced large proton currents across the plasma membrane, but did not activate any other observable conductance. In intact hepatocytes FCCP inhibits thapsigargin-activated store-operated Ca²⁺ entry, but in patch clamping under the conditions of strong Ca²⁺ buffering it has no effect on store-operated Ca²⁺ current (I_SOC). These results indicate that there is no direct connection between mitochondria and activation of I_SOC in liver cells and support the notion of indirect regulation of I_SOC by mitochondrial Ca²⁺ buffering.     

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells

A network model for the determination of tumor metabolic fluxes from ¹³C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-¹³C₂]glucose under normoxic conditions at 37 °C and monitored by ¹³C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.     

BCL(X)L and BCL2 increase the metabolic fitness of breast cancer cells: a single-cell imaging study

The BCL2 family of proteins regulate apoptosis by controlling mitochondrial outer membrane permeability. However, the effects on mitochondrial structure and bioenergetics have also been reported. Here we comprehensively characterized the effects of BCL2 and BCL(X)L on cellular energetics in MCF7 breast cancer cells using time-lapse confocal single-cell imaging and mitochondrial and cytosolic FRET reporters. We found that BCL2 and BCL(X)L increase the metabolic robustness of MCF7 cells, and that this was associated with increased mitochondrial NAD(P)H and ATP levels. Experiments with the F₁F₀ synthase inhibitor oligomycin demonstrated that BCL2 and in particular BCL(X)L, while not affecting ATP synthase activity, more efficiently coupled the mitochondrial proton motive force with ATP production. This metabolic advantage was associated with an increased resistance to nutrient deprivation and enhanced clonogenic survival in response to metabolic stress, in the absence of profound effects on cell death. Our data suggest that a primary function of BCL(X)L and BCL2 overexpression in tumor cells is to increase their resistance to metabolic stress in the tumor microenvironment, independent of cell death signaling.Subject terms: Cancer metabolism, Cancer metabolism     

PA‐MSHA inhibits proliferation and induces apoptosis through the up‐regulation and activation of caspases in the human breast cancer cell lines

To investigate the effects of PA‐MSHA (Pseudomonas aeruginosa‐mannose sensitive hemagglutinin) on inhibiting proliferation of breast cancer cell lines and to explore its mechanisms of action in human breast cancer cells. MCF‐10A, MCF‐7, MDA‐MB‐468, and MDA‐MB‐231HM cells were treated with PA‐MSHA or PA (Heat‐killed P. aeruginosa) at different concentrations and different times. Changes of cell super‐microstructure were observed by transmission electron microscopy. Cell cycle distribution and apoptosis induced by PA‐MSHA were measured by flow cytometry (FCM) with PI staining, ANNEXIN V‐FITC staining and Hoechst33258 staining under fluorescence microscopy. Western blot was used to evaluate the expression level of apoptosis‐related molecules. A time‐dependent and concentration‐dependent cytotoxic effect of PA‐MSHA was observed in MDA‐MB‐468 and MDA‐MB‐231HM cells but not in MCF‐10A or MCF‐7 cells. The advent of PA‐MSHA changed cell morphology, that is to say, increases in autophagosomes, and vacuoles in the cytoplasm could also be observed. FCM with PI staining, ANNEXIN V‐FITC and Hoechst33258 staining showed that the different concentrations of PA‐MSHA could all induce the apoptosis and G₀–G₁ cell cycle arrest of breast cancer cells. Cleaved caspase 3, 8, 9, and Fas protein expression levels were strongly associated with an increase in apoptosis of the breast cancer cells. There was a direct relationship with increased concentrations of PA‐MSHA but not of PA. Completely different from PA, PA‐MSHA may impart antiproliferative effects against breast cancer cells by inducing apoptosis mediated by at least a death receptor‐related cell apoptosis signal pathway, and affecting the cell cycle regulation machinery. J. Cell. Biochem. 108: 195–206, 2009. © 2009 Wiley‐Liss, Inc.     

