Dissipation Of Delta Psi Research Articles

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 FOF1-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 FO and F1 subunits of the mitochondrial FOF1-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 (Δψm). Similarly, we found that oligomycin attenuated apoptosis and collapse of the Δψm, 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 FO subunit of the mitochondrial FOF1-ATP(synth)ase in ErPC3-induced apoptosis and dissipation of Δψm as well as ROS generation by ErPC3 and TSPO.

Bacterial Porin Disrupts Mitochondrial Membrane Potential and Sensitizes Host Cells to Apoptosis

The bacterial PorB porin, an ATP-binding β-barrel protein of pathogenic Neisseria gonorrhoeae, triggers host cell apoptosis by an unknown mechanism. PorB is targeted to and imported by host cell mitochondria, causing the breakdown of the mitochondrial membrane potential (ΔΨm). Here, we show that PorB induces the condensation of the mitochondrial matrix and the loss of cristae structures, sensitizing cells to the induction of apoptosis via signaling pathways activated by BH3-only proteins. PorB is imported into mitochondria through the general translocase TOM but, unexpectedly, is not recognized by the SAM sorting machinery, usually required for the assembly of β-barrel proteins in the mitochondrial outer membrane. PorB integrates into the mitochondrial inner membrane, leading to the breakdown of ΔΨm. The PorB channel is regulated by nucleotides and an isogenic PorB mutant defective in ATP-binding failed to induce ΔΨm loss and apoptosis, demonstrating that dissipation of ΔΨm is a requirement for cell death caused by neisserial infection.

BNIP3 mediates cell death by different pathways following localization to endoplasmic reticulum and mitochondrion

BNIP3 (Bcl-2/adenovirus E1B 19-kDa interacting protein 3) is a BH3-only proapoptotic member of the Bcl-2 family. Because the interaction of Bcl-2 proteins with intracellular Ca(2+) stores has been linked to apoptosis, the role of Ca(2+) transfer between endoplasmic reticulum (ER) and mitochondria in BNIP3-mediated cell death was determined in a rat dopaminergic neuronal cell line, Mes 23.5. BNIP3 mutants were constructed to target either ER or mitochondria. Localization of BNIP3 to the ER membrane facilitated release of Ca(2+) and subsequently increased uptake of Ca(2+) into mitochondria. Excessive accumulation of mitochondrial Ca(2+) decreased mitochondrial membrane potential (DeltaPsi(m)), resulting in execution of a caspase-independent cell death. Reduction of ER Ca(2+) induced by ER-targeted BNIP3 and the subsequent cell death was blocked by the antiapoptotic protein, Bcl-2. On the other hand, mitochondria-targeted BNIP3 initiated apoptosis by a Ca(2+)-independent mechanism by inducing mitochondrial pore transition and dissipation of DeltaPsi(m). The disruption of DeltaPsi(m) and cell death was not blocked by Bcl-2 overexpression. These findings show that BNIP3 undergoes a dual subcellular localization and initiates different cell death signaling events in the ER and mitochondria. Bcl-2 counters the BNIP3-initiated mobilization of ER Ca(2+) depletion to reduce the level of apoptosis.

Effects of Tl+ on ion permeability, membrane potential and respiration of isolated rat liver mitochondria

It is known that permeability of the inner mitochondrial membrane is low to most univalent cations (K(+), Na(+), H(+)) but high to Tl(+). Swelling, state 4, state 3, and 2,4-dinitrophenol (DNP)-stimulated respiration as well as the membrane potential (DeltaPsi(mito)) of rat liver mitochondria were studied in media containing 0-75 mM TlNO(3) either with 250 mM sucrose or with 125 mM nitrate salts of other monovalent cations (KNO(3), or NaNO(3), or NH(4)NO(3)). Tl(+) increased permeability of the inner mitochondrial membrane to K(+), Na(+), and H(+), that was manifested as stimulation of the swelling of nonenergized and energized mitochondria as well as via an increase of state 4 and dissipation of DeltaPsi(mito). These effects of Tl(+) increased in the order of sucrose <K(+) <Na(+) <or= NH(4)(+). They were stimulated by inorganic phosphate and decreased by ADP, Mg(2+), and cyclosporine A. Contraction of energized mitochondria, swollen in the nitrate media, was markedly inhibited by quinine. It suggests participation of the mitochondrial K(+)/H(+) exchanger in extruding of Tl(+)-induced excess of univalent cations from the mitochondrial matrix. It is discussed that Tl(+) (like Cd(2+) and other heavy metals) increases the ion permeability of the inner membrane of mitochondria regardless of their energization and stimulates the mitochondrial permeability transition pore in low conductance state. The observed decrease of state 3 and DNP-stimulated respiration in the nitrate media resulted from the mitochondrial swelling rather than from an inhibition of respiratory enzymes as is the case with the bivalent heavy metals.

The effect of oxygenation level on cerebral post‐traumatic apoptotsis is modulated by the 18‐kDa translocator protein (also known as peripheral‐type benzodiazepine receptor) in a rat model of cortical contusion

Hyperbaric hyperoxia has been shown to reduce apoptosis in brain injury. As the 18-kDa translocator protein (TSPO), also known as peripheral-type benzodiazepine receptor, is closely associated with the mitochondrial transition pore and because of its role in mitochondrial respiration and apoptosis, we hypothesized that reduction of apoptosis by hyperoxia may involve the TSPO. TSPO and transferase-mediated dUTP nick end labelling (TUNEL) immunopositivity was first assessed in cortical contusion, created by dynamic cortical deformation, by immunohistochemistry in rats exposed to normoxia [(dynamic cortical deformation (DCD)], normobaric hyperoxia or hyperbaric hyperoxia [hyperbaric oxygen therapy (HBO)]. In a second step, transmembrane mitochondrial potential (Deltapsi(M)) and caspase 9 activity were assessed in the injured area in comparison with the noninjured hemisphere. Measurements were performed in DCD and HBO groups. A third group receiving both HBO and the TSPO ligand PK11195 was investigated as well. TSPO correlated quantitatively and regionally with TUNEL immunopositivity in the perilesional area. Hyperoxia reduced both the number of TSPO expressing and TUNEL positive cells in the perilesional area, and this effect proved to be pressure dependent. After contusion, we demonstrated a dissipation of Deltapsi(M) in isolated mitochondria and an elevation of caspase 9 activity in tissue homogenates from the contused area, both of which could be substantially reversed by hyperbaric hyperoxia. This protective effect of hyperoxia was reversed by PK11195. The present findings suggest that the protective effect of hyperoxia may be due to a negative regulation of the proapoptotic function of mitochondrial TSPO, including conservation of the mitochondrial membrane potential.

NO mobilizes intracellular Zn2+ via cGMP/PKG signaling pathway and prevents mitochondrial oxidant damage in cardiomyocytes.

Our aim was to determine if NO prevents mitochondrial oxidant damage by mobilizing intracellular free zinc (Zn(2+)). Zn(2+) levels were determined by imaging enzymatically isolated adult rat cardiomyocytes loaded with Newport Green DCF. Mitochondrial membrane potential (DeltaPsi(m)) was assessed by imaging cardiomyocytes loaded with tetramethylrhodamine ethyl ester (TMRE). S-nitroso-N-acetylpenicillamine (SNAP) dramatically increased Zn(2+), which was blocked by both ODQ and NS2028, two specific inhibitors of guanylyl cyclase. The protein kinase G (PKG) inhibitor KT5823 blocked the effect of SNAP while the PKG activator 8-Br-cGMP mimicked the action of SNAP, indicating that the cGMP/PKG pathway is responsible for the effect of SNAP. The increased Zn(2+) was prevented by 5-hydroxydecanoate (5HD) but was mimicked by diazoxide, implying that mitochondrial K(ATP) channel opening may account for this effect. Since chelation of Zn(2+) blocked the preventive effect of SNAP on H(2)O(2)-induced loss of DeltaPsi(m) and exogenous zinc (1 microM ZnCl(2)) prevented dissipation of DeltaPsi(m), Zn(2+) may play a critical role in the protective effect of NO. The MEK (mitogen-activated protein kinase or extracellular signal-regulated kinase) inhibitor PD98059 blocked the preventive effects of SNAP and zinc on DeltaPsi(m), indicating that extracellular signal-regulated kinase (ERK) mediates the protective effect of both these compounds on mitochondrial oxidant damage. A Western blot analysis further showed that ZnCl(2) significantly enhances phosphorylation of ERK, confirming the involvement of ERK in the action of Zn(2+). In isolated cardiomyocytes, NO mobilizes endogenous zinc by opening mitochondrial K(ATP) channels through the cGMP/PKG pathway. In these cells, Zn(2+) may be an important mediator of the action of NO on the mitochondrial death pathway.