Terpene Conjugates of the Nigella sativa Seed‐Oil Constituent Thymoquinone with Enhanced Efficacy in Cancer Cells

Thymoquinone (TQ; 1 ) is a weak anticancer constituent of black seed oil. Derivatives bearing terpene‐terminated 6‐alkyl residues were tested in cells of human HL‐60 leukemia, 518A2 melanoma, multidrug‐resistant KB‐V1/Vbl cervix, and MCF‐7/Topo breast carcinomas, as well as in non‐malignant human foreskin fibroblasts. Derivatives with a short four‐atom spacer between quinone and cyclic monoterpene moieties were more antiproliferative than analogues with longer spacers. 6‐(Menthoxybutyryl)thymoquinone ( 3a ) exhibited single‐digit micromolar IC₅₀ (72 h) values in all four cell lines. It was seven times more active than TQ ( 1 ) in 518A2 melanoma cells and four times in KB‐V1/Vbl cervix carcinoma cells, while only half as toxic in the fibroblasts. Compound 3a was also not a substrate for the P‐gp and BCRP drug transporters of the resistant cancer cells. The caryophyllyl and germacryl conjugates 3e and 3f specifically inhibited the growth of the resistant MCF‐7 breast carcinoma cells. Conjugation of TQ with the triterpene betulinic acid via the OH group as in 3g led to a loss in activity, while conjugation via the carboxylic acid afforded compound 4 with nanomolar IC₅₀ (72 h) activity against HL‐60 cells. All anticancer‐active derivatives of TQ ( 1 ) induced apoptosis associated with DNA laddering, a decrease in mitochondrial membrane potential and a slight increase in reactive oxygen species.     

In vitro changes in mitochondrial potential,aggresome formation and caspase activity by a novel 17‐β‐estradiol analogue in breast adenocarcinoma cells

2‐Methoxyestradiol, a natural metabolite of estradiol, exerts antiproliferative and antitumour properties in vitro and in vivo. Because of its low oral bioavailability, several promising analogues of 2‐methoxyestradiol have been developed. In this study, the in vitro influence of the compound, 2‐ethyl‐3‐O‐sulphamoyl‐estra‐1,3,5(10)16‐tetraene (C19), a non‐commercially available 17‐β‐estradiol analogue, was tested on the breast adenocarcinoma MCF‐7 cell line. The in vitro influence of 24 h exposure to 0.18 μM of C19 on MCF‐7 cells was evaluated on cell morphology, cell cycle progression and possible induction of apoptosis and autophagy. Polarization‐optical transmitted light differential interference contrast and fluorescence microscopy revealed the presence of cells blocked in metaphase, occurrence of apoptotic bodies and compromised cell density in C19‐treated cells. Hallmarks of autophagy, namely an increase in the number of acidic vacuoles and lysosomes, were also observed in C19‐treated samples. An increase in the number of cells present in the sub‐G₁ fraction, as well as a reduction in mitochondrial membrane potential was observed. No significant alterations in caspase 8 activity were observed. A twofold increase in aggresome formation was observed in C19‐treated cells. C19 induced both apoptosis and autophagy in MCF‐7 cells. Copyright © 2013 John Wiley & Sons, Ltd.     

An in vitro study of apoptotic like death in Leishmania donovani promastigotes by withanolides

The aim of this study was to isolate and evaluate the withanolides in inducing apoptotic like death in Leishmania donovani in vitro. Withanolides were fractionated and isolated from the leaves of Withania somnifera and LC-MS/MS analysis of two fractions namely, F5 and F6 of ethanolic extracts, obtained through column chromatography with silica gel, was performed. The antileishmanial effect of withanolides on L. donovani promastigotes was assessed in vitro using PI dye exclusion test. The effect of withanolides on promastigote morphology was determined by scanning electron microscopy. To understand their mode of action against L. donovani, DNA fragmentation, quantification of parasites at sub G₀/G₁ phase, determination of phosphatidylserine externalization, measurement of reactive oxygen species (ROS) and mitochondrial membrane potential (Ψ_m) were done. Results showed that LC–MS/MS analysis confirmed the presence of withanolides in isolated fractions. Treatment with withanolides resulted in morphological alterations from spindle to round shape and loss of flagella/cell integrity in promastigotes. Moreover, it induced DNA nicks, cell cycle arrest at sub G₀/G₁ phase and externalization of phosphatidylserine in dose and time dependent manner via increase in ROS and decrease in Ψ_m. Results of this study indicate that withanolides induce apoptotic like death through the production of ROS from mitochondria and disruption of Ψ_m in promastigotes of L donovani.     