The effect of metabolites on the pH gradient and membrane potential of the bean peribacteroid membrane

The effects of malate, succinate, and glutamate on the kinetics of changes in the pH gradient (delta pH) and membrane potential (delta psi) on the peribacteroid membrane (PBM) of the symbiosomes of bean root nodules varying in age were recorded spectrophotometrically. Addition of all the tested metabolites to potassium-free incubation medium stimulated a passive acidification of the peribacteroid space (PBS) and dissipation of delta psi in PBM of young developing nodules in the presence of the K+/H+ antiporter nigericin in the medium. However, in mature nodules with a high nitrogen-fixing activity, only malate and succinate (but not glutamate) increased delta pH during both passive and ATP-dependent PBS acidification. Dicarboxylates also caused dissipation of both delta pH in the presence of nigericin in the medium and delta psi generated on PBM by H+-ATPase. A decrease in the effects of metabolites on delta pH and the absent activity of the PBM H+ pump were observed in the aging nodules. The obtained data on the changes in deltapH and dlta psi caused by the metabolites in question suggest that PBM is permeable for all these metabolites only in young nodules. Only malate and succinate (but not glutamate) are transported through PBM in mature nodules; and the rate of metabolite translocation through PBM in aging nodules is decreased.

B-Type Natriuretic Peptide Enhances Mild Hypoxia-Induced Apoptotic Cell Death in Cardiomyocytes

In the case of left ventricle remodeling after myocardial infarction, cardiomyocyte apoptosis is attributed to increased cardiac workload by the stimulus such as chronic hypoxia. B-Type natriuretic peptide, being known as a reliable prognostic of cardiovascular pathology, plays an important role in the myocardial infarction. However, the action of B-type natriuretic peptide on cardiomyocytes undergoing apoptosis is unclear. In the present study, B-type natriuretic peptide have exhibited the enhancive effects on the mild hypoxia-induced cardiomyocyte apoptosis with the manifestation of facilitating phosphatidylserine evagination and increasing typical fragmented nuclei. In addition, B-type natriuretic peptide aggravated the dissipation of delta psi(m), the depletion of intracellular ATP and the increase of caspase-3 activity. 8-Bromo-cGMP, which increased cGMP independent of B-type natriuretic peptide, could mimic B-type natriuretic peptide's effects; whereas cGMP-dependent protein kinase inhibitor, Rp-8-br-cGMP inhibited that. Further study revealed the enhancive effect of BNP on down-regulation of Bcl-2 mRNA expression in the presence of mild hypoxia. In conclusion, the present study demonstrated that B-type natriuretic peptide aggravated the cardiomyocyte apoptosis by influencing hypoxia-induced mitochondrial death pathway, which is true at least in this oxygen deprivation model; and this effect was partially realized through intracellular cGMP.

Involvement of mitochondrial aggregation in arsenic trioxide (As2O3)‐induced apoptosis in human glioblastoma cells

Arsenic trioxide (As(2)O(3)) is effective against acute promyelocytic leukemia and has potential as a novel treatment against malignant solid tumors. As(2)O(3) induces differentiation and inhibits growth. It also causes mitochondrial damage mediated by the production of reactive oxygen species (ROS) and the dissipation of mitochondrial transmembrane potential (DeltaPsi(m)), leading to apoptosis. Mitochondria might be the key target of antitumor activity by As(2)O(3); however, its mechanisms have not been completely elucidated. Using two human glioblastoma cell lines, A172 and T98G, we found that As(2)O(3) induced apoptosis in A172 cells but not in T98G cells. As(2)O(3)-induced ROS production was observed in both cell lines; however, the dissipation of DeltaPsi(m), Bax oligomerization and caspase activation occurred only in As(2)O(3)-sensitive A172 cells. To determine the mechanisms of As(2)O(3)-induced apoptosis after ROS generation, we examined the change of mitochondrial morphology. As we reported previously, mitochondrial aggregation occurs before cytochrome c release during apoptosis, thus playing a role in cell death progression. We observed mitochondrial aggregation in As(2)O(3)-sensitive A172 cells but not in T98G cells treated with As(2)O(3). Using laser scanning cytometry, we quantitatively confirmed the results, which indicate that mitochondrial aggregation plays an important role in regulating sensitivity to As(2)O(3)-induced apoptosis. We propose a sequential process involving ROS generation, mitochondrial aggregation, Bax oligomerization and DeltaPsi(m) dissipation, and caspase activation during As(2)O(3)-induced apoptosis.

Nitrate Uptake in the Halotolerant Cyanobacterium Aphanothece halophytica is energy-dependent driven by ΔpH

The energetics of nitrate uptake by intact cells of the halotolerant cyanobacterium Aphanothece halophytica were investigated. Nitrate uptake was inhibited by various protonophores suggesting the coupling of nitrate uptake to the proton motive force. An artificially-generated pH gradient across the membrane (DeltapH) caused an increase of nitrate uptake. In contrast, the suppression of DeltapH resulted in a decrease of nitrate uptake. The increase of external pH also resulted in an enhancement of nitrate uptake. The generation of the electrical potential across the membrane (Deltapsi) resulted in no elevation of the rate of nitrate uptake. On the other hand, the valinomycin-mediated dissipation of Deltapsi caused no depression of the rate of nitrate uptake. Thus, it is unlikely that Deltapsi participated in the energization of the uptake of nitrate. However, Na(+)-gradient across the membrane was suggested to play a role in nitrate uptake since monensin which collapses Na(+)-gradient strongly inhibited nitrate uptake. Exogenously added glucose and lactate stimulated nitrate uptake in the starved cells. N, N'-dicyclohexylcarbodiimide, an inhibitor of ATPase, could alsoinhibit nitrate uptake suggesting that ATP hydrolysis was required for nitrate uptake. All these results indicate that nitrate uptake in A. halophytica is ATP-dependent, driven by DeltapH and Na(+)-gradient.

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.

Involvement of Sox-4 in the cytochrome c-dependent AIF-independent apoptotic pathway in HeLa cells induced by Δ12-prostaglandin J2

Delta(12)-Prostaglandin (PG) J(2) is known to elicit an anti-neoplastic effects via apoptosis induction. Previous study showed Delta(12)-PGJ(2)-induced apoptosis utilized caspase cascade through cytochrome c-dependent pathways in HeLa cells. In this study, the cellular mechanism of Delta(12)-PGJ(2)- induced apoptosis in HeLa cells, specifically, the role of two mitochondrial factors; bcl-2 and apoptosis-inducing factor (AIF) was investigated. Bcl-2 attenuated Delta(12)-PGJ(2)-induced caspase activation, loss of mitochondrial transmembrane potential (Deltapsi(m)), nuclear fragmentation, DNA laddering, and growth curve inhibition for approximately 24 h, but not for longer time. AIF was not released from mitochondria, even if the Deltapsi(m) was dissipated. One of the earliest events observed in Delta(12)-PGJ(2)-induced apoptotic events was dissipation of Deltapsi(m), the process known to be inhibited by bcl-2. Pre-treatment of z-VAD- fmk, the pan-caspase inhibitor, resulted in the attenuation of ym depolarization in Delta(12)-PGJ(2)-induced apoptosis. Up-regulation of Sox-4 protein by Delta(12)-PGJ(2) was observed in HeLa and bcl-2 overexpressing HeLa B4 cell lines. Bcl-2 overexpression did not attenuate the expression of Sox-4 and its expression coincided with other apoptotic events. These results suggest that Delta(12)-PGJ(2) induced Sox-4 expression may activate another upstream caspases excluding the caspase 9-caspase 3 cascade of mitochondrial pathway. These and previous findings together suggest that Delta(12)-PGJ(2)-induced apoptosis in HeLa cells is caspase-dependent, AIF-independent events which may be affected by Sox-4 protein expression up-regulated by Delta(12)-PGJ(2).