Inhibitory Activities of Butanol Fraction from Butea monosperma (Lam.) Taub. Bark Against Free Radicals,Genotoxins and Cancer Cells

The present study was undertaken to investigate antioxidant, antigenotoxic, and antiproliferative activity of butanol fraction (Bmbu) from bark of medicinal plant Butea monosperma. Antioxidant potency of Bmbu was examined by various in vitro assays. It was also investigated for antigenotoxic activity using Escherichia coli. PQ37 employing SOS chromotest. Further, cytotoxic and apoptosis inducing activity of Bmbu was evaluated in MCF‐7 breast cancer cells. Bmbu showed potent free radical scavenging ability in ABTS assay (IC₅₀ 56.70 μg/ml) and anti‐lipid peroxidation ability (IC₅₀ 40.39 μg/ml). 4NQO and H₂O₂ induced genotoxicity was suppressed by Bmbu in SOS chromotest by 74.26% and 82.02% respectively. It also inhibited the growth of MCF‐7 cells with GI₅₀ value of 158.71 μg/ml. Induction of apoptosis in MCF‐7 cells by Bmbu treatment was deciphered using confocal microscopy, flow cytometry, and neutral comet assay. Bmbu treatment increased cell population in sub‐G₁ phase (69.6%) indicating apoptotic cells. Further, Bmbu treatment resulted in increased reactive oxygen species generation and decreased mitochondrial membrane potential indicating involvement of mitochondrial dependent pathway of apoptosis. HPLC profiling showed the presence of polyphenols such as ellagic acid, catechin, quercetin, and gallic acid as its major constituents. Consequently, it is suggested that the phytoconstituents from this plant may be further exploited for development of novel drug formulation with possible therapeutic implication.     

PRKCB/protein kinase C,beta and the mitochondrial axis as key regulators of autophagy

Autophagy is the major intracellular system of degradation, and it plays an essential role in various biological events. Recent observations indicate that autophagy is modulated in response to the energy status of the mitochondrial compartment. However, the exact signaling mechanism that controls autophagy under these conditions remains unclear. In this study, we report that the activation of protein kinase C β (PRKCB), a member of the classical PRKCs, negatively modulates the mitochondrial energy status and inhibits autophagy. Furthermore, cells treated with a pharmacological PRKCB inhibitor, and prkcb knockout MEFs showed an increase in autophagy both in vitro and in vivo, as well as an increased mitochondrial membrane potential (Ψ_m), suggesting a strong involvement of mitochondrial energy in the modulation of the autophagy machinery. Finally, we show that factors that increase the Ψ_m oppose the PRKCB-dependent inhibition of autophagy. Altogether, these data underscore the importance of PRKCB in the regulation of autophagy; moreover, the finding that a pharmacological modulation of the Ψ_m modifies autophagy levels may be useful in fighting pathologies (including various types of cancer and neurodegenerative disorders) that are characterized by reduced levels of autophagy.     

Corilagin inhibits breast cancer growth via reactive oxygen species‐dependent apoptosis and autophagy

Corilagin is a component of Phyllanthus urinaria extract and has been found of possessing anti‐inflammatory, anti‐oxidative, and anti‐tumour properties in clinic treatments. However, the underlying mechanisms in anti‐cancer particularly of its induction of cell death in human breast cancer remain undefined. Our research found that corilagin‐induced apoptotic and autophagic cell death depending on reactive oxygen species (ROS) in human breast cancer cell, and it occurred in human breast cancer cell (MCF‐7) only comparing with normal cells. The expression of procaspase‐8, procaspase‐3, PARP, Bcl‐2 and procaspase‐9 was down‐regulated while caspase‐8, cleaved PARP, caspase‐9 and Bax were up‐regulated after corilagin treatment, indicating apoptosis mediated by extrinsic and mitochondrial pathways occurred in MCF‐7 cell. Meanwhile, autophagy mediated by suppressing Akt/mTOR/p70S6K pathway was detected with an increase in autophagic vacuoles and LC3‐II conversion. More significantly, inhibition of autophagy by chloroquine diphosphate salt (CQ) remarkably enhanced apoptosis, while the caspase inhibitor z‐VAD‐fmk failed in affecting autophagy, suggesting that corilagin‐induced autophagy functioned as a survival mechanism in MCF‐7 cells. In addition, corilagin induced intracellular reactive oxygen species (ROS) generation, when reduced by ROS scavenger NAC, apoptosis and autophagy were both down‐regulated. Nevertheless, in SK‐BR3 cell which expressed RIP3, necroptosis inhibitor Nec‐1 could not alleviate cell death induced by corilagin, indicating necroptosis was not triggered. Subcutaneous tumour growth in nude mice was attenuated by corilagin, consisting with the results in vitro. These results imply that corilagin inhibits cancer cell proliferation through inducing apoptosis and autophagy which regulated by ROS release.     

Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes

Background

Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear.

Methodology/Principal Findings

This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

Conclusions/Significance

The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism.     

The Proton Permeability of Liposomes Made from Mitochondrial Inner Membrane Phospholipids: Comparison with Isolated Mitochondria

Unilamellar liposomes with native phospholipid fatty acid composition were prepared from rat liver mitochondrial inner membrane phospholipids by extrusion in medium containing 50 mm potassium. They were diluted into low potassium medium to establish a transmembrane potassium gradient. A known membrane potential was imposed by addition of valinomycin, and proton flux into liposomes was measured. Valinomycin in the range 10 pm–1nm was sufficient to fully establish membrane potential. Valinomycin concentrations above 3 nm catalyzed additional proton flux and were avoided. At 300 pm valinomycin, proton flux depended nonlinearly on membrane potential. At 160 mV membrane potential the flux was 30 nmol H⁺/min/mg phospholipid—approximately 5% of the proton leak flux under comparable conditions in isolated mitochondria, indicating that leak pathways through bulk phospholipid bilayer account for only a small proportion of total mitochondrial proton leak. Received: 5 August 1996/Revised: 1 October 1996     

Potential involvement of F0F1-ATP(synth)ase and reactive oxygen species in apoptosis induction by the antineoplastic agent erucylphosphohomocholine in glioblastoma cell lines

Erucylphosphohomocholine (ErPC3, Erufosine?) was reported previously to induce apoptosis in otherwise highly apoptosis-resistant malignant glioma cell lines while sparing their non-tumorigenic counterparts. We also previously found that the mitochondrial 18 kDa Translocator Protein (TSPO) is required for apoptosis induction by ErPC3. These previous studies also suggested involvement of reactive oxygen species (ROS). In the present study we further investigated the potential involvement of ROS generation, the participation of the mitochondrial respiration chain, and the role of the mitochondrial F_OF₁-ATP(synth)ase in the pro-apoptotic effects of ErPC3 on U87MG and U118MG human glioblastoma cell lines. For this purpose, cells were treated with the ROS chelator butylated hydroxyanisole (BHA), the mitochondrial respiration chain inhibitors rotenone, antimycin A, myxothiazol, and the uncoupler CCCP. Also oligomycin and piceatannol were studied as inhibitors of the F_O and F₁ subunits of the mitochondrial F_OF₁-ATP(synth)ase, respectively. BHA was able to attenuate apoptosis induction by ErPC3, including mitochondrial ROS generation as determined with cardiolipin oxidation, as well as collapse of the mitochondrial membrane potential (Δψ_m). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Δψ_m, normally induced by ErPC3, including the accompanying reductions in cellular ATP levels. Other inhibitors of the mitochondrial respiration chain, as well as piceatannol, did not show such effects. Consequently, our findings strongly point to a role for the F_O subunit of the mitochondrial F_OF₁-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Δψ_m as well as ROS generation by ErPC3 and TSPO.     

Mitochondrial inhibitors activate influx of external Ca in sea urchin sperm

Sea urchin sperm have a single mitochondrion which, aside from its main ATP generating function, may regulate motility, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and possibly the acrosome reaction (AR). We have found that acute application of agents that inhibit mitochondrial function via differing mechanisms (CCCP, a proton gradient uncoupler, antimycin, a respiratory chain inhibitor, oligomycin, a mitochondrial ATPase inhibitor and CGP37157, a Na⁺/Ca²⁺ exchange inhibitor) increases [Ca²⁺]_i with at least two differing profiles. These increases depend on the presence of extracellular Ca²⁺, which indicates they involve Ca²⁺ uptake and not only mitochondrial Ca²⁺ release. The plasma membrane permeation pathways activated by the mitochondrial inhibitors are permeable to Mn²⁺. Store-operated Ca²⁺ channel (SOC) blockers (Ni²⁺, SKF96365 and Gd²⁺) and internal-store ATPase inhibitors (thapsigargin and bisphenol) antagonize Ca²⁺ influx induced by the mitochondrial inhibitors. The results indicate that the functional status of the sea urchin sperm mitochondrion regulates Ca²⁺ entry through SOCs. As neither CCCP nor dicycloexyl carbodiimide (DCCD), another mitochondrial ATPase inhibitor, eliminate the oligomycin induced increase in [Ca²⁺]_i, apparently oligomycin also has an extra mitochondrial target.