Mitochondrial Thioredoxin System: EFFECTS OF TrxR2 OVEREXPRESSION ON REDOX BALANCE, CELL GROWTH, AND APOPTOSIS

Thioredoxin-2 (Trx2) is a mitochondrial protein-disulfide oxidoreductase essential for control of cell survival during mammalian embryonic development. This suggests that mitochondrial thioredoxin reductase-2 (TrxR2), responsible for reducing oxidized Trx2, may also be a key player in the regulation of mitochondria-dependent apoptosis. With this in mind, we investigated the effects of overexpression of TrxR2, Trx2, or both on mammalian cell responses to various apoptotic inducers. Stable transfectants of mouse Neuro2A cells were generated that overexpressed TrxR2 or an EGFP-TrxR2 fusion protein. EGFP-TrxR2 was enzymatically active and was localized in mitochondria. TrxR2 protein level and TrxR activity could be increased up to 6-fold in mitochondria. TrxR2 and EGFP-TrxR2 transfectants showed reduced growth rates as compared with control cells. This growth alteration was not due to cytotoxic effects nor related to changes in basal mitochondrial transmembrane potential (DeltaPsi(m)), reactive oxygen species production, or to other mitochondrial antioxidant components such as Trx2, peroxyredoxin-3, MnSOD, GPx1, and glutathione whose levels were not affected by increased TrxR2 activity. In response to various apoptotic inducers, the extent of DeltaPsi(m) dissipation, reactive oxygen species induction, caspase activation, and loss of viability were remarkably similar in TrxR2 and control transfectants. Excess TrxR2 did not prevent trichostatin A-mediated neuronal differentiation of Neuro2A cells nor did it protect them against beta-amyloid neurotoxicity. Neither massive glutathione depletion nor co-transfection of Trx2 and TrxR2 in Neuro2A (mouse), COS-7 (monkey), or HeLa (human) cells revealed any differential cellular resistance to prooxidant or non-oxidant apoptotic stimuli. Our results suggest that neither Trx2 nor TrxR2 gain of function modified the redox regulation of mitochondria-dependent apoptosis in these mammalian cells.

Glibenclamide interferes with mitochondrial bioenergetics by inducing changes on membrane ion permeability.

The interference of glibenclamide, an antidiabetic sulfonylurea, with mitochondrial bioenergetics was assessed on mitochondrial ion fluxes (H+, K+, and Cl-) by passive osmotic swelling of rat liver mitochondria in K-acetate, KNO3, and KCl media, by O2 consumption, and by mitochondrial transmembrane potential (Deltapsi). Glibenclamide did not permeabilize the inner mitochondrial membrane to H+, but induced permeabilization to Cl- by opening the inner mitochondrial anion channel (IMAC). Cl- influx induced by glibenclamide facilitates K+ entry into mitochondria, thus promoting a net Cl-/K+ cotransport, Deltapsi dissipation, and stimulation of state 4 respiration rate. It was concluded that glibenclamide interferes with mitochondrial bioenergetics of rat liver by permeabilizing the inner mitochondrial membrane to Cl- and promoting a net Cl-/K+ cotransport inside mitochondria, without significant changes on membrane permeabilization to H+.

Reactive oxygen disrupts mitochondria in MA-10 tumor Leydig cells and inhibits steroidogenic acute regulatory (StAR) protein and steroidogenesis.

Reactive oxygen species (ROS) are involved in a variety of pathophysiological conditions of the testis, and oxidative stress is known to inhibit ovarian and testicular steroidogenesis. The site of ROS-mediated inhibition of steroidogenesis in the corpus luteum and MA-10 tumor Leydig cells was shown to be the hormone-sensitive mitochondrial cholesterol transfer step. The purpose of this study was to examine the effects of ROS on steroidogenic acute regulatory (StAR) protein in MA-10 cells and determine the extent to which MA-10 cell mitochondria are sensitive to oxidative stress. cAMP-stimulated progesterone production was inhibited in a dose-dependent manner in MA-10 cells exposed to H(2)O(2). StAR protein, but not mRNA levels, was decreased in parallel to changes in progesterone production. Even at the highest concentrations of H(2)O(2) tested, there was no effect on P450 side-chain cleavage enzyme protein levels. Oxidative stress from exposure to exogenous xanthine oxidase and xanthine resulted in the inhibition of both progesterone production and StAR protein expression. The mature 30- and 32-kDa intramitochondrial forms of StAR were decreased relative to the 37-kDa extramitochondrial precursor form of StAR, indicating that the ROS-mediated inhibition of StAR protein was due, in part, to the inhibition of mitochondrial import and processing. Vital staining with the fluorescent dye tetramethylrhodamine ethyl ester was used to visualize changes in the mitochondrial electrochemical gradient-dependent membrane potential (Deltapsim). ROS caused a significant dissipation of Deltapsi(m) and time-dependent loss of tetramethylrhodamine ethyl ester fluorescence. The inhibitory effects of H(2)O(2) were transient. There was no evidence for ROS-induced cell death, and following H(2)O(2) removal in the presence of continuous treatment with 8-bromo-cAMP, StAR protein levels and progesterone production were restored. In addition, there was no loss of cell viability following treatment with H(2)O(2) or xanthine/xanthine oxidase as determined by trypan blue exclusion. H(2)O(2) did not cause a significant decrease in total cellular ATP levels. These data indicate that oxidative stress-mediated perturbation of the mitochondria and dissipation of Deltapsi(m) results in the inhibition of StAR protein expression and its import, processing, and cholesterol transfer activity. These findings confirm earlier studies demonstrating the requirement for maintenance of an intact Deltapsi(m) for StAR protein function in cholesterol transport. The significant reduction in the 32- to 30-kDa mature forms of StAR, cessation of cholesterol transport, and loss of Deltapsi(m) are consistent with mitochondrial perturbation because of oxidative stress. This mechanism likely contributes to a host of pathophysiological events evident in testicular disorders such as infection, reperfusion injury, aging, cryptorchidism, and varicocele.

Reactive oxygen species induce swelling and cytochrome c release but not transmembrane depolarization in isolated rat brain mitochondria.

1 In this study, we have used isolated brain mitochondria to investigate the effects of superoxide anions (O(2)(-)) on mitochondrial parameters related to apoptosis, such as swelling, potential, enzymatic activity, NAD(P)H, cytochrome c release, and caspase activity. 2 Addition of the reactive oxygen species (ROS) generator KO(2) produced brain mitochondrial swelling, which was blocked by cyclosporin A (CSA), and which was Ca(2+) independent. 3 Calcium induced mitochondrial swelling only at high concentrations and in the presence of succinate. This correlated with the increase in O(2)(-) production detected with hydroethidine in mitochondrial preparations exposed to Ca(2+) and the fact that ROS were required for Ca(2+)-induced mitochondrial swelling. 4 Superoxide anions, but not Ca(2+), decreased citrate synthase and dehydrogenase enzymatic activities and dropped total mitochondrial NAD(P)H levels. 5 Calcium, but not O(2)(-), triggered a rapid loss of mitochondrial potential. Calcium-induced Deltapsi(m) dissipation was inhibited by Ruthenium Red, but not by CSA. 6 Calcium- and superoxide-induced mitochondrial swelling released cytochrome c and increased caspase activity from isolated mitochondria in a CS A-sensitive manner. 7 In summary, superoxide potently triggers mitochondrial swelling and the release of proteins involved in activation of postmitochondrial apoptotic pathways in the absence of mitochondrial depolarization.

Chemotherapy enhances TNF-related apoptosis-inducing ligand DISC assembly in HT29 human colon cancer cells.

Cytokines such as Fas-ligand (Fas-L) and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAIL) can induce human colon cancer cell apoptosis through engagement of their death domain receptors. All the cancer cells are not sensitive to these cytokines. We have shown recently that low doses of cytotoxic drugs could restore TRAIL-induced cell death in resistant colon cancer cell lines. The present work further explores the death pathway triggered by the cytotoxic drug/TRAIL combination in HT-29 colon cancer cells (www.alexis-corp.com). Clinically relevant concentrations of cisplatin, doxorubicin and 5-fluorouracil synergize with TRAIL to trigger HT-29 cell death. Activation of this pathway leads to apoptosis that involves both caspases and the mitochondria. An increased recruitment of Fas-associated death domain (FADD) and procaspase-8 to the TRAIL-induced death-inducing signaling complex (DISC) was shown in cells exposed to anticancer drugs. Following caspase-8 activation at the DISC level, the mitochondria-dependent death pathway is activated, as demonstrated by the cleavage of Bid, the dissipation of DeltaPsi(m), the release of mitochondrial proteins in the cytosol and the inhibitory effect of Bcl-2 expression. Importantly, besides mitochondrial potentiation, we show here that cytotoxic drugs sensitize HT-29 colon cancer cells to TRAIL-induced cell death by enhancing FADD and procaspase-8 recruitment to the DISC, a novel mechanism whose efficacy could depend partly on Bcl-2 expression level.

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).

Differential Regulation of Doxorubicin-induced Mitochondrial Dysfunction and Apoptosis by Bcl-2 in Mammary Adenocarcinoma (MTLn3) Cells

Various anticancer drugs cause mitochondrial perturbations in association with apoptosis. Here we investigated the involvement of caspase- and Bcl-2-dependent pathways in doxorubicin-induced mitochondrial perturbations and apoptosis. For this purpose, we set up a novel three-color flow cytometric assay using rhodamine 123, annexin V-allophycocyanin, and propidium iodide to assess the involvement of the mitochondria in apoptosis caused by doxorubicin in the breast cancer cell line MTLn3. Doxorubicin-induced apoptosis was preceded by up-regulation of CD95 and CD95L and a collapse of mitochondrial membrane potential (Deltapsi) occurring prior to phosphatidylserine externalization. This drop in Deltapsi was independent of caspase activity, since benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone did not inhibit it. Benzyloxycarbonyl-Val-Ala-dl-Asp-fluoromethylketone also blocked activation of caspase-8, thus excluding an involvement of the death receptor pathway in Deltapsi dissipation. Furthermore, although overexpression of Bcl-2 in MTLn3 cells inhibited apoptosis, dissipation of Deltapsi was still observed. No decrease in Deltapsi was observed in cells undergoing etoposide-induced apoptosis. Immunofluorescent analysis of Deltapsi and cytochrome c localization on a cell-to-cell basis indicates that the collapse of Deltapsi and cytochrome c release are mutually independent in both normal and Bcl-2-overexpressing cells. Together, these data indicate that doxorubicin-induced dissipation of the mitochondrial membrane potential precedes phosphatidylserine externalization and is independent of a caspase- or Bcl-2-controlled checkpoint.

Zinc causes loss of membrane potential and alters production of reactive oxygen species in isolated brain mitochondria

Excessive accumulation of intracellular free zinc is lethal to neurons. The precise mechanism by which zinc kills neurons remains unclear, however, a number of reports suggest that zinc may impair neuronal metabolism. The present study examined how zinc affects the activity of isolated rat brain mitochondria. Using spectrofluorometry and polarography, we determined the effects of zinc on mitochondrial membrane potential (Δψ), reactive oxygen species (ROS) generation, and respiration. In mitochondria respiring on either glutamate + malate (G + M), succinate, or glycerol 3‐phosphate (G3P), addition of submicromolar levels of zinc caused dissipation of delta psi. Zinc‐induced depolarization was rapidly reversed by the addition of the membrane‐impermeant metal chelator EGTA. In all three substrate conditions, ruthenium red (200 nm) partially protected mitochondria from zinc‐induced depolarization, suggesting that the calcium uniporter may mediate mitochondrial zinc uptake. Conversely, addition of cyclosporin A (1 µM) afforded no protection, arguing against zinc‐induced permeability transition. ROS production was enhanced by zinc in mitochondria fueled by either G + M or G3P, but zinc inhibited succinate‐driven ROS production. Zinc inhibited respiration in phosphorylating mitochondria regardless of substrate conditions, and zinc also inhibited respiration in uncoupled mitochondria using G + M. Together, these results suggest that zinc has several deleterious effects on mitochondria that could contribute to neuronal injury.Acknowledgements: Supported by NS34138 and the American Heart Association